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Sample records for coli expression purification

  1. Solubilization and purification of Escherichia coli expressed GST ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    May 18, 2009 ... prognosis, monitoring of therapy and diagnosis. VEGF has been identified as the target for the treatment of cancer. Though prokaryotic expression of VEGF has been done, the solubilization and purification is time consuming and empirical. In this study, VEGF165 and VEGF121 were cloned into. pGEX-4T-1 ...

  2. Expression and purification of recombinant hemoglobin in Escherichia coli

    DEFF Research Database (Denmark)

    Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela

    2011-01-01

    BACKGROUND: Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe...

  3. Recombinant expression, refolding, purification and characterization of Pseudomonas aeruginosa protease IV in Escherichia coli.

    Science.gov (United States)

    Zhao, Mingzhi; Cai, Man; Wu, Feilin; Zhang, Yao; Xiong, Zhi; Xu, Ping

    2016-10-01

    Several protease IV enzymes are widely used in proteomic research. Specifically, protease IV from Pseudomonas aeruginosa has lysyl endopeptidase activity. Here, we report the recombinant expression, refolding, activation, and purification of this protease in Escherichia coli. Proteolytic instability of the activated intermediate, a major obstacle for efficient production, is controlled through ammonium sulfate precipitation. The purified protease IV exhibits superior lysyl endopeptidase activity compared to a commercial product. Copyright © 2016. Published by Elsevier Inc.

  4. Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

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    L. Avilán

    1997-12-01

    Full Text Available We cloned the streptokinase (STK gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

  5. Homologous high-throughput expression and purification of highly conserved E coli proteins

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    Duchmann Rainer

    2007-06-01

    Full Text Available Abstract Background Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD. Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies. Results As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP, N-utilizing substance A (NusA, or glutathione S-transferase (GST to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS. Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500

  6. Purification and characterization of Streptococcus mutans glucosyltransferase (GtfC) expressed in Escherichia coli.

    Science.gov (United States)

    Chia, J S; Hsieh, C C; Yang, C S; Chen, J Y

    1995-11-01

    Streptococcus mutans constitutively expresses three glucosyltransferases, i.e., GtfB, GtfC, and GtfD; which synthesize glucan polymers from sucrose. To obtain individual GTF without complexing with one another, a purification strategy was developed to recover recombinant GTF expressed from Escherichia coli. The recombinant GtfC was aggregated and associated with the insoluble fraction in E. coli homogenates. GtfC was solublized with the 8M urea, renatured to its biologically active form by serial dialysis against sodium phosphate buffer, and subsequently purified to homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography. The GtfC enzyme preparation was purified 16.3-fold and the molecular weight was estimated to be 140 kDa. GtfC synthesized water insoluble glucan in a primer independent manner and its enzymatic activities could be enhanced by dextran. Purified GtfC had a pH optimum of 6.5, a K(m) of 9.26 mM for sucrose and a pI of 5.5. Distinct from the previous reports, results from this study offers an alternative for the purification of the recombinant GTFs free from any detergent contamination to make it more suitable for utilization in vivo.

  7. Expression and purification of an anti-clenbuterol single chain Fv antibody in Escherichia coli.

    Science.gov (United States)

    Wang, Hong; Liu, Xixia; He, Yongsheng; Dong, Jiexian; Sun, Yuanming; Liang, Yan; Yang, Jinyi; Lei, Hongtao; Shen, Yudong; Xu, Xiaoyan

    2010-07-01

    Recombinant antibodies with desirable characteristics that can replace polyclonal or monoclonal antibodies are important for enzyme-linked immunosorbent assay (ELISA) of residues of clenbuterol (CBL), an illicit veterinary drug. Here, we report our work on expression and purification of a mouse-derived anti-CBL single chain Fv (scFv) antibody in Escherichia coli (E. coli). An expression plasmid pBV220-CBL was constructed and transformed into E. coli BL21 (DH3) strain cells. After induction by temperature, the 6x His-tagged anti-CBL scFv antibodies were expressed with the yield of 31%. The solubilized inclusion bodies were extracted, denatured and then purified by Ni-NTA column chromatography. The purified recombinant target protein was analyzed by high performance liquid chromatography, SDS-PAGE and Western blotting, respectively. The results showed the prepared anti-CBL scFv antibodies posed HRP-anti-His-tag antibody-recognized activity and their purity was up to 96%. Moreover, an indirect competitive ELISA based on the anti-CBL scFv antibodies revealed that the limit of detection for CBL was 0.5 ng/ml and the linear range was 1.5-10.6 ng/ml. Taken together, these findings suggest that the prepared recombinant antibody can be used for future immunoassay detection for CBL. Copyright 2010 Elsevier Inc. All rights reserved.

  8. Efficient expression and purification of recombinant glutaminase from Bacillus licheniformis (GlsA) in Escherichia coli.

    Science.gov (United States)

    Sinsuwan, Sornchai; Yongsawatdigul, Jirawat; Chumseng, Suchintana; Yamabhai, Montarop

    2012-05-01

    Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation of glutamic acid and ammonium ion from glutamine. This enzyme functions in cellular metabolism of every organism by supplying nitrogen required for the biosynthesis of a variety of metabolic intermediates, while glutamic acid plays a role in both sensory and nutritional properties of food. So far there have been only a few reports on cloning, expression and characterization of purified glutaminases. Microbial glutaminases are enzymes with emerging potential in both the food and the pharmaceutical industries. In this research a recombinant glutaminase from Bacillus licheniformis (GlsA) was expressed in Escherichia coli, under the control of a ptac promoter. The recombinant enzyme was tagged with decahistidine tag at its C-terminus and could be conveniently purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The enzyme could be induced for efficient expression with IPTG, yielding approximately 26,000 units from 1-l shake flask cultures. The enzyme was stable at 30°C and pH 7.5 for up to 6h, and could be used efficiently to increase glutamic acid content when protein hydrolysates from soy and anchovy were used as substrates. The study demonstrates an efficient expression system for the production and purification of bacterial glutaminase. In addition, its potential application for bioconversion of glutamine to flavor-enhancing glutamic acid has been demonstrated. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography

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    Geisler Markus

    1998-01-01

    Full Text Available In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993 et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 &mgr;M and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

  10. Expression, purification, crystallization and preliminary diffraction data characterization of Escherichia coli ribonuclease II (RNase II)

    Energy Technology Data Exchange (ETDEWEB)

    McVey, Colin E. [Division of Biological Chemistry, ITQB - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras (Portugal); Amblar, Mónica; Barbas, Ana; Cairrão, Fátima [Division of Biology, ITQB - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras (Portugal); Coelho, Ricardo; Romão, Célia [Division of Biological Chemistry, ITQB - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras (Portugal); Arraiano, Cecília M. [Division of Biology, ITQB - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras (Portugal); Carrondo, Maria A.; Frazão, Carlos, E-mail: frazao@itqb.unl.pt [Division of Biological Chemistry, ITQB - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras (Portugal)

    2006-07-01

    Diffraction data from E. coli RNase II crystals of wild type and of an inactive mutant and its SeMet-derivative form were obtained to 2.44 and 2.74 Å resolution, providing a set of preliminary phases. An improved purification protocol allowed higher reproducibility in the crystallization of the mutant form. RNA degradation is important in the post-transcriptional control of gene expression. The processing, degradation and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a 643-amino-acid enzyme that degrades single-stranded RNA from its 3′-end, releasing ribonucleoside 5′-monophosphates. RNase II was expressed both as the wild type and as a D209N mutant form. The latter was also produced as an SeMet derivative. The various protein forms were crystallized using the vapour-diffusion method. Wild-type RNase II was crystallized in two crystal forms, both of which belonged to space group P2{sub 1}. X-ray diffraction data were collected to 2.44 and 2.75 Å resolution, with unit-cell parameters a = 56.8, b = 125.7, c = 66.2 Å, β = 111.9° and a = 119.6, b = 57.2, c = 121.2 Å, β = 99.7°, respectively. The RNase II D209N mutant gave crystals that belonged to space group P6{sub 5}, with unit-cell parameters a = b = 86.3, c = 279.2 Å, and diffracted to 2.74 Å. Diffraction data from the mutant and its SeMet derivative enabled the determination of a partial Se-atom substructure by SIRAS.

  11. In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli.

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    Aytak Novinrooz

    Full Text Available E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC, and fatal hemolytic uremic syndrome (HUS and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA, and B subunit of E. coli heat labile enterotoxin (LTB which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+ expression vector and transferred to E. coli BL21(DE3 cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3 cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG. The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient

  12. Use of polymeric membranes for purification of an E. coli expressed biotherapeutic protein.

    Science.gov (United States)

    Muthukumar, S; Rathore, Anurag S

    2016-01-01

    Polymers have had a significant impact on the field of bioseparations in the past few decades. Most recently, membrane chromatography has emerged as an efficient alternative to the conventional packed-bed chromatography by eliminating the diffusion-related limitations associated with the traditional resin beads. In this article, we examine six membrane adsorbers for purification of granulocyte colony-stimulating factor (GCSF), an Escherichia coli-based biotherapeutic. These adsorbers differ either in their base matrix or in the surface chemistry. The role of interactions between the filter surfaces and the protein molecules in effecting these separations is the focus of the article.

  13. Expression Optimizing and Purification of Recombinant Human Leukemia Inhibitory Factor Produced in E. coli Strain BL21

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    Houman Kahroba

    2015-02-01

    Full Text Available Background: Leukemia inhibitory factor (LIF is a glycoprotein, categorized as a subfamily of interleukin 6 cytokines which is known in many mammolals. A pluripotent cytokine with a wide biological function range has numerous effects on target cells. The LIF regulates neuron survival, hematopoiesis and seen in LIF-/- knockout mice affects blastocyst implantation, also acts as pre-inflammolatory cytokine, and regulates immolune response. Further, it is able to maintain stem cells poly potency. The main object of present work was expression, optimizing, and purification of recombinant human leukemia inhibitory factor (rhLIF. Materials and Methods: In this experimental study, Pet28 (+ carrying the LIF gene and kanamycin resistance marker was cloned in E. coli strain BL21. The induction was optimized by altering 3 factors including the temperature, the induction time, and the concentration of the Isopropyl β-D-1-thiogalactopyranoside (IPTG as inducer. The purification of the recombinant human LIF (rhLIF was done by single step affinity chromatography. After the purification, method accuracy was proved by Sodium dodecyl sulfate (SDS -PAGE electrophoresis and Western blotting. Results: Optimizing of the expression was reached by changing various parameters, and purification has been done successful. Conclusion: rhLIF undergoes modification by glycosylation to get its full functionality. The produced rhLIF in prokaryotic host in this work is lacking of glycosylation. However, its proper function should be evaluated in further studies.

  14. [Cloning of the fimA gene of Porphyromonas gingivalis and its expression and purification in Escherichia coli].

    Science.gov (United States)

    Liu, Wei; Yu, Fei; Chen, Wei-Min; He, Wei

    2009-12-01

    OBJECTIVE; To clone the fimA gene of Porphyromonas gingivalis (P. gingivalis) and detect its expression in Escherichia coli (E. coli). The fimA gene was obtained by PCR from the genome of P. gingivalis to construct a prokaryotic expression plasmid pT-BAD/fimA. pT-BAD/fimA was transformed into E. coli BL21 (DE3) competent cells and the recombination protein was characterized by means of matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. The bound protein was eluted with different concentrations of imidazole (250, 200, 150, 100, 50 micromol x L(-1)) respectively. DNA sequencing showed that the fragment was 99.9% consistent with that of the published. After induction with L-arabinose, a new 3.8 x 10(4) protein appeared on SDS-PAGE gel. The protein was further identified by MALDI-TOF-MS. Purity of 95% of the target protein was purified by Ni-NTA Purification System after eluted with 100 micromol x L(-1) imidazole. The fimA gene of P. gingivalis was cloned successfully and its protein was expressed correctly in E. coli. A high purity of protein FimA was obtained and it could be applied for follow-up researches.

  15. Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli.

    Science.gov (United States)

    Chiang, Chung-Jen; Chen, Hong-Chen; Chao, Yun-Peng; Tzen, Jason T C

    2005-06-15

    Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.

  16. Purification and Characterization of Recombinant Staphylococcus haemolyticus DNA Gyrase and Topoisomerase IV Expressed in Escherichia coli

    OpenAIRE

    Bronstein, Joel C.; Olson, Stacey L.; LeVier, Kristin; Tomilo, Mark; Weber, Peter C.

    2004-01-01

    The subunits of DNA gyrase and topoisomerase IV from Staphylococcus haemolyticus were expressed in Escherichia coli, purified to homogeneity, and used to reconstitute active enzymes that were sensitive to known topoisomerase inhibitors. This represents the first description of a method for isolating type II topoisomerases of a coagulase-negative staphylococcal species.

  17. Data on enhanced expression and purification of camelid single domain antibodies from Escherichia coli classical inclusion bodies.

    Science.gov (United States)

    Maggi, Maristella; Scotti, Claudia

    2017-06-01

    Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in E. coli classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007).

  18. High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli

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    Huang Yadong

    2010-02-01

    Full Text Available Abstract Background Fibroblast growth factor 21 (FGF21 is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21 in Escherichia coli (E. coli is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the FGF21 with SUMO (Small ubiquitin-related modifier by polymerase chain reaction (PCR, and expressed the fused gene in E. coli BL21(DE3. Results By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC was shown to be higher than 96% with low endotoxin level (in vivo animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ injection. Conclusions This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.

  19. Expression and purification of L-asparaginase from Escherichia coli and the inhibitory effects of cyclic dipeptides.

    Science.gov (United States)

    Zhang, Yanan; Li, Dan; Li, Yan

    2017-09-01

    L-asparaginase, a key enzyme involved in nitrogen metabolism, is an effective anti-tumour agent. Cyclic dipeptides, a group of compounds, contain several important biological functions. In this paper, we proposed a novel method for L-asparaginase expression and purification from Echerichia coli and determined the effect of cyclic dipeptides on the enzymatic activity of recombinant L-asparaginase. The gene ansB encoding L-asparaginase was amplified from the genome of E. coli BL21 (DE3) by polymerase chain reaction and sub-cloned into pET-15b vector to construct expressing plasmid pET-15b-ansB. The expression of recombinant protein was purified by affinity chromatography using a nickel resin followed by anion exchange chromatography. The purity and quality of the recombinant L-asparaginase were optimised. The results indicated that km for the recombinant L-asparaginase was 3.02 × 10(-4) mol/L. Both cyclo-(Pro-Tyr) and cyclo-(Pro-Phe) could inhibit the activity of recombinant L-asparaginase at the level of 10(-5) mol/L.

  20. Expression and purification of functional human mu opioid receptor from E.coli.

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    Yanbin Ma

    Full Text Available N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3-0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K(D of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.

  1. Expression and purification of functional human mu opioid receptor from E.coli.

    Science.gov (United States)

    Ma, Yanbin; Kubicek, Jan; Labahn, Jörg

    2013-01-01

    N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3-0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K(D) of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.

  2. Purification and characterization of recombinant protein kinase CK2 from Zea mays expressed in Escherichia coli

    DEFF Research Database (Denmark)

    Riera, Marta; Pages, Montserrat; Issinger, Olaf Georg

    2003-01-01

    Recombinant protein kinase subunits rmCK2alpha-1 and rmCK2beta-1 from Zea mays were expressed separately in Escherichia coli and assembled to a fully active tetrameric holoenzyme complex in vitro. The obtained maize holoenzyme was purified to homogeneity, biochemically characterized, and compared...... between the maize holoenzyme and the catalytic subunit from CK2 maize shows that the incorporation of the catalytic subunit into the holoenzyme leads to a 14-fold activation in the case of ATP and 8-fold activation in the case of GTP. The maize holoenzyme is about 10 times more sensitive towards CK2...

  3. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

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    Yi Ma

    2016-01-01

    Full Text Available Human epidermal growth factor (hEGF is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  4. High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli.

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    Cipáková, Ingrid; Hostinová, Eva; Gasperík, Juraj; Velebný, Vladimír

    2004-09-01

    In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with isolation procedures and toxicity caused by rhBD-1 to the host organism. As a result, the majority of fusion protein (LMM-rhBD-1(43)) was obtained in the soluble state, isolated by a low salt-high salt treatment of total cell protein. The rhBD-1(43) was cleaved from the fusion with Protease 4 and purified on CM Sepharose Fast Flow column with the yield of approximately 1 mg rhBD-1(43) from 6 g of wet weight cells. Purified rhBD-1(43) showed antimicrobial activity against E. coli ML-35p at a concentration of 129 microM. The procedure of rhBD-1 expression and purification we present can provide a reliable and simple method for production of different cationic peptides for biological studies.

  5. Cloning, Purification, and Partial Characterization of Bacillus subtilis Urate Oxidase Expressed in Escherichia coli

    Science.gov (United States)

    Pfrimer, Pollyana; de Moraes, Lidia Maria Pepe; Galdino, Alexsandro Sobreira; Salles, Loise Pedrosa; Reis, Viviane Castelo Branco; De Marco, Janice Lisboa; Prates, Maura Vianna; Bloch, Carlos; Torres, Fernando Araripe Gonçalves

    2010-01-01

    Urate oxidase (EC 1.7.3.3) is an enzyme involved in purine metabolism which is used in the treatment of gout and as diagnostic reagent for detection of uric acid. In order to produce this enzyme in large quantities for biotechnological purposes, the gene coding for the Bacillus subtilis urate oxidase was cloned and heterologously expressed in Escherichia coli. Time course induction in E. coli showed an induced protein with an apparent molecular mass of ∼60 kDa. Soluble recombinant enzyme was purified in a single-step procedure using Ni-NTA column. The enzyme was purified 2.1-fold with a yield of 56% compared to the crude extract. MALDI-TOF analysis revealed an ion with a mass of 58675 Da which is in agreement with the expected mass of the recombinant protein. The purified enzyme showed an optimal pH and temperature of 8.0 and 37°C, respectively, and retained 90% of its activity after 72 hours of incubation at −20°C and 4°C. PMID:20168977

  6. High-level expression in Escherichia coli, purification and mosquito-larvicidal activity of the binary toxin from Bacillus sphaericus.

    Science.gov (United States)

    Promdonkoy, Boonhiang; Promdonkoy, Peerada; Panyim, Sakol

    2008-12-01

    The mosquito-larvicidal binary toxin produced by Bacillus sphaericus consists of two polypeptides: BinA and BinB. Both proteins function together, and maximum toxicity is obtained when both are present in equimolar ratio. Cloning and expression of each component separately in heterologous hosts led to low toxicity of the crystal proteins. To improve the expression level, the purification process, and the activity of the binary toxin, the binA and binB genes were separately cloned in Escherichia coli. Each gene was fused in frame to the glutathione S-transferase (GST) gene to be expressed as GST-fusion protein (GST-BinA and GST-BinB). A high expression level was observed from both constructs, and the fusion proteins exhibited high toxicity to Culex quinquefasciatus larvae. High-purity toxin could be obtained by affinity chromatography. The result suggests that GST moiety facilitates high protein production and enables better solubility of the toxin inclusions inside the larval gut, leading to higher toxicity of the fusion protein.

  7. Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli.

    Science.gov (United States)

    Wen, Yong; Shi, Xunlong; Yuan, Zhongyi; Zhou, Pei

    2004-11-01

    The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-beta-D-galactoside (IPTG) was found to be 5 microM. The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55 degrees C, respectively. The Km and Vmax are 17.6 microM and 23.8 U/mg, respectively. Therefore, the high yield and high specific activity of recombinant KcPGA produced in E. coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications.

  8. Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in Esherichia coli

    Directory of Open Access Journals (Sweden)

    Li-Xi Niu

    2015-04-01

    Full Text Available Human enterokinase (synonym: enteropeptidase, EC 3.4.21.9 light chain (hEKL gene was designed and artificially synthesized with built-in codon blas towards Escherichia colicodon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3. Recombinant hEKL protein with a maltose binding protein (MBP tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (> 95% by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate. Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen. From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104U/mg.

  9. An improved method for purification and refolding of recombinant HIV Vif expressed in Escherichia coli.

    Science.gov (United States)

    Duarte, Carlos A; Palomino, Mickel

    2017-02-08

    Virion infectivity factor (Vif) is a 23 kDa protein that protects HIV-1 from deamination of its proviral DNA by APOBEC3G. The active form of Vif is a multimer that interacts simultaneously with CBF-beta, the elongin B and C subunits, Cullin 5, and APOBEC3G to form a ubiquitin ligase complex targeting the latter for degradation. Vif clearly represents an attractive target for developing novel antiviral drugs for the therapy of HIV/AIDS, and this goal requires a source of well folded, readily available protein. For that purpose, we have cloned Vif in the pET28a expression vector, expressing the resulting His-tagged recombinant protein in the BL21(DE3) Escherichia coli strain. After lysis, Vif was solubilized from the insoluble fraction with 6 M guanidinium chloride and purified by denaturing immobilized-metal affinity chromatography, refolding the protein afterwards by dialysis. The use of 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.2 and the presence of EDTA improved Vif refolding yields by reducing the formation of insoluble aggregates. The purified protein was bound by two monoclonal antibodies against sequential and conformational epitopes located at the C and N terminus, respectively. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  10. One-step purification of E. coli elongation factor Tu

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.; Degn, B

    1993-01-01

    The tuf A gene, encoding the E. coli elongation factor Tu, was cloned in the pGEX gene fusion system. Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose. This allows purification of EF-Tu in a one...

  11. Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158

    Directory of Open Access Journals (Sweden)

    Seetha Ram Kotra

    2014-03-01

    Full Text Available Objective:Synthetic cationic antimicrobial peptide (SC-AMP is an important and upcoming therapeutic molecule against onventional antibiotics. In this study, an attempt was made to purify the SC-AMP without the enzymatic cleavage of the affinity tag, by using an intein-based system. Methods:The intein sequence was amplified from pTYB11 vector using PCR methodologies and the N-terminal of intein was ligated with SC-AMP. The designed construct, intein-SC-AMP was cloned into MCS region of cold shock expression vector, pCOLDI and the recombinant peptide was purified on a chitin affinity column by cleaving intein with 50 mM DTT without applying enzymatic cleavage. Later the peptide was quantified and its antibacterial activity of the purified peptide was studied using well diffusion method. Results: Initially, intein-SC-AMP was expressed as a fusion protein in both IPTG inducible E. coli BL21(DE3 and salt inducible E. coli GJ1158. Single step purification using CBD (chitin binding domain - intein tag in salt inducible E. coli GJ1158, yields the SC-AMP in the soluble form at a oncentration of 208 mg/L. The antibacterial activity and minimal inhibitory concentration (MIC of the purified SC-AMP was studied against both Gram positive and Gram negative microorganisms. Conclusion: For the first time, single step purification of soluble SC-AMP was carried out using chitin-binding domain affinity tag in salt inducible E. coli GJ1158 without an application of enzymatic cleavage. J Microbiol Infect Dis 2014;4(1:13-19

  12. Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli.

    Science.gov (United States)

    Farshadpour, Fatemeh; Taherkhani, Reza; Makvandi, Manoochehr; Rajabi Memari, Hamid; Samarbafzadeh, Ali Reza

    2014-07-01

    Hepatitis E virus (HEV) is a causative agent of acute hepatitis among people of different age groups and has high mortality rate of up to 30% among pregnant women. Therefore, primary prevention of HEV infection is essential. The aim of this study was to obtain the highly purified truncated open reading frames 2 (ORF2) protein, which might be a future HEV vaccine candidate. The truncated orf2 gene (orf2.1), encoding the 112-660 amino acid of HEV capsid protein sequence, was optimized, synthesized, and cloned into pBluescript II SK(+) vector. After subcloning into expression vector pET-30a (+), a 193-nucleotide fragment was deleted from the construct and the recombinant plasmid pET-30a-ORF2.2 (orf2.2 encodes 112-608 amino acid sequence of HEV capsid protein) was constructed and used for transformation of Escherichia coli BL21 cells. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG) and optimizing the conditions of expression, the target protein was highly expressed and purified by Ni(2+)-chelate affinity chromatography. The expressed and purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The subcloning was confirmed by PCR, restriction enzyme digestion, and DNA sequencing of recombinant plasmid pET30a-ORF2.2. The results obtained from optimizing the expression conditions showed that the highest expression of the protein was obtained by adding IPTG at a final concentration of 1 mM at 37℃ for four hours. The expression and purification of truncated ORF2 protein was confirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed a protein band of about 55 kDa. SDS-PAGE of the purified protein revealed that the highest amount of target protein in elution buffer at the pH of 4.5 was obtained. The yield of the purified protein was about 1 mg/L of culture media. In this study, the optimized truncated ORF2 protein was expressed in E. coli successfully and the highly purified

  13. Purification and partial characterization of intact and truncated chitinase from Bacillus thuringiensis HZP7 expressed in Escherichia coli.

    Science.gov (United States)

    Sha, Li; Shao, Ensi; Guan, Xiong; Huang, Zhipeng

    2016-02-01

    To ascertain the effect of chitin-binding domain (ChBD) and fibronectin type III domain (FN3) on the characterization of the intact chitinase from Bacillus thuringiensis. An intact chitinase gene (chi74) from B. thuringiensis HZP7 and its truncated genes (chi54, chi63 and chi66) were expressed in Escherichia coli BL21. The expression products were analyzed after purification. All chitinases were active from pH 4-7.5 and from 20 to 80 °C with identical optimal: pH 5.5 and 60 °C. The activity of colloid chitin degradation for Chi74 was the highest, followed by Chi66, Chi63 and Chi54. Ag(+) reduced the activity of Chi74, Chi54, Chi63 and Chi66, but Mg(2+) enhanced them. The effect of Ag(+) and Mg(2+) was more significant on the activity of Chi54 than on the activities of Chi63, Chi66 and Chi74. ChBDChi74 and FN3Chi74 domains play a role in exerting enzymatic activity and can improve the stability of chitinase.

  14. Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli

    NARCIS (Netherlands)

    Schulze, Egbert; Westphal, Adrie H.; Veenhuis, Marten; Kok, Arie de

    1992-01-01

    Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been

  15. Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Lisa Gerner

    2016-09-01

    Full Text Available Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2 has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 ‘apo’, CaMKK2 (165-501 in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, “Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2” [1].

  16. Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

    Science.gov (United States)

    Khademi, Farzad; Yousefi-Avarvand, Arshid; Derakhshan, Mohammad; Meshkat, Zahra; Tafaghodi, Mohsen; Ghazvini, Kiarash; Aryan, Ehsan; Sankian, Mojtaba

    2017-10-01

    The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

  17. Rapid purification of high activity Taq DNA polymerase expressed in ...

    African Journals Online (AJOL)

    A simplified method is described here for the preparation of a thermostable Taq DNA polymerase enzyme from Escherichia coli (E. coli) strain DH5a carrying the pTTQ18 expression vector transformed with the Taq polymerase gene. Standard purifications were done with 1 litre batch cultures of E. coli cells and produced ...

  18. High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli.

    Science.gov (United States)

    Saez, Natalie J; Nozach, Hervé; Blemont, Marilyne; Vincentelli, Renaud

    2014-07-30

    Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive

  19. Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori.

    Science.gov (United States)

    Li, Bao-Cun; Zhang, Shuang-Quan; Dan, Wen-Bing; Chen, Yu-Qing; Cao, Peng

    2007-07-01

    The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni(2+)-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K(12)D(31), Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.

  20. [Prokaryotic expression, purification and identification of NY-ESO-1/GST fusion protein in E.coli].

    Science.gov (United States)

    Tang, Lei; Song, Chao-jun; Sun, Yuan-jie; Li, Na; Wei, Yu-ying; Sun, Yi; Yang, Kun

    2012-10-01

    To construct an expression plasmid for NY-ESO-1 gene and identify the expression of recombinant protein NY-ESO-1/GST in E.coli. NY-ESO-1 segment was amplified from the testis cDNA library by RT-PCR and cloned into the prokaryotic expression vector pGEX4T-1 downstream tagged by GST to construct the expression plasmid pGEX-4T1-NY-ESO-1. The recombinant vector was transformed to BL21 (DE3) and NY-ESO-1/GST fusion protein was induced expression by IPTG. The protein was purified by urea elution and identified by SDS-PAGE and Western blotting. The NY-ESO-1 segment was successfully amplified and its sequence was identical with that published in GenBank. The BL21 (DE3) pLysS containing the pGEX-4T1-NY-ESO-1 expressed a M(r); 44 000 fusion protein under the induction of IPTG. The purity of the protein was 90%. Western blotting proved that NY-ESO-1/GST had a specific reaction with anti-GST mAb. The prokaryotic expression vector of NY-ESO-1 has been constructed and the fusion protein NY-ESO-1/GST of high purity is successfully expressed.

  1. Purification and characterization of Pseudomonas aeruginosa LasR expressed in acyl-homoserine lactone free Escherichia coli cultures.

    Science.gov (United States)

    Corral Lugo, Andrés; Daddaoua, Abdelali; Ortega, Alvaro; Morel, Bertrand; Díez Peña, Ana Isabel; Espinosa-Urgel, Manuel; Krell, Tino

    2017-02-01

    Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 μM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL. The α-helical content as determined by CD spectroscopy was found to be in agreement with the corresponding value derived from the homology model. Analytical ultracentrifugation studies show that LasR is a mixture of monomers and dimers and that AHL binding does not alter its oligomeric state. Thermal unfolding studies indicate that LasR has a significant thermal stability and that AHL binding does not significantly alter the unfolding temperature. Two LasR-DNA complexes were observed in electrophoretic mobility shift assays using the hcnABC promoter that has two lux boxes. Taken together, data indicate that the presence of AHLs is not a requisite for correct LasR protein folding. The protein is able to bind AHL ligands in a reversible manner, revising initial concepts of this regulator. The availability of AHL-free protein will permit further studies to determine more precisely its mode of action. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli

    Directory of Open Access Journals (Sweden)

    Hussin A. Rothan

    2012-03-01

    Full Text Available Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL was higher than that of E-ADP (0.04 mg/mL and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.

  3. Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli.

    Science.gov (United States)

    Hadj Sassi, Azza; Trigui-Lahiani, Hèla; Abdeljalil, Salma; Gargouri, Ali

    2017-02-01

    Pectin lyase (pnl) is the only pectinase able to hydrolyze directly the highly methylated pectin without liberating the toxic methanol and without disturbing ester content responsible for specific aroma of juices. The cDNA of Penicillium occitanis pnl (mature form) was cloned into pET-21a as expression vector and over-expressed into Esherichia coli. Most of recombinant pnl was expressed as inclusion bodies. Pnl activity was confirmed by colorimetric assay. To enhance the solubility yield of the expressed pnl, the effects of induction temperature, host strain and expression level were optimized. Maximal production of functional pnl was obtained after induction by 0.4mM IPTG at 30°C and 150rpm for 16h. Interestingly, the use of Origami host strain, having an oxidized cytoplasm favoring disulfide bonds formation required for the active conformation of the enzyme, has significantly improved the yield of the soluble active form of recombinant pnl. This pnl was successfully purified through a single step purification using His-Trap affinity column chromatography. This work is the first to report pnl expression into Origami strain. Alternatively, the inclusion bodies were isolated, denatured by high concentration of urea and gradually refolded by successive dialysis, leading to their transformation into soluble and active form. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion system.

    Science.gov (United States)

    Rao, Shengqi; Zang, Xiangyu; Yang, Zhenquan; Gao, Lu; Yin, Yongqi; Fang, Weiming

    2016-02-01

    A bioactive peptide precursor (BPP-1, 14.3 kDa/115AA), a newly designed polypeptide that may exert a potential antihypertensive effect in vivo, is composed of many different ACE inhibitory peptides and antioxidant peptides tandemly linked according to the restriction sites of gastrointestinal proteases. In this report, we present a novel method to obtain soluble BPP-1 in Escherichia coli using cationic elastin-like polypeptide and SUMO (cELP-SUMO) tags. The cELP-SUMO-tagged fusion protein was expressed in soluble form at 20 °C for 20 h. After purification based on the inverse transition cycling (ITC) method, the purified cELP-SUMO-CFPP fusion protein was subsequently cleaved by a SUMO protease to release the mature BPP-1. After a subsequent simple salt precipitation process, approximately 167.2 mg of recombinant BPP-1 was obtained from 1 l of bacterial culture with at least 92% purity. The molecular mass (Mr) of the recombinant BPP-1 was confirmed by MALDI-TOF MS to equal 14,347. The purified BPP-1 was subjected to simulated gastrointestinal digestion, and the resulting hydrolysates exhibited notable ACE inhibitory and antioxidant activities in vitro. This report provides the first description of the soluble production of a bioactive peptide multimer with potential ACE inhibitory and antioxidant activities in E. coli using a cELP-SUMO tag. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Expression, purification and preliminary diffraction studies of PhnP

    DEFF Research Database (Denmark)

    Podzelinska, Kateryna; He, Shumei; Soares, Alexei

    2008-01-01

    PhnP belongs to a 14-gene operon that supports the growth of Escherichia coli on alkylphosphonates as a sole source of phosphorus; however, the exact biochemistry of phosphonate degradation by this pathway is poorly understood. The protein was recombinantly expressed in Escherichia coli and purif...

  6. Gene synthesis, expression in Escherichia coli, purification and characterization of the recombinant bovine acyl-CoA-binding protein

    DEFF Research Database (Denmark)

    Mandrup, S; Højrup, P; Kristiansen, K

    1991-01-01

    for recombinant ACBP was obtained by extracting induced E. coli cells with 1 M-acetic acid. Recombinant ACBP was purified to homogeneity by successive use of gel-filtration chromatography, ion-exchange chromatography and reverse-phase h.p.l.c. Recombinant ACBP differed from native ACBP by lacking the N...

  7. Cloning, expression, and purification of the His(6)-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR

    DEFF Research Database (Denmark)

    Dabrowski, Slawomir; Ahring, Birgitte Kiær

    2003-01-01

    The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E coli BL21(DE3)pLysS and E. coli Rosetta(DE3)p......LysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni2+-IDA-Sepharose, dialyzed...

  8. Expression, purification, and initial characterization of a recombinant form of plant PEP-carboxylase kinase from CAM-induced Mesembryanthemum crystallinum with enhanced solubility in Escherichia coli.

    Science.gov (United States)

    Ermolova, Natalia V; Ann Cushman, Mary; Taybi, Tahar; Condon, Shirley A; Cushman, John C; Chollet, Raymond

    2003-05-01

    Plant phosphoenolpyruvate-carboxylase kinase (PEPC-kinase [PpcK]) is the smallest Ser/Thr kinase identified to date, having a molecular mass of approximately 32,000. This novel, monomeric kinase is dedicated to the phosphorylation of plant PEPC, thereby regulating this target enzyme's activity and allosteric properties. Although several recombinant, non-fusion PpcK proteins have been produced recently in Escherichia coli, these are plagued by their high degree of insolubility. Here, we report the use of the native, E. coli NusA protein and a related E. coli expression vector (pET-43a(+) [Novagen]) for enhancing the solubility of this recalcitrant Ser/Thr kinase at least 10-fold by its production as a dual 6xHis-tagged NusA/McPpcK1 fusion protein, which accounts for approximately 10% of the soluble protein fraction from induced cells. Capture of this fusion protein from the centrifuged cell extract by immobilized metal (Ni(2+)) affinity-chromatography, its "on-bead" cleavage by thrombin, and subsequent elution yielded milligram quantities of a "free," approximately 36-kDa form of PpcK for further purification by fast-protein liquid chromatography on blue dextran-agarose or preparative SDS-PAGE. Steady-state kinetic analysis of the former, active preparation revealed that this dedicated kinase discriminates against neither various isoforms of plant PEPC nor certain mutant forms of recombinant C(4) PEPC. Alternatively, the latter, electrophoretically homogeneous sample of the approximately 36-kDa polypeptide was used as antigen for polyclonal-antibody production in rabbits. The antibodies against the recombinant McPpcK1 from Mesembryanthemum crystallinum cross-reacted on Western blots with an enriched preparation of the maize-leaf kinase, but not with the parent crude extract, thus directly documenting this protein's extremely low abundance in vivo. However, these antibodies were effective in immunoprecipitating 32P-based PpcK activity from crude, desalted extracts of

  9. Simple method for purification of enterotoxigenic Escherichia coli fimbriae.

    Science.gov (United States)

    Curtis, Brittany; Grassel, Christen; Laufer, Rachel S; Sears, Khandra T; Pasetti, Marcela F; Barry, Eileen M; Simon, Raphael

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Escherichia coli enterotoxin. Purification and partial characterization.

    Science.gov (United States)

    Dorner, F

    1975-11-25

    Enterotoxin, a diarrheagenic protein elaborated by pathogenic Escherichia coli strains has been isolated from the supernatant of fermenter cultures of E. coli strain P263, a porcine enteropathogen. Purification steps involving Bio-Gel agarose A-5m, Sephadex G-75 chromatography, and preparative isotachophoresis were used in the isolation. The resulting product appears to be pure according to immunoelectrophoretic, disc electrophoretic, ultracentrifugal, and immunologic criteria. The entertoxin has an apparent molecular weight of 102,000 as judged by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and its isoelectric point is 6.90. The isolated product is highly active in inducing experimental diarrhea in adult rabbits and piglets. It also elicits, in small dosage, a marked increase in adenylate cyclase activity in broken cell preparations of cat heart tissue. The enterotoxin activity is acid-labile and is destroyed by heating at 65 degrees for 30 min. It is suggested that the heat-stable enterotoxin material is derived from heat-labile enterotoxin by forming a complex with endotoxin or capsular material present in the culture supernatant.

  11. PURIFICATION AND CELLULAR-LOCALIZATION OF WILD-TYPE AND MUTATED DIHYDROLIPOYLTRANSACETYLASES FROM AZOTOBACTER-VINELANDII AND ESCHERICHIA-COLI EXPRESSED IN ESCHERICHIA-COLI

    NARCIS (Netherlands)

    SCHULZE, E; WESTPHAL, AH; VEENHUIS, M; DEKOK, A

    1992-01-01

    Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been

  12. One-step purification of rat heart-type fatty acid-binding protein expressed in Escherichia coli

    NARCIS (Netherlands)

    Schaap, F. G.; Specht, B.; van der Vusse, G. J.; Börchers, T.; Glatz, J. F.

    1996-01-01

    Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14-15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat

  13. Purification and properties of Rhizobial DehL expressed in ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-06-17

    Jun 17, 2008 ... Full Length Research Paper. Purification and properties of Rhizobial DehL expressed in Escherichia coli. Fahrul Huyop1*, Nooraini Abdul Rashid1, Roswanira A. B. Wahab2 and Ronald A. Cooper3. 1Industrial Biotechnology Department, University Technology Malaysia, 81310 Skudai, Johor, Malaysia.

  14. Cloning, high-level expression, purification and characterization of a ...

    African Journals Online (AJOL)

    The staphylokinase (Sak) is emerging as an important thrombolytic agent for the treatment of patients suffering from cardiovascular disease. Hence in this study, we reported the cloning, high-level expression, purification and characterization of the Sak variant SakøC from Staphylococcus aureus QT08 in Escherichia coli ...

  15. CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

    Directory of Open Access Journals (Sweden)

    Kusdianawati Kusdianawati

    2015-09-01

    Full Text Available Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from Lactobacillus plantarum S34 in Escherichia coli. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. plantarum S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3 pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. plantarum S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His6tag had successfully been expressed in E. coli BL21 (DE3 pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1. Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

  16. Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli

    Science.gov (United States)

    Richard, Christelle; Drider, Djamel; Elmorjani, Khalil; Marion, Didier; Prévost, Hervé

    2004-01-01

    Divercin V41, a class IIa bacteriocin with strong antilisterial activity, is produced by Carnobacterium divergens V41. To express a recombinant version of divercin V41, we constructed a synthetic gene that encodes the mature divercin V41 peptide and then overexpressed the gene in pET-32b by using the T7 RNA polymerase promoter in the Escherichia coli Origami (DE3)(pLysS) strain. The DvnRV41 peptide was expressed as a translational fusion protein with thioredoxin and accumulated in the cell cytoplasm in a soluble anti-Listeria active form. The fusion protein was then purified and cleaved to obtain pure, soluble, folded DvnRV41 (462 μg per 20 ml of culture). This paper describes the first design of a synthetic bacteriocin gene and the first bacteriocin expressed in the E. coli cytoplasm. PMID:15205430

  17. Expression, purification and characterization of the interferon ...

    Indian Academy of Sciences (India)

    Earlier, we had reported that expression of recombinant human RNase L caused RNA-degradation and cell-growth inhibition in E. coli without the need for exogenous 2-5A. Expression of human RNase L in E. coli usually leads to problems of leaky expression, low yield and degradation of the recombinant protein, which ...

  18. Construction of high level prokaryotic expression and purification system of PD-L1 extracellular domain by using Escherichia coli host cell machinery.

    Science.gov (United States)

    Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

    2017-10-01

    Programmed cell death 1 ligand 1 (PD-L1) is a trans-membrane protein highly expressed on the membrane of cancer cell, which binds inhibitory receptor of PD-1 on the T cells and attenuates anti-tumor immune response.The strategy of blocking PD1 and PD-L1 interaction has been widely used for anti-cancer drug development. The DNA encoding extracellular domain of PD-L1 was cloned and expressed with the pET30(+) and Escherichia coli BL21(DE3) system. Cloning of PD-L1 extracellular domain was confirmed by PCR and enzymatic digestion. Sequence analysis of cloned targeted genes showed 100% homology of original sequence. The recombinant protein was expressed using 1mM/mL IPTG and purified by affinity chromatography on a column of Ni-NTA and confirmed by SDS-PAGE and western blot analysis. Results showed that our constructed pET30(+)/PDL1-ECD system efficiently produces desired recombinant protein with molecular weight of 38.1kDa. The prokaryotic expression system provides an easy method to express PD-L1 extracellular domain that further facilitate the role of PD-1/PD-L1 binding inhibition and helps in valuable drug and antibodies production. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  19. Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

    Science.gov (United States)

    Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

    2014-02-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

  20. [The expression of gene related to salt tolerance from Sinorhizobium meliloti 042BM in Escherichia coli and purification of its fusion protein].

    Science.gov (United States)

    Ge, Shi-chao; Wang, Lei; Li, Xiao-hong; Qi, Su-wei; Yang, Su-sheng

    2005-06-01

    A 1.9kb DNA fragment related to salt tolerance of S. meliloti strain 042BM containing two open reading frames were obtained by PCR amplification and ligated into shuttle vector pBBR1-MCS2. The complementation experiment showed that ORF2 is related to salt tolerance and named as rstA gene. Then the gene was cloned into the expression vector pThio-HisA, B and C, respectively, and recombinant expression vectors pGSA, pGB and pGC were constructed, and transformed into E. coli Top10. Inducing by IPTG and analyzing with SDS-PAGE, the fusion protein encoded by pGSA was obtained,and it is 36% content of whole cell protein. It was isolated and purified by affinity chromography on ProBond, and the inclusion body precipitated by saturated sulfate ammonium, and 95% purity of fusion protein was obtained. The final product displayed a single band with a corresponding molecular weight 43kD in SDS-PAGE, and was verified by the Western blot.

  1. Overproduction in Escherichia coli and purification of Epstein-Barr virus EBNA-1.

    Science.gov (United States)

    Duellman, Sarah J; Burgess, Richard R

    2006-06-01

    Epstein-Barr virus nuclear antigen 1 (EBNA-1) is a multi-functional protein of the Epstein-Barr virus (EBV). Due to its low abundance in EBV-transformed cells, overproduction in a foreign host is preferred to obtain purified EBNA-1 protein. The EBNA-1 gene possesses a large number of Escherichia coli rare codons (23%). By using E. coli BL21(DE3)Rosetta2 cells that augment the low-abundance tRNA genes, the expression level of EBNA-1 in E. coli was greatly enhanced. EBNA-1 was then purified by applying the whole cell extract soluble fraction to a Ni-NTA Superflow column and eluting with an imidazole gradient. The improved overexpression in E. coli followed by a one-step Ni-NTA purification resulted in a sufficient amount of pure EBNA-1 protein to test DNA binding activity, and prepare and test EBNA-1-specific monoclonal antibodies (mAbs).

  2. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Kawahara, Kazuki; Oki, Hiroya; Fukakusa, Shunsuke; Maruno, Takahiro; Kobayashi, Yuji; Motooka, Daisuke; Taniguchi, Tooru; Honda, Takeshi; Iida, Tetsuya; Nakamura, Shota; Ohkubo, Tadayasu

    2015-06-01

    Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.

  3. Expression and purification of the functional ectodomain of human anthrax toxin receptor 2 in Escherichia coli Origami B cells with assistance of bacterial Trigger Factor.

    Science.gov (United States)

    Jacquez, Pedro; Lei, Ningjing; Weigt, David; Xiao, Chuan; Sun, Jianjun

    2014-03-01

    The ectodomain of anthrax toxin receptor 2 (ANTXR2) is composed of a von Willebrand factor A (VWA) domain that binds to anthrax toxin protective antigen (PA) and a newly defined immunoglobulin-like (Ig) domain, in which the disulfide bonds are required for PA pore formation and for the folding of ANTXR2. While the VWA domain has been well characterized, the structure and function of the whole ectodomain (VWA-Ig) are poorly defined, which is mainly due to the limited production of the soluble recombinant protein of the ectodomain. In the present study, the ANTXR2 ectodomain was fused to the C-terminus of bacterial Trigger Factor (TF), a chaperone that mediates the ribosome-associated, co-translational folding of newly synthesized polypeptides in Escherichia coli. Under the control of a cold shock promoter, the fusion protein was overly expressed as a dominant soluble protein at a low temperature in the oxidative cytoplasm of Origami B cells, where formation of the disulfide bonds is favored. Through a series of chromatography, the ANTXR2 ectodomain was purified into homogeneity. The purified ectodomain is functional in binding to PA and mediating PA pore formation on the liposomal membranes, and the yield is applicable for future biochemical and structural characterization. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Expression and Purification of Sperm Whale Myoglobin

    Science.gov (United States)

    Miller, Stephen; Indivero, Virginia; Burkhard, Caroline

    2010-01-01

    We present a multiweek laboratory exercise that exposes students to the fundamental techniques of bacterial expression and protein purification through the preparation of sperm whale myoglobin. Myoglobin, a robust oxygen-binding protein, contains a single heme that gives the protein a reddish color, making it an ideal subject for the teaching…

  5. A novel flavin adenine dinucleotide (FAD) containing d-lactate dehydrogenase from the thermoacidophilic crenarchaeota Sulfolobus tokodaii strain 7: purification, characterization and expression in Escherichia coli.

    Science.gov (United States)

    Satomura, Takenori; Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2008-07-01

    Dye-linked D-lactate dehydrogenase activity was found in the crude extract of a continental thermoacidophilic crenarchaeota, Sulfolobus tokodaii strain 7, and was purified 375-fold through four sequential chromatography steps. With a molecular mass of about 93 kDa, this enzyme was a homodimer comprised of identical subunits with molecular masses of about 48 kDa. The enzyme retained its full activity after incubation at 80 degrees C for 10 min and after incubation at pHs ranging from 6.5 to 10.0 for 30 min at 50 degrees C. The preferred substrate for this enzyme was D-lactate, with 2,6-dichloroindophenol serving as the electron acceptor. Using high-performance liquid chromatography (HPLC), the enzyme's prosthetic group was determined to be flavin adenine dinucleotide (FAD). Its N-terminal amino acid sequence was MLEGIEYSQGEEREDFVGFKIKPKI. Using that sequence and previously reported genome information, the gene encoding the enzyme (ST0649) was identified. It was subsequently cloned and expressed in Escherichia coli and found to encode a polypeptide of 440 amino acids with a calculated molecular weight of 49,715. The amino acid sequence of this dye-linked D-lactate dehydrogenase showed higher homology (39% identity) with that of a glycolate oxidase subunit homologue from Archaeoglobus fulgidus, but less similarity (32% identity) to D-lactate dehydrogenase from A. fulgidus. Taken together, our findings indicate that the dye-linked D-lactate dehydrogenase from S. tokodaii is a novel type of FAD containing D-lactate dehydrogenase.

  6. Production and Purification Immunoglobulin against E. coli in Egg Yolk

    Directory of Open Access Journals (Sweden)

    Mohammadreza Nassiri

    2016-08-01

    Full Text Available Introduction Chicken is the only avian species in which polyclonal antibodies, like IgG is transported from the hen to the egg yolk in a similar manner as the transport of mammalian IgG from the mother to the fetus. Immunoglobulin Y in the chicken is transported to the egg and accumulates in the egg yolk in large quantities. IgY is an egg yolk antibody that has been used widely for treatment and prevention of infections in humans and animal. IgY is used for passive protection of the pathogen infections such as Escherichia coli, bovine and human rotavirus, bovine coronavirus, salmonella, staphylococcus and Pseudomonas. IgY is a promising candidate as an alternative to antibiotics. Eschericha coli strains of serotype O157: H7 belongs to a family of pathogenic E. coli called enterohemorrhagic E. coli (EHEC strains responsible for hemorrhagic colitis, bloody or non-bloody diarrhea, and hemolytic uremic syndrome in humans. This strain of E. coli pathogenises by adhering to host intestinal epithelium and forming bacterial colonies. The purpose of this study was to produce and purify immunoglobulin Y against E. coli O157:H7 and develop specific polyclonal anti E. coli antibody in the egg yolk. Materials and Methods Sixteen-week-old laying hens (Mashhad, Iran were kept in individual cages with food and water ad libitum. Immunization of hens was performed by intramuscularly injecting killed E. coli O157: H7 with an equal volume of Freund’s complete adjuvant into two sides of chest area (Sigma, USA for the first immunization. Two booster immunizations followed up using complete and incomplete Freund’s adjuvants in two weeks interval. Freund’s adjuvant without antigen was injected to the control group. Two weeks after the last injection, the eggs were collected daily for eight weeks, marked and stored at 4 ºC. In order to IgY purification, eggs were collected. Purification of IgY from egg yolk was based on Polson and using PEG6000. Finally, the

  7. THE CATALYTIC DOMAIN OF THE DIHYDROLIPOYL TRANSACETYLASE COMPONENT OF THE PYRUVATE-DEHYDROGENASE COMPLEX FROM AZOTOBACTER-VINELANDII AND ESCHERICHIA-COLI - EXPRESSION, PURIFICATION, PROPERTIES AND PRELIMINARY-X-RAY ANALYSIS

    NARCIS (Netherlands)

    SCHULZE, E; WESTPHAL, AH; OBMOLOVA, G; MATTEVI, A; HOL, WGJ; DEKOK, A

    1991-01-01

    Partial sequences of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli, containing the catalytic domain, were cloned in pUC plasmids and over-expressed in E. coli TG2. A high expression of a homogeneous protein was

  8. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly...... intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely...

  9. Cloning, expression, purification and antigenic evaluation of ...

    African Journals Online (AJOL)

    Streptococcus pyogenes produce an extracellular hyaluronidase which is associated with the spread of the organism during infection. Enzyme hyaluronidase is capable of degrading hyaluronic acid. The aim of the present study was to clone and express antigenic regions of the hylA of S.pyogenes in Escherichia coli.

  10. Purification of Biotransformation Products of Cis-Isoflavan-4-ol by Biphenyl Dioxygenase of Pseudomonas pseudoalcaligenes KF707 Strain Expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Tri Ratna Sulistiyani

    2013-04-01

    Full Text Available Isoflavone has multiple beneficial effects on human health, especially through its antioxidant and anticancer activities. The biotransformation of isoflavone using byphenyl dioxygenase could be performed to extend the diversity of flavonoids and to improve their biological and physiological properties. Biotransformation of two enantiomers (3R, 4R-cis-isoflavan-4-ol and (3S, 4S-cis-isoflavan-4-ol by E. coli JM109 (pJHF108 carrying a biphenyl dioxygenase gene from P. pseudoalcaligenesKF707 produced two products, designated as CM1 andCM2. The products had a retention time of 11.9 and 14.6 min, respectively, and the same absorption peaks at 204, 220, and 275 nm. CM1 and CM2 had [M-H2O+H]+ at m/z 225. Based on the molecular mass and hydrolysis products, we proposed that epoxidation occurred on cis-isoflavan-4-ol. Chloroform extraction instead of ethyl acetate extraction was performed to improve the stability of cismetabolites, CM1 and CM2.

  11. Structure of the β-Galactosidase Gene from Thermus sp. Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein

    Science.gov (United States)

    Vian, Alejandro; Carrascosa, Alfonso V.; García, José L.; Cortés, Estrella

    1998-01-01

    The nucleotide sequence of both the bgaA gene, coding for a thermostable β-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a Km value of 1.6 mM with o-nitrophenyl-β-d-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C. PMID:9603833

  12. High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli

    DEFF Research Database (Denmark)

    Hiszczynska-Sawicka, E.; Brillowska-Dabrowska, A.; Dabrowski, Slawomir

    2003-01-01

    This report describes a simple, highly efficient and reproducible method for obtaining large quantities of highly pure recombinant Toxoplasma gondii antigens, which can be used for diagnostic application. The obtained T gondii SAG1, GRA1, and GRA7 antigens (as fusion proteins), expressed in Esche......This report describes a simple, highly efficient and reproducible method for obtaining large quantities of highly pure recombinant Toxoplasma gondii antigens, which can be used for diagnostic application. The obtained T gondii SAG1, GRA1, and GRA7 antigens (as fusion proteins), expressed...

  13. Data of expression and purification of recombinant Taq DNA polymerase

    Directory of Open Access Journals (Sweden)

    Na Fang

    2016-12-01

    Full Text Available Polymerase chain reaction (PCR technique is widely used in many experimental conditions, and Taq DNA polymerase is critical in PCR process. In this article, the Taq DNA polymerase expression plasmid is reconstructed and the protein product is obtained by rapid purification, (“Rapid purification of high-activity Taq DNA polymerase” (Pluthero, 1993 [1], “Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli” (Desai and Pfaffle, 1995 [2]. Here we present the production data from protein expression and provide the analysis results of the production from two different vectors. Meanwhile, the purification data is also provided to show the purity of the protein product.

  14. EXPRESSION, PURIFICATION, AND KINETIC CHARACTERIZATION OF THE MANNITOL TRANSPORT DOMAIN OF THE PHOSPHOENOLPYRUVATE-DEPENDENT MANNITOL PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI - KINETIC EVIDENCE THAT THE ESCHERICHIA-COLI MANNITOL TRANSPORT PROTEIN IS A FUNCTIONAL DIMER

    NARCIS (Netherlands)

    Boer, H.; Duurkens, Hinderika; Schuurman-Wolters, Geesina; Dijkstra, A; Robillard, George

    1994-01-01

    The overexpression of the membrane-bound C domain of the mannitol transport protein EII(Mtl) of Escherichia coli has been achieved. This protein, IICMtl, consisting of the first 346 amino acids, was purified from membrane vesicles and still bound mannitol with a high affinity. Gel filtration

  15. Expression, Purification, and Kinetic Characterization of the Mannitol Transport Domain of the Phosphoenolpyruvate-dependent Mannitol Phosphotransferase System of Escherichia coli. Kinetic Evidence that the E. coli Mannitol Transport Protein is a Function

    NARCIS (Netherlands)

    Hoeve-Duurkens, Ria H. ten; Schuurman-Wolters, Gea K.; Dijkstra, Arnoud; Robillard, George T.

    1994-01-01

    The overexpression of the membrane-bound C domain of the mannitol transport protein EIIMtl of Escherichia coli has been achieved. This protein, IICMtl, consisting of the first 346 amino acids, was purified from membrane vesicles and still bound mannitol with a high affinity. Gel filtration

  16. Extração, purificação e caracterização físico-química da proteína verde fluorescente recombinante (GFPuv expressa em Escherichia coli Extraction, purification and physical-chemical characterization of recombinant green fluorescent protein (GFPuv expressed in Escherichia coli cells

    Directory of Open Access Journals (Sweden)

    Eb Chiarini

    2003-12-01

    Full Text Available O aumento do uso da proteína verde fluorescente (GFPuv como ferramenta de pesquisas biotecnológicas requer um estudo mais cuidadoso das propriedades bioquímicas e físicas da molécula de GFPuv. Este trabalho teve como objetivo a aplicação de métodos físicos e químicos para o isolamento, a extração da GFPuv de células de Escherichia coli DH5-±, purificação da proteína, e o estudo da estabilidade desta em diferentes valores de pH. Células de E. coli expressando GFPuv foram submetidas a quatro ciclos sucessivos de congelamento e descongelamento intercalados por sonicação (CDS, para promover a permeação seletiva da GFPuv. Os permeados foram submetidos à extração por partição em três fases (TPP e posterior purificação por eluição da proteína em coluna cromatográfica de interação hidrofóbica (HIC.Obteve-se rendimento semelhante em GFPuv no 1º ciclo de permeação seletiva (CDS e por extração (TPP associada à purificação (HIC para os quais impurezas não foram visualizadas por eletroforese. As estruturas moleculares da GFPuv extraída e purificada mostraram-se inalteradas em valores de pH entre 6,0 e 9,8, e foram confirmadas nos espectros de emissão e de excitação.The recombinant green fluorescent protein (GFPuv has been used as a marker in several research fields. The purpose of the present work was to evaluate the influence of the selective physical permeation procedure applied to the cells of Escherichia coli for the extraction of GFPuv in relation to the chemical procedures of extraction and purification. Transformed cells (0.92-1.44 mg/mL of E. coli DH5-a expressing GFPuv were submitted to four cycles (1º, 2º, 3º, 4º of freezing (-20 ºC/ 0.83 ºC/ min/thawing interlaid by sonication (3 pulses/6 s/25 vibrations. The intracellular permeate with GFPuv in buffer solution (Tris-HCl 25 mM pH 8.0 + b-mercaptoethanol (1 mM + PMSF (0.1 mM was submitted to the three-phase partitioning (TPP method and

  17. Expression and purification of recombinant Shiga toxin 2B from ...

    African Journals Online (AJOL)

    The two step purification trains were used to purify native Stx2B. First step purification was Ni-immobilized metal ion affinity chromatography (IMAC) column, followed by second step using HaloLink resin. The native Stx2B was obtained after column cleavage of halo-tag using HaloTEV protease. Maximum protein expression ...

  18. Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

    Science.gov (United States)

    Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei

    2017-07-01

    It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

  19. Recombinant allergen Lol p II: expression, purification and characterization.

    Science.gov (United States)

    Tamborini, E; Brandazza, A; De Lalla, C; Musco, G; Siccardi, A G; Arosio, P; Sidoli, A

    1995-05-01

    Pollen from perennial rye grass (Lolium perenne) is a major cause of type I allergies worldwide. It contains complex mixtures of proteins, among which Lol p II is a major allergen. Previously, we have reported the cloning and sequencing of Lol p II and its expression in fusion with the heavy chain of human ferritin as carrier polypeptide (Sidoli et al., 1993, J. biol. Chem. 268, 21819-21825). Here, we describe the expression, purification and characterization of a recombinant Lol p II overproduced as a non-fusion protein in the periplasm of E. coli. The recombinant allergen was expressed in high yields and was easily purified in milligram amounts. It competed with the natural Lol p II for binding to specific IgE, and it induced allergic responses in skin prick tests, indicating to be immunologically analogous to the natural protein. Biochemical analyses indicate that recombinant Lol p II is a highly stable and soluble monomeric molecule which behaves like a small globular protein.

  20. Expression of a soluble truncated Vargula luciferase in Escherichia coli.

    Science.gov (United States)

    Hunt, Eric A; Moutsiopoulou, Angeliki; Broyles, David; Head, Trajen; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K

    2017-04-01

    Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase.

    Science.gov (United States)

    Chesterman, Chelsy; Jia, Zongchao

    2016-09-01

    Escherichia coli tyrosine kinase (Etk) is a membrane bound kinase in gram-negative bacteria that regulates the export of capsular polysaccharides (CPS). The molecular mechanism behind CPS regulation remains unclear, despite access to a crystal structure of the cytoplasmic kinase domain of Etk. In this study, an efficient protocol to produce full length Etk solubilized in n-dodecyl-β-d-maltoside has been established with high yield. We have determined that detergent solubilized Etk retains kinase activity, but the protein is prone to aggregation, degradation, and has been unsuccessful in protein crystallization trials. In response, we designed and characterized truncations of Etk that do not aggregate and have led to successful crystallization experiments. In this article, we discuss our optimized expression and purification protocol for Etk, the design of Etk protein truncations, and the behavior of Etk during purification in a range of stabilizing detergents. These efforts have successfully produced protein suitable for crystallization. Our results will be a useful guide for future structural and functional studies of the bacterial tyrosine kinase family. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence.

    Science.gov (United States)

    Gromek, Katarzyna A; Meddaugh, Hannah R; Wrobel, Russell L; Suchy, Fabian P; Bingman, Craig A; Primm, John G; Fox, Brian G

    2013-06-01

    Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Leppla, Stephen H., E-mail: sleppla@niaid.nih.gov [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  4. Expression, Purification, and Characterisation of Dehydroquinate Synthase from Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Leonardo Negron

    2011-01-01

    Full Text Available Dehydroquinate synthase (DHQS catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30∘C followed by a heat treatment at 70∘C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry and is active over broad pH and temperature ranges. The kinetic parameters are KM (3-deoxy-D-arabino-heptulosonate 7-phosphate 3.7 μM and kcat 3.0 sec-1 at 60∘C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness Cd2+, Co2+, Zn2+, and Mn2+. High activity of a DHQS in the presence of Cd2+ has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd2+ for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay.

  5. Application of HaloTag technology to expression and purification of cannabinoid receptor CB2.

    Science.gov (United States)

    Locatelli-Hoops, Silvia; Sheen, Fangmin C; Zoubak, Lioudmila; Gawrisch, Klaus; Yeliseev, Alexei A

    2013-05-01

    Expression of milligram quantities of functional, stable G protein-coupled receptors (GPCR) for high-resolution structural studies remains a challenging task. The goal of this work was to evaluate the usefulness of the HaloTag system (Promega) for expression and purification of the human cannabinoid receptor CB(2), an important target for development of drugs for treatment of immune disorders, inflammation, and pain. Here we investigated expression in Escherichia coli cells of the integral membrane receptor CB(2) as a fusion with the 34 kDa HaloTag at N- or C-terminal location, either in the presence or in the absence of the N-terminal maltose-binding protein (MBP). The CB(2) was flanked at both ends by the tobacco etch virus (TEV) protease cleavage sites to allow for subsequent removal of expression partners. Expression by induction with either IPTG (in E. coli BL21(DE3) cell cultures) or by auto-induction (in E. coli KRX cells) were compared. While the N-terminal location of the HaloTag resulted in high levels of expression of the fusion CB(2), the recombinant receptor was not functional. However, when the HaloTag was placed in the C-terminal location, a fully active receptor was produced irrespective of induction method or bacterial strain used. For purification, the fusion protein was captured onto HaloLink resin in the presence of detergents. Treatment with specific TEV protease released the CB(2) upon washing. To our knowledge, this study represents the first example of expression, surface immobilization and purification of a functional GPCR using HaloTag technology. Published by Elsevier Inc.

  6. Purification and Crystallization of ZITB, A Zinc Transporter from Escherichia Coli

    Energy Technology Data Exchange (ETDEWEB)

    Kao, K.; Fu, D.

    2004-01-01

    Cellular zinc homeostasis is essential to human health. Zinc transporters transport zinc ions into and out of cells to maintain cellular zinc concentrations in a narrow range. Several membrane proteins have been shown to facilitate transmembrane fluxes of zinc ions, however, structures of these zinc transporters are unknown. The purpose of this work is to express, purify and crystallize a Zinc transporter, ZitB for crystallographic studies. ZitB was over-expressed as a His-tagged membrane protein using a pET15b expression vector hosted in E. coli BL21 cells. Purification of ZitB was achieved by preparation of ZitB-containing membrane vesicles, followed by detergent extraction, and completed with Ni-NTA metal affinity and size exclusion chromatography. The molecular identity of the purified ZitB was confirmed by mass spectrometry, which showed the expected molecular weight of 35.2kDa. Crystallization trials of ZitB were conducted at 20 oC, using a series of low molecular weight PEGs as precipitants. Micro-crystals were grown in 25% PEG 1K, whereas only amorphous precipitations were observed in PEG 400 and 600. In conclusion, this work yielded highly purified ZitB protein and defined an initial crystallization condition for ZitB.

  7. Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells.

    Science.gov (United States)

    Li, Xiaoxiang; Wang, Xiaoli; Xiong, Shaoling; Zhang, Jing; Cai, Litao; Yang, Yanyan

    2007-10-01

    A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells.

  8. Teaching molecular biology to undergraduate biology students: An illustration of protein expression and purification*.

    Science.gov (United States)

    Sommer, César Adolfo; Silva, Flávio Henrique; Novo, Maria Teresa Marques

    2004-01-01

    Practical classes on protein expression and purification were given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering." The heterologous expression of the green fluorescent protein (GFP)* of Aequorea victoria is an interesting system for didactic purposes because it can be viewed easily during experiments. The students were provided with basic information about the molecular features and applications of the GFP in molecular biology, the available heterologous expression systems, and the theoretical and experimental details of GFP expression in Escherichia coli and its purification. E. coli BL21-competent cells were transformed with the pET28a expression vector containing the GFP gene fused to a histidine (His) tag. During the induction of a transformed clone by isopropylthiogalactoside, a time course for GFP expression was analyzed by SDS-PAGE, and the expression was also visualized by the increasing green fluorescence of the bacterial culture. After cellular disruption, protein purification was illustrated by affinity chromatography of the His-tagged protein in a nickel column. Eluted fractions containing imidazole in increasing concentrations were analyzed visually and also by SDS-PAGE, demonstrating the role of imidazole in protein recovery by competition with nonspecific proteins and the His-tagged protein. The results obtained and the experimental factors involved in protein expression, solubilization, and folding were discussed following the laboratory experiments. These practical classes allowed several current approaches to molecular biology to be demonstrated rapidly and helped underscore some of the topics taught during the course. Copyright © 2004 International Union of Biochemistry and Molecular Biology, Inc.

  9. Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA.

    Science.gov (United States)

    Gruss, Fabian; Hiller, Sebastian; Maier, Timm

    2015-01-01

    TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family.

  10. Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration

    Directory of Open Access Journals (Sweden)

    Wang Yiran

    2009-06-01

    Full Text Available Abstract Background Thermostable enzymes from thermophilic microorganisms are playing more and more important roles in molecular biology R&D and industrial applications. However, over-production of recombinant soluble proteins from thermophilic microorganisms in mesophilic hosts (e.g. E. coli remains challenging sometimes. Results An open reading frame TM0438 from a hyperthermophilic bacterium Thermotoga maritima putatively encoding 6-phosphogluconate dehydrogenase (6PGDH was cloned and expressed in E. coli. The purified protein was confirmed to have 6PGDH activity with a molecular mass of 53 kDa. The kcat of this enzyme was 325 s-1 and the Km values for 6-phosphogluconate, NADP+, and NAD+ were 11, 10 and 380 μM, respectively, at 80°C. This enzyme had half-life times of 48 and 140 h at 90 and 80°C, respectively. Through numerous approaches including expression vectors, hosts, cultivation conditions, inducers, and codon-optimization of the 6pgdh gene, the soluble 6PGDH expression levels were enhanced to ~250 mg per liter of culture by more than 500-fold. The recombinant 6PGDH accounted for >30% of total E. coli cellular proteins when lactose was used as a low-cost inducer. In addition, this enzyme coupled with glucose-6-phosphate dehydrogenase for the first time was demonstrated to generate two moles of NADPH per mole of glucose-6-phosphate. Conclusion We have achieved a more than 500-fold improvement in the expression of soluble T. maritima 6PGDH in E. coli, characterized its basic biochemical properties, and demonstrated its applicability for NADPH regeneration by a new enzyme cocktail. The methodology for over-expression and simple purification of this thermostable protein would be useful for the production of other thermostable proteins in E. coli.

  11. Refolding and purification of recombinant human (Pro)renin receptor from Escherichia coli by ion exchange chromatography.

    Science.gov (United States)

    Wang, Fei; Guo, Jinjin; Bai, Quan; Wang, Lili

    2014-01-01

    Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anion-exchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 × 10(8) L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful tool for the purification and renaturation of rhRnR. © 2014 American Institute of Chemical Engineers.

  12. A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

    Directory of Open Access Journals (Sweden)

    Bottomley Stephen P

    2006-03-01

    Full Text Available Abstract Background In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. Results The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. Conclusion The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high

  13. Expression of tung tree diacylglycerol acyltransferase 1 in E. coli

    Directory of Open Access Journals (Sweden)

    Klasson K Thomas

    2011-07-01

    Full Text Available Abstract Background Diacylglycerol acyltransferases (DGATs catalyze the final and rate-limiting step of triacylglycerol (TAG biosynthesis in eukaryotic organisms. Database search has identified at least 59 DGAT1 sequences from 48 organisms, but the expression of any DGAT1 as a full-length protein in E. coli had not been reported because DGAT1s are integral membrane proteins and difficult to express and purify. The objective of this study was to establish a procedure for expressing full-length DGAT1 in E. coli. Results An expression plasmid containing the open reading frame for tung tree (Vernicia fordii DGAT1 fused to maltose binding protein and poly-histidine affinity tags was constructed and expressed in E. coli BL21(DE3. Immunoblotting showed that the recombinant DGAT1 (rDGAT1 was expressed, but mostly targeted to the membranes and insoluble fractions. Extensive degradation also occurred. Nonetheless, the fusion protein was partially purified from the soluble fraction by Ni-NTA and amylose resin affinity chromatography. Multiple proteins co-purified with DGAT1 fusion protein. These fractions appeared yellow in color and contained fatty acids. The rDGAT1 was solubilized from the insoluble fraction by seven detergents and urea, with SDS and Triton X-100 being the most effective detergents. The solubilized rDGAT1 was partially purified by Ni-NTA affinity chromatography. PreScission protease digestion confirmed the identity of rDGAT1 and showed extensive precipitation following Ni-NTA affinity purification. Conclusions This study reports the first procedure for expressing full-length DGAT1 from any species using a bacterial expression system. The results suggest that recombinant DGAT1 is degraded extensively from the carboxyl terminus and associated with other proteins, lipids, and membranes.

  14. Purification of human transcription factors Nanog and Sox2, each in complex with Skp, an Escherichia coli periplasmic chaperone.

    Science.gov (United States)

    Ha, Sung Chul; Pereira, Jose Henrique; Jeong, Jin Hee; Huh, Jin Hoe; Kim, Sung-Hou

    2009-10-01

    Nanog and Sox2 are key transcriptional factors involved in self-renewal and pluripotency of stem cells in human and other mammals. Nanog and Sox2 contain homeodomain (HD) and high-mobility group (HMG) DNA-binding domain, respectively, for targeting them to their regulatory regions and the other regions with transactivation function by providing sites for recruiting other transcriptional regulators. To gain insights in the biochemical and biophysical characteristics of the other regions of Nanog and Sox2, we have tried to overproduce and purify full length wild-type human Nanog and Sox2 expressed in Escherichia coli. Interestingly, we found that Nanog and Sox2 were individually stabilized by tight interaction with Skp, an E. coli periplasmic chaperone, thereby enabling stable over-expression and purification of Nanog and Sox2, each in complex with Skp. Purified Skp complexes of Nanog and Sox maintained DNA-binding activity toward its cognate DNA sequence. A similar approach may be applicable for some other mammalian proteins that are unstable or difficult to over-express in E. coli.

  15. Expression, purification and characterization of the interferon ...

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... recombinant human RNase L caused RNA-degradation and cell-growth inhibition in E. coli without the need for exogenous 2-5A. ..... agarose-TBE gel showing RNA degradation profile at 30°C for 10 min at different enzyme concentrations: RNA (2 μg/20 μL) of mouse kidney total RNA; 2-5A (10.0 nM); ...

  16. Recombinant E. coli expressing Vitreoscilla haemoglobin prefers ...

    Indian Academy of Sciences (India)

    Supplementary section 2. KEGG pathway analysis of proteins that were differentially expressed under aerobic and micro- aerobic conditions in VHb expressing E. coli. Pathway. Aerobiosis. Up. Down. Up. Microaerobiosis. Down. Biosynthesis of secondary metabolites (ecx01110). sdhB, pckA, gnd, icdA. purC, glyA. pckA ...

  17. Expression, Purification and Characterisation of Tryptophan Hydroxylases

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo

    2007-01-01

    hjernen. Unormalheder i serotonins funktion som signalstof i hjernen, menes at spille en rolle i flere psykiske lidelser såsom depression, obsessiv-kompulsiv lidelse (OCD) og skizofreni. Flere varianter af TPH er fremstillet i Escherichia coli og der er udviklet en simpel metode til at oprense store...... og co-substratet tetrahydrobiopterin spalter dioxygen (O2) så det ene oxygen-atom indsættes i aminosyren tryptophan. Der findes to forskellige former af TPH: TPH-1 findes flere forskellige steder i kroppen, mens TPH-2 findes i hjernen hvor det har afgørende betydning for mængden af serotonin i...... røntgenkrystallografi. Denne struktur er af stor betydning for en øget forståelse af dette vigtige enzym....

  18. Expression and Purification of Soluble, Biologically Active ...

    African Journals Online (AJOL)

    Purpose: To investigate Pichia pastoris expression system for producing clinically usable, high-quality dipeptidyl peptidase 4 recombinant protein. Methods: The yeast, Pichia pastoris, expression system was used for the production of the human recombinant dipeptidyl peptidase 4 as a secreted form. The full-length human ...

  19. Expression, purification and characterization of a peroxidase from ...

    African Journals Online (AJOL)

    The cDNA was subcloned in bacterial expression vector pET28a and expressed as N-terminal histidine tagged fusion protein in Escherichia coli (Rosetta gami). After induction with IPTG, a molecular weight of 42 kDa fusion protein was obtained and purified by Ni-NTA affinity chromatography. New Zealand white rabbit was ...

  20. Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1

    Directory of Open Access Journals (Sweden)

    Hartinger Doris

    2010-08-01

    Full Text Available Abstract Background Fumonisin B1 is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B1, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B1. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity. Results When expressed from a T7 promoter at 30°C, the aminotransferase accumulated invariably in inclusion bodies in DE3 lysogens of the E. coli strains BL21, HMS174, Rosetta 2, Origami 2, or Rosetta-gami. Omission of the isopropyl-beta-D-thiogalactopyranoside (IPTG used for induction caused a reduction of expression level, but no enhancement of solubility. Likewise, protein production but not solubility correlated with the IPTG concentration in E. coli Tuner(DE3. Addition of the solubility enhancers betaine and sorbitol or the co-enzyme pyridoxal phosphate showed no effect. Maltose-binding protein, used as an N-terminal fusion partner, promoted solubility at 30°C or less, but not at 37°C. Low enzyme activity and subsequent aggregation in the course of purification and cleavage indicated that the soluble fusion protein contained incorrectly folded aminotransferase. Expression in E. coli ArcticExpress(DE3, which co-expresses two cold-adapted chaperonins, at 11°C finally resulted in production of appreciable amounts of active enzyme. Since His tag-mediated affinity purification from this strain was hindered by co-elution of chaperonin, two steps of chromatography with optimized imidazole concentration in the

  1. Expression and purification of recombinant Shiga toxin 2B from ...

    African Journals Online (AJOL)

    sunny t

    expression of Stx2B economically was obtained using 1 mM IPTG for 4 h at 37°C. Protein identity was confirmed by a band at ~11.4 kDa using ... of recombinant Shiga toxin B subunit (rStxB) protein in. BALB/c mice was evaluated. Animal ..... tag-based purification of PKCƔ Kinase free tag protein. (Ohana et al., 2011) which ...

  2. Expression, purification and characterization of recombinant ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... A recombinant targeting bifunctional hirudin was expressed in the yeast Pichia pastoris. In order to decrease the side effects of hirudin and increase its activity to prevent arterial thrombus, we fused a factor Xa (FXa) recognition sequence into N' of hirudin, while maintaining the activity of natural hirudin.

  3. Cloning, expression, purification and antigenic evaluation of ...

    African Journals Online (AJOL)

    Yomi

    2012-01-31

    Jan 31, 2012 ... Key word: Hyaluronidase gene, cloning, expression of recombinant gene, antigenic region. INTRODUCTION. Group A streptococcus (Streptococcus pyogenes) is an important species of gram-positive pathogenic extra- cellular bacteria. This bacteria can produce wide range of infectious diseases like ...

  4. Expression and Purification of Soluble, Biologically Active ...

    African Journals Online (AJOL)

    except that glycerol was replaced by methanol. ... the culture reached the log growth phase. ... Methanol as expression inducer ... Fig 1: The schematic diagram of the human DDP4/CD26 molecule (A), cloning strategy for the human rDDP4(31-.

  5. Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli.

    Science.gov (United States)

    Gurunathan, Sangiliyandi; Kalishwaralal, Kalimuthu; Vaidyanathan, Ramanathan; Venkataraman, Deepak; Pandian, Sureshbabu Ram Kumar; Muniyandi, Jeyaraj; Hariharan, Nellaiah; Eom, Soo Hyun

    2009-11-01

    The application of nanoscale materials and structures, usually ranging from 1 to 100 nanometers (nm), is an emerging area of nanoscience and nanotechnology. Nanomaterials may provide solutions to technological and environmental challenges in the areas of solar energy conversion, catalysis, medicine, and water-treatment. The development of techniques for the controlled synthesis of nanoparticles of well-defined size, shape and composition, to be used in the biomedical field and areas such as optics and electronics, has become a big challenge. Development of reliable and eco-friendly processes for synthesis of metallic nanoparticles is an important step in the field of application of nanotechnology. One of the options to achieve this objective is to use 'natural factories' such as biological systems. This study reports the optimal conditions for maximum synthesis of silver nanoparticles (AgNPs) through reduction of Ag(+) ions by the culture supernatant of Escherichia coli. The synthesized silver nanoparticles were purified by using sucrose density gradient centrifugation. The purified sample was further characterized by UV-vis spectra, fluorescence spectroscopy and TEM. The purified solution yielded the maximum absorbance peak at 420 nm and the TEM characterization showed a uniform distribution of nanoparticles, with an average size of 50 nm. X-ray diffraction (XRD) spectrum of the silver nanoparticles exhibited 2theta values corresponding to the silver nanocrystal. The size-distribution of nanoparticles was determined using a particle-size analyzer and the average particle size was found to be 50 nm. This study also demonstrates that particle size could be controlled by varying the parameters such as temperature, pH and concentration of AgNO(3).

  6. Recombinant bacterial expression and purification of human fragile X mental retardation protein isoform 1.

    Science.gov (United States)

    Evans, Timothy L; Mihailescu, Mihaela-Rita

    2010-12-01

    The loss of expression of the fragile X mental retardation protein (FMRP) leads to fragile X syndrome. FMRP has two types of RNA binding domains, two K-homology domains and an arginine-glycine-glycine box domain, and it is proposed to act as a translation regulator of specific messenger RNA. The interest to produce sufficient quantities of pure recombinant FMRP for biochemical and biophysical studies is high. However, the recombinant bacterial expression of FMRP has had limited success, and subsequent recombinant eukaryotic and in vitro expression has also resulted in limited success. In addition, the in vitro and eukaryotic expression systems may produce FMRP which is posttranslationally modified, as phosphorylation and arginine methylation have been shown to occur on FMRP. In this study, we have successfully isolated the conditions for recombinant expression, purification and long-term storage of FMRP using Escherichia coli, with a high yield. The expression of FMRP using E. coli renders the protein devoid of the posttranslational modifications of phosphorylation and arginine methylation, allowing the study of the direct effects of these modifications individually and simultaneously. In order to assure that FMRP retained activity throughout the process, we used fluorescence spectroscopy to assay the binding activity of the FMRP arginine-glycine-glycine box for the semaphorin 3F mRNA and confirmed that FMRP remained active. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. [Prokaryotic expression, purification and biological activity analysis of recombinant β-Lactamase protein].

    Science.gov (United States)

    Zhou, Xiao-liang; Shi, Pei-ji; Wang, Hao

    2011-01-01

    To prepare RGD4CβL fusion protein using prokaryotic expression system and evaluate the biological activity of the RGD4CβL. RGD4CβL gene was cloned into pColdII to contruct β-Lactamase prokaryotic expression vector. After transformation, the recombinant vector was induced to express recombinant protein RGD4CβL by IPTG in E.coli BL(DE3). The recombinant protein was purified by Ni-NTA resin under denaturing condition and then dialyzed to renature. The tumor cell targeting ability of the recombinant protein was analyzed by flow cytometric analysis. After cleavage and purification, β-Lactamase moiety showed the expected size of 42 000 on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein specially targeted breast cancer cell line MCF-7. This research successfully estiblished a method for prokaryotic expression and purification of β-lactamase. These results suggest the potential use of the protein as an agent for ADEPT.

  8. Expression, purification, and characterization of formaldehyde dehydrogenase from Pseudomonas aeruginosa.

    Science.gov (United States)

    Zhang, Wangluo; Chen, Shuai; Liao, Yuanping; Wang, Dingli; Ding, Jianfeng; Wang, Yingming; Ran, Xiaoyuan; Lu, Daru; Zhu, Huaxing

    2013-12-01

    As a member of zinc-containing medium-chain alcohol dehydrogenase family, formaldehyde dehydrogenase (FDH) can oxidize toxic formaldehyde to less active formate with NAD(+) as a cofactor and exists in both prokaryotes and eukaryotes. Most FDHs are well known to be glutathione-dependent in the catalysis of formaldehyde oxidation, but the enzyme from Pseudomonas putida is an exception, which is independent of glutathione. To identify novel glutathione-independent FDHs from other bacterial strains and facilitate the corresponding structural and enzymatic studies, high-level soluble expression and efficient purification of these enzymes need to be achieved. Here, we present molecular cloning, expression, and purification of the FDH from Pseudomonas aeruginosa, which is a Gram-negative pathogenic bacterium causing opportunistic human infection. The FDH of P. aeruginosa shows high sequence identity (87.97%) with that of P. putida. Our results indicated that coexpression with molecular chaperones GroES, GroEL, and Tig has significantly attenuated inclusion body formation and improved the solubility of the recombinant FDH in Escherichiacoli cells. A purification protocol including three chromatographic steps was also established to isolate the recombinant FDH to homogeneity with a yield of ∼3.2 mg from 1L of cell culture. The recombinant P. aeruginosa FDH was properly folded and biologically functional, as demonstrated by the mass spectrometric, crystallographic, and enzymatic characterizations of the purified proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Folding and purification of a recombinantly expressed interferon regulatory factor, IRF-4.

    Science.gov (United States)

    Moellering, B J; Yoshinaga, S K; Hui, A; Delaney, J M; Hara, S; Narhi, L O; Westcott, K R

    1999-06-01

    Interferon regulatory factor 4 (IRF-4), an intracellular, multidomain protein, is a member of the interferon regulatory factor family and a lymphoid-specific transcription factor that can form a ternary complex with DNA and the transcription factor PU.1. Recombinant human IRF-4 was expressed in Escherichia coli and purified from the soluble cell extract and the insoluble inclusion bodies. The inclusion bodies were solubilized with guanidinium-hydrochloride and sequentially buffer exchanged into urea- and then NaCl-containing solutions. This two-step process for the removal of the denaturants was the critical step to allow for the correct folding of IRF-4. Following purification through immobilized metal affinity, hydrophobic interaction, and gel permeation chromatographies, the renatured protein was shown to be structurally and physically equivalent to a sample of IRF-4 produced in the soluble fraction of E. coli cells. This was confirmed by near and far UV circular dichroism analysis, including thermal stability analysis. The purified IRF-4 was also shown to be capable of binding DNA in a PU.1-dependent manner by electrophoretic mobility shift analysis. The protein folding and purification methods are suitable for producing large quantities of full-length IRF-4. Copyright 1999 Academic Press.

  10. Prokaryotic expression, purification, and polyclonal antibody production of a hydrophobin from Grifola frondosa.

    Science.gov (United States)

    Wang, Zefang; Feng, Shuren; Huang, Yujian; Qiao, Mingqiang; Zhang, Baohua; Xu, Haijin

    2010-06-15

    Hydrophobins are small fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach. A new hydrophobin gene hgfI was identified recently from the edible mushroom Grifola frondosa. In this paper, the cloning, expression, purification, and polyclonal antibody preparation of the HGFI were described. The hgfI gene was cloned into pET-28a expression plasmid at the EcoRI and NdeI restriction sites and then transformed into Escherichia coli BL21 strain. SDSPAGE analysis showed that recombinant HGFI protein was satisfactorily expressed by optimizing the concentration and induction time of IPTG. The expressed recombinant HGFI protein was purified by electroelution because its inclusion body was insoluble in traditional processing method. After a desalting procedure with Sephadex G-25, the recombinant HGFI protein was used to immunize adult rabbits following standard protocol. ELISA and western blot analysis indicated that the produced antiserum could detect both HGFI protein expressed in the prokaryotic (E. coli) and in the eukaryotic cells (G. frondosa). Furthermore, the antiserum was used to determine the localization of HGFI protein in G. frondosa cells using an immunofluorescence technique. The results demonstrated that HGFI protein was localized in the cell wall, especially at the budding position of hypha. The polyclonal antibody against HGFI will facilitate further production and functional study of HGFI protein.

  11. A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

    Science.gov (United States)

    Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

    2013-11-01

    Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pHpurification step. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Global gene expression in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Schembri, Mark; Kjærgaard, K.; Klemm, Per

    2003-01-01

    It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance...... to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared...... the transition to biofilm growth, and these included genes expressed under oxygen-limiting conditions, genes encoding (putative) transport proteins, putative oxidoreductases and genes associated with enhanced heavy metal resistance. Of particular interest was the observation that many of the genes altered...

  13. Expression and purification of coat protein of citrus tristeza virus ...

    African Journals Online (AJOL)

    Six colonies of TOP10 E. coli were selected and checked for the appropriate insertion of cp gene with PCR using T7F (5' TAA TAC GAC TCA CTA TAG GG 3') as forward primer and CTVCP2 as reverse primer. Two colonies having appropriate insertion were selected for transformation into BLD21 star (DE3) expression E.

  14. Purification and properties of Rhizobial DehL expressed in ...

    African Journals Online (AJOL)

    The Rhizobium sp. DehL was produced by heterologous expression of the cloned gene in Escherichia coli. DehL enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 61 and 31 kDa by gel filtration and DS-polyacrylamide gel electrophoresis (SDSPAGE), respectively, ...

  15. Cloning and Expression of Functional Reteplase in Escherichia coli TOP10.

    Science.gov (United States)

    Khodabakhsh, Fatemeh; Dehghani, Zohreh; Zia, Mohammad Farid; Rabbani, Mohammad; Sadeghi, Hamid Mir Mohammad

    2013-07-01

    Production of tissue Plasminogen Activator protein (t-PA) in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli (E. coli) bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBAD/gIII plasmid which allowed secretion of this protein into the periplasmic space and would allow the correct formation of disulfide bonds in protein structure. The presence of reteplase cDNA in pBAD/gIII plasmid was confirmed by restriction digestion and sequencing. After induction of the expression of this protein by adding 0.0002% L-Arabinose to the medium, the proteins in periplasmic space as well as the inclusion bodies formed inside the cell were extracted. Subsequently, these proteins were purified and detected by Western blot method. Our results showed that the amount of reteplase extracted from periplasmic space was much lower than the extracted inclusion bodies and large quantities of the recombinant protein were present as inclusion bodies. Therefore, it was more efficient to use inclusion body extraction method for protein isolation and purification. We produced active reteplase after its expression in E. coli TOP10 and isolation of inclusion bodies produced the best results for purification and extraction of this protein.

  16. Expression, purification and characterization of Oryza sativa L. NAD ...

    African Journals Online (AJOL)

    The cDNA fragment of a rice NAD-malic enzyme (OsNAD-ME1) was cloned and constructed into expression vector (pGEX-6p-3). OsNAD-ME1 was successfully expressed as a GST fusion protein in Escherichia coli BL21. The optimal concentration of IPTG for inducement was 1 mmol/L and the optimal culture temperature ...

  17. An efficient strategy for heterologous expression and purification of active peptide hainantoxin-IV.

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    Full Text Available Hainantoxin-IV (HNTX-IV from the venom of the spider Selenocosmia hainana is a potent antagonist that specifically inhibits the tetrodotoxin-sensitive (TTX-S sodium channels. The toxin peptide consists of 35 amino acids and adopts a typical inhibitory cystine knot (ICK motif. To obtain adequate HNTX-IV peptides for further insight into the structure-activity relationships of the toxin, a novel strategy including cloning, expression and purification was developed in an E. coli expression system. For this purpose, a seamless restriction-free (RF cloning method was employed for the construction of an expression vector to avoid introducing unwanted sequences into the target gene. Furthermore, the solubility of recombinant HNTX-IV could be promoted efficiently by the combination of a glutathione S-transferase (GST tag and a small ubiquitin-related modifier (SUMO tag. Finally, an affinity-chromatography-free purification strategy was developed by cut-off dialysis tubing combined with trichloroacetic acid (TCA extraction. Further HPLC purification yielded recombinant, tag-free HNTX-IV with high yield and purity. The molecular weight of recombinant HNTX-IV (rHNTX-IV is identical to its theoretical value according to Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS analysis. The recombinant toxin has similar activity (IC50 value of 120 nM on the tetrodotoxin-sensitive (TTX-S sodium channels in adult rat dorsal root ganglion (DRG neurons to native toxins. In the report, an efficient and cost-effective strategy for producing rHNTX-IV was developed, which paved the way for the further study of structure-activity relationships of rHNTX-IV and its pharmaceutical applications.

  18. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Directory of Open Access Journals (Sweden)

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  19. Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.

    Science.gov (United States)

    Babu, Mohan; Kagan, Olga; Guo, Hongbo; Greenblatt, Jack; Emili, Andrew

    2012-11-12

    Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems(1, 2). Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria(1-6). In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes(1, 2, 7). Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast(8, 9), and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system(10). Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large

  20. Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14

    Science.gov (United States)

    Leong, L. E.-C.; Walker, P. A.; Porter, A. G.

    1992-08-01

    The protease (3C pro) from human rhinovirus serotype-14 (HRV-14) has been cloned and efficiently expressed in E. coli. A straightforward single-step purification of the recombinant 3C pro has been achieved by fusing the protein to the car☐y-terminus of the glutathione-S-transferase from Schistosoma japonicum. Modifications made to the 5' end of the PCR fragment coding for the 3C pro have allowed the specific cleavage of the fusion protein using thrombin to yield mature 3C pro with the correct amino-terminal amino acid. This protease has been shown to be active when assayed using synthetic peptides corresponding to the natural cleavage recognition sequences within the polyprotein. Other substrates are being developed for this protease for possible use in the screening of inhibitors of 3C pro. Sufficient protease 3C pro has been purified for initial attempts at crystallization.

  1. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Energy Technology Data Exchange (ETDEWEB)

    Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  2. Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis.

    Science.gov (United States)

    Sugimoto, S; Iwase, T; Sato, F; Tajima, A; Shinji, H; Mizunoe, Y

    2011-12-01

    Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression-secretion system. The esp gene was fused with the N-terminal Sec-dependent signal sequence of the B. choshinensis cell wall protein and a C-terminal hexa-histidine-tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity. The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20-ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1-l culture). Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  3. Expression, Purification, Characterization and In Vitro Activity of ...

    African Journals Online (AJOL)

    HP

    The amplified PCR product was ligated into a modified pET15b (Sma I) expression vector[14], resulting in the recombinant vector pET15b-mSOD1. The recombinant plasmid constructs were confirmed by direct sequencing (Yinjun, Shanghai, China). The pET15b-mSOD1 plasmid was transformed into E. coli Rosetta-gami ...

  4. High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

    Science.gov (United States)

    Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

    2018-02-01

    Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni2+. Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Modular broad-host-range expression vectors for single-protein and protein complex purification.

    Science.gov (United States)

    Fodor, Barna D; Kovács, Akos T; Csáki, Róbert; Hunyadi-Gulyás, Eva; Klement, Eva; Maróti, Gergely; Mészáros, Lívia S; Medzihradszky, Katalin F; Rákhely, Gábor; Kovács, Kornél L

    2004-02-01

    A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.

  6. The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

    Science.gov (United States)

    Mlynek, Georg; Lehner, Anita; Neuhold, Jana; Leeb, Sarah; Kostan, Julius; Charnagalov, Alexej; Stolt-Bergner, Peggy; Djinović-Carugo, Kristina; Pinotsis, Nikos

    2014-06-01

    Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

  7. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

    Energy Technology Data Exchange (ETDEWEB)

    Tykvart, J.; Sacha, P.; Barinka, C.; Knedlik, T.; Starkova, J.; Lubkowski, J.; Konvalinka, J. (Gilead); (NCI); (Czech Academy)

    2012-02-07

    Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

  8. Cloning and expression of Pseudomonas aeruginosa flagellin in Escherichia coli.

    OpenAIRE

    Kelly-Wintenberg, K; Montie, T. C.

    1989-01-01

    The flagellin gene was isolated from a Pseudomonas aeruginosa PAO1 genomic bank by conjugation into a PA103 Fla- strain. Flagellin DNA was transferred from motile recipient PA103 Fla+ cells by transformation into Escherichia coli. We show that transformed E. coli expresses flagellin protein. Export of flagellin to the E. coli cell surface was suggested by positive colony blots of unlysed cells and by isolation of flagellin protein from E. coli supernatants.

  9. Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus

    Directory of Open Access Journals (Sweden)

    Rybak M. Yu.

    2015-06-01

    Full Text Available D-Tyr-tRNATyr-deacylase (DTD is a conservative enzyme, found in all domains of life, which ensures an additional checkpoint in the recycling of misaminoacylated D-Tyr-tRNATyr. DTD is capable of accelerating the hydrolysis of the ester linkage of D-Tyr-tRNATyr producing a free tRNA and D-tyrosine, thereby preventing an incorrect incorporation of D-amino acids into proteins. Deacylase distinguishes between D- and L-aminoacyl moieties and does not hydrolyze L-aminoacylated tRNA. The structural bases of this specificity and the mechanism of D-aminoacyl-tRNA hydrolysis are poorly understood. Aim. To clone D-Tyr-tRNATyr-deacylase from T. thermophilus (DTDTT, optimize the conditions for its expression in E.coli and develop an efficient purification procedure yielding the high quality enzyme suitable for the structural and functional studies. Methods. For amplification of DTD gene from T. thermophilus genomic DNA and its cloning into the pProEXHTb expression vector modern techniques were applied. Purification of the recombinant DTD protein was done with three types of column chromatography. His-tag was cleaved out from DTD by TEV protease. The cleavage was confirmed by Western blot analysis with anti-His-tag antibodies. Molecular weight of purified DTDTT was determined by the gel-filtration. Results. The expression construct pProEXHTb, containing DTD sequence from T. thermophilus, was obtained and successfully expressed in the BL21(DE3pLysS E.coli strain. The protein of interest was purified to homogeneity by the combination of affinity (Ni-NTA, anion-exchange (Q-Sepharose and size-exclusion (Superdex S 200 chromatographies. 2 mg of more than 90% pure recombinant DTD can be obtained from 1 L of bacterial culture. Molecular weight of purified DTD from T. thermophilus was determined to be 32 kDa, suggesting its dimeric structure. Conclusions. The pProEXHTb expression vector can be used for expression of DTD from T. thermophilus. The preparative amounts of

  10. Escherichia coli expression, refolding and characterization of human laforin.

    Science.gov (United States)

    Castanheira, Pedro; Moreira, Susana; Gama, Miguel; Faro, Carlos

    2010-06-01

    Laforin is a unique human dual-specificity phosphatase as it contains an amino terminal carbohydrate binding module (CBM). Laforin gene mutations lead to Lafora disease, a progressive myoclonus epilepsy with an early fatal issue. Previous attempts to produce recombinant laforin faced various difficulties, namely the appearance of protein inclusion bodies, the contamination with bacterial proteins and a high tendency of the protein to aggregate, despite the use of fusion tags to improve solubility and ease the purification process. In this work, we have expressed human laforin in Escherichia coli in the form of inclusion bodies devoid of any fusion tags. After a rapid dilution refolding step, the protein was purified by two chromatographic steps, yielding 5-7mg of purified protein per liter of bacterial culture. The purified protein was shown to have the kinetic characteristics of a dual-specificity phosphatase, and a functional carbohydrate binding module. With this protocol, we were able for the first time, to produce and purify laforin without fusion tags in the amounts traditionally needed for the crystallographic structural studies paving the way to the understanding of the molecular mechanisms of laforin activity and to the development of novel therapies for Lafora disease.

  11. HETEROLOGOUS EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE LIP11 FROM Yarrowia lipolytica in Pichia pastoris X33

    Directory of Open Access Journals (Sweden)

    Arti Kumari

    2013-04-01

    Full Text Available A gene encoding Lip 11 from Yarrowia lipolytica MSR80 was cloned and expressed into methanol inducible expression vector pPICZ�A and successfully transformed into Pichia pastoris X33. The recombinant mut+ clones were selected on zeocine-YPD plates and high-yield clones was identified by tributyrin agar plate screening. The clone produced 32110 U/L of enzyme after 48 h expression in BMMY medium following induction with 0.5% methanol. Lip 11 was purified by affinity chromatography using Ni+ - NTA column with a purification fold of 27 and yield of 59%. It was expressed as glycosylated protein of molecular mass 47 kDa. Biochemical characterization revealed that it was more thermostable with improved Kd value of 5.5 x 10–3 at 70 °C and better catalytic efficiency than the previously expressed E.coli recombinant enzyme.

  12. Expression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio carchariae

    Energy Technology Data Exchange (ETDEWEB)

    Songsiriritthigul, Chomphunuch [School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand); National Synchrotron Research Center, Nakhon Ratchasima 30000 (Thailand); Yuvaniyama, Jirundon [Center for Excellence in Protein Structure and Function and Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand); Robinson, Robert C. [Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673 (Singapore); Vongsuwan, Archara [School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand); Prinz, Heino [Max-Planck Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany); Suginta, Wipa, E-mail: wipa@sut.ac.th [School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand)

    2005-10-01

    This article describes the high-level expression, purification and crystallization as well as preliminary X-ray diffraction study of a family 18 chitinase, chitinase A from V. carchariae. Chitinase A of Vibrio carchariae was expressed in Escherichia coli M15 host cells as a 575-amino-acid fragment with full enzymatic activity using the pQE60 expression vector. The yield of the highly purified recombinant protein was approximately 70 mg per litre of bacterial culture. The molecular mass of the expressed protein was determined by HPLC/ESI–MS to be 63 770, including the hexahistidine tag. Crystals of recombinant chitinase A were grown to a suitable size for X-ray structure analysis in a precipitant containing 10%(v/v) PEG 400, 0.1 M sodium acetate pH 4.6 and 0.125 M CaCl{sub 2}. The crystals belonged to the tetragonal space group P422, with two molecules per asymmetric unit and unit-cell parameters a = b = 127.64, c = 171.42 Å. A complete diffraction data set was collected to 2.14 Å resolution using a Rigaku/MSC R-AXIS IV{sup ++} detector system mounted on an RU-H3R rotating-anode X-ray generator.

  13. Prokaryotic Expression, Purification and Immunogenicity in Rabbits of the Small Antigen of Hepatitis Delta Virus

    Directory of Open Access Journals (Sweden)

    Vera L. Tunitskaya

    2016-10-01

    Full Text Available Hepatitis delta virus (HDV is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg, its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 μg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 μg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.

  14. Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol.

    Directory of Open Access Journals (Sweden)

    Elizaveta O Boldinova

    Full Text Available Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37ºC. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

  15. Expression of green fluorescent protein (GFPuv) in Escherichia coli ...

    African Journals Online (AJOL)

    The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by ...

  16. Switchable gene expression in Escherichia coli using a miniaturized photobioreactor

    National Research Council Canada - National Science Library

    Lee, Jae Myung; Lee, Junhyeong; Kim, Taesung; Lee, Sung Kuk

    2013-01-01

    We present a light-switchable gene expression system for both inducible and switchable control of gene expression at a single cell level in Escherichia coli using a previously constructed light-sensing system. The λ...

  17. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Nie,Y.; Hobbs, J.; Vigues, S.; Olson, W.; Conn, G.; Munger, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

  18. Solubilization and purification of Escherichia coli expressed GST ...

    African Journals Online (AJOL)

    Vascular endothelial growth factor (VEGF) is a potent mitogen for tumor angiogenesis. Clinically, VEGF detection in human blood can be expected to be used in the very near future for cancer screening, prognosis, monitoring of therapy and diagnosis. VEGF has been identified as the target for the treatment of cancer.

  19. Solubilization and purification of Escherichia coli expressed GST ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... and secreted by almost all solid tumors. VEGF is a potential and unique tumor marker in tumor diagnosis and prognosis. Current data show that: (i) ... 8.0;150 mM sodium chloride; and 1 mM EDTA) and pelleted by centrifugation. The pelleted bacteria were then resuspended in lysis buffer (STE + lysozyme ...

  20. Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

    Science.gov (United States)

    2016-08-01

    Concentration Determination ................................................................4  2.10  Enzyme -Linked Immunosorbent Assay (ELISA...pET21b using the Nde1 and Xho1 restriction sites, which resulted in the addition of a C-terminal hexahistidine tag. This plasmid was sequence-verified...Center for Biotechnology Information (Bethesda, MD) databases. 2.2 Recombinant Protein Expression in E. coli BL21 DE3 cells were transformed with

  1. Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know.

    Science.gov (United States)

    Hayat, Seyed Mohammad Gheibi; Farahani, Najmeh; Golichenari, Behrouz; Sahebkar, Amir Hosein

    2018-01-31

    Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli. A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed. Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Purification

    DEFF Research Database (Denmark)

    Andersen, Astrid Oberborbeck

    2017-01-01

    of categories can be understood as practices of purification. However, a purely technical grip on water is never possible. Unruly elements, like weather, contamination, urban dwellers, and competing interests, interfere and make processes of intervention unstable. Water is never completely cleaned, and, equally......In Arequipa, Peru’s second largest city, engineers work hard to control water flows and provide different sectors with clean and sufficient water. In 2011, only 10 percent of the totality of water used daily by Arequipa’s then close to 1 million people—in households, tourism, industry, and mining......—was treated before it was returned to the river where it continues its flow downstream towards cultivated fields and, finally, into the Pacific Ocean. It takes specialized knowledge and manifold technologies to manage water and sustain life in Arequipa, and engineers are central actors for making water flow...

  3. Telomerase repeat amplification protocol (TRAP) activity upon recombinant expression and purification of human telomerase in a bacterial system.

    Science.gov (United States)

    Hansen, Debra T; Thiyagarajan, Thirumagal; Larson, Amy C; Hansen, Jeffrey L

    2016-07-01

    Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a ∼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Expression, Purification and Preliminary Diffraction Studies of CmlS

    Energy Technology Data Exchange (ETDEWEB)

    Latimer, R.; Podzelinska, K; Soares, A; Bhattacharya, A; Vining, L; Jia, Z; Zechel, D

    2009-01-01

    CmlS, a flavin-dependent halogenase (FDH) present in the chloramphenicol-biosynthetic pathway in Streptomyces venezuelae, directs the dichlorination of an acetyl group. The reaction mechanism of CmlS is of considerable interest as it will help to explain how the FDH family can halogenate a wide range of substrates through a common mechanism. The protein has been recombinantly expressed in Escherichia coli and purified to homogeneity. The hanging-drop vapour-diffusion method was used to produce crystals that were suitable for X-ray diffraction. Data were collected to 2.0 Angstroms resolution. The crystal belonged to space group C2, with unit-cell parameters

  5. Production of engineered human pancreatic ribonucleases, solving expression and purification problems, and enhancing thermostability.

    Science.gov (United States)

    Canals, A; Ribó, M; Benito, A; Bosch, M; Mombelli, E; Vilanova, M

    1999-10-01

    Human pancreatic ribonuclease, the homolog of bovine pancreatic ribonuclease, has a significant therapeutic potential. Its study has been hindered by the difficulty of obtaining the enzyme in a pure and homogeneous form, either from human source or using heterologous expression. Engineering of different variants of human pancreatic ribonuclease has allowed us to study and overcome some problems encountered during its heterologous production in an Escherichia coli system and its purification from inclusion bodies. The 5'-end region of the mRNA that encodes the enzyme is critical for obtaining high expression levels. The results also suggest the importance of the proline 50 residue in the recovery yields of human pancreatic ribonuclease. All the variants produced are pure and homogeneous. Their homogeneity has been demonstrated by cation-exchange and reversed-phase chromatography and by mass spectrometry analysis. Moreover, enhancement of human pancreatic ribonuclease thermal stability is observed when residues R4, K6, Q9, D16, and S17 are changed to the corresponding residues of bovine seminal ribonuclease. Copyright 1999 Academic Press.

  6. [Cloning, expression, purification and identification of Der f6 gene and its immunological characteristics from the dust house mite].

    Science.gov (United States)

    Zhu, Yong-feng; Liu, Zhi-gang; Gao, Bo

    2006-08-01

    To construct, purify and characterize a recombinant expression plasmid containing Der f6 gene of Dermatophagoides farinae. A pair of primers was designed according to the known sequence of Der f6 gene. The live mites identified and cultured locally were picked and the total RNA was extracted. The Der f6 gene fragment was amplified by RT-PCR, and cloned into pMD18-T vector, and then transferred into E. coli Top10. The target gene obtained from the recombinant plasmid by digestion with EcoR I and Xho I was connected to the prokaryotic expression vector pET-24a. The expressed recombinant plasmid containing Der f6 gene was constructed by cloning target gene into pET-24a and first transferred into E. coli Top10, then into E. coli B121 (DE3). The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting, and was purified by immobilized metal ion affinity chromatography (IMAC). The two recombinant plasmids, pMD18-T-Der f6 and pET24a-Der f6, were constructed. SDS-PAGE showed a correct molecular weight of the recombinant Der f6 protein. After purification by affinity chromatography, the protein showed only one strip on SDS-PAGE gel and appropriate combination ability with IgE in sera of allergic patients. The Der f6 gene has been cloned into plasmid pMD18-T vector and sub-cloned into the expression vector pET-24a, the recombinant plasmid pET24a-Der f6 has been expressed in E. coli BL21 (DE3), purified by IMAC, and showed appropriate IgE-combined ability.

  7. Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis

    Directory of Open Access Journals (Sweden)

    Schlegel Brigitte

    2004-03-01

    Full Text Available Abstract Background High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination. Results 88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains were quickly identified as being well folded and suitable for structural analysis. Conclusion The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.

  8. High-yield recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli.

    Science.gov (United States)

    Feng, Xingjun; Xu, Wenshan; Qu, Pei; Li, Xiaochong; Xing, Liwei; Liu, Di; Jiao, Jian; Wang, Jue; Li, Zhongqiu; Liu, Chunlong

    2015-01-01

    The antimicrobial peptide fowlicidin-2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin-2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin-2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin-2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET-32a(+), which features fusion protein thioredoxin at the N-terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria-Bertani (LB) medium. After isopropyl-β-D-thiogalactopyranoside (IPTG) induction, the fowlicidin-2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse-phase high-performance liquid chromatography (RP-HPLC), ∼6.0 mg of fowlicidin-2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram-positive and Gram-negative bacteria, and even drug-resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large-scale production. © 2015 American Institute of Chemical Engineers.

  9. Differential expression of the Escherichia coli autoaggregation factor antigen 43

    DEFF Research Database (Denmark)

    Schembri, Mark; Hjerrild, Louise; Gjermansen, Morten

    2003-01-01

    Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most Escherichia coli strains. Due to its excellent cell-to-cell aggregation characteristics, Ag43 expression confers clumping and fluffing of cells and promotes biofilm formation. Ag43 expression is repressed by the cellular redox......-forming potential of E. coli. Finally, we demonstrated that Ag43-mediated cell aggregation confers significant protection against hydrogen peroxide killing....

  10. A chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants

    Directory of Open Access Journals (Sweden)

    Frank eSainsbury

    2016-02-01

    Full Text Available The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to rapidly purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues.

  11. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2008-06-01

    Full Text Available Abstract Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP, glycoprotein 1 (GP1, and glycoprotein 2 (GP2. Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP fusions in the Rosetta strains of Escherichia coli (E. coli using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC. Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA. Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

  12. Production of Computationally Designed Small Soluble- and Membrane-Proteins: Cloning, Expression, and Purification.

    Science.gov (United States)

    Tripathy, Barsa; Acharya, Rudresh

    2017-01-01

    This book chapter focuses on expression and purification of computationally designed small soluble proteins and membrane proteins that are ordinarily difficult to express in good amounts for experiments. Over-expression of such proteins can be achieved by using the solubility tag such as maltose binding protein (MBP), Thioredoxin (Trx), and Gultathione-S-transferase (GST) fused to the protein of interest. Here, we describe and provide the protocols for cloning, expression and purification of such proteins using the solubility tag.

  13. Enhanced gentamicin killing of Escherichia coli by tet gene expression.

    OpenAIRE

    Merlin, T L; Corvo, D L; Gill, J H; Griffith, J K

    1989-01-01

    Time-kill studies were performed to determine the effect of tetracycline resistance (tet) gene expression on gentamicin killing of Escherichia coli. Expression of tet increased gentamicin killing in laboratory strains and clinical isolates. A role for tetracycline in inducing tet expression and increasing the bactericidal activity of aminoglycosides is suggested.

  14. Heterologous expression and secretion of an antifungal Bacillus subtilis chitosanase (CSNV26) in Escherichia coli.

    Science.gov (United States)

    Kilani-Feki, Olfa; Frikha, Fakher; Zouari, Imen; Jaoua, Samir

    2013-07-01

    The aims of the study were the production improvement, the purification, the characterization and the activity investigation of chitosanase CSNV26 of Bacillus subtilis (V26). The gene csnV26 encoding for this protein was amplified and cloned in the pBAD vector then expressed in Escherichia coli (Top10). The SDS-PAGE and zymogram analysis of the recombinant protein showed that it has two active forms sized 27 and 31 kDa, corresponding to the protein with and without signal peptide. This protein has the particularity of being secreted by Top10-pBAD-csnV26 with a high yield of 6.2 g/l. The HPLC purification of CSNV26 from supernatant confirmed the presence of the two sizes. The investigation of the CSNV26 thermostability showed that the pure protein is highly stable keeping 68 % of its activity after 30-min treatment at 100 °C, contrarily to the protein present within the supernatant of E. coli and B. subtilis (V26). The molecular dynamics study of the predicted structure of protein in both forms showed that the presence of the peptide signal in the form of 31 kDa gave it a remarkable thermal stability. The antifungal activity of CSNV26 was evidenced on Rhizopus nigricans and Rhizopus oryzae. Indeed, it has provoked an alteration and embrittlement of their hyphae with onset of protoplast.

  15. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    Science.gov (United States)

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-06

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

  16. Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

    Science.gov (United States)

    Bataille, Laure; Dieryck, Wilfrid; Hocquellet, Agnès; Cabanne, Charlotte; Bathany, Katell; Lecommandoux, Sébastien; Garbay, Bertrand; Garanger, Elisabeth

    2015-06-01

    Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein.

    Science.gov (United States)

    Nguyen, Anh Ngoc; Song, Jung-A; Nguyen, Minh Tan; Do, Bich Hang; Kwon, Grace G; Park, Sang Su; Yoo, Jiwon; Jang, Jaepyeong; Jin, Jonghwa; Osborn, Mark J; Jang, Yeon Jin; Thi Vu, Thu Trang; Oh, Heung-Bum; Choe, Han

    2017-11-23

    Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a β-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.

  18. Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli.

    Science.gov (United States)

    Vallejo, Luis Felipe; Brokelmann, Maren; Marten, Sabine; Trappe, Susanne; Cabrera-Crespo, Joaquin; Hoffmann, Andrea; Gross, Gerhard; Weich, Herbert A; Rinas, Ursula

    2002-03-28

    Eschericha coli was genetically engineered to produce recombinant human bone morphogenetic protein-2 (rhBMP-2) in a non-active aggregated form using a temperature-inducible expression system. High concentrations of both biomass (75 g cell dry weight per liter of culture broth) and inactive rhBMP-2 (8.6 gl(-1)) were obtained by applying a high-cell-density cultivation procedure. After washing and solubilizing the inclusion bodies, rhBMP-2 was refolded and dimerized at concentrations up to 100 mgl(-1) by means of a simple dilution method with yields exceeding 50%. Finally, a one-step purification procedure based on affinity chromatography was implemented to isolate the rhBMP-2 dimer. With the established renaturation and purification protocols, yields of more than 10 mg rhBMP-2 dimer per gram cell dry weight were obtained corresponding to 750 mg rhBMP-2 dimer per liter of culture broth. The purified rhBMP-2 dimer showed biological activity equivalent to CHO produced rhBMP-2 as tested by the induction of alkaline phosphatase activity in C2C12 cells.

  19. Teaching Molecular Biology to Undergraduate Biology Students: An Illustration of Protein Expression and Purification

    Science.gov (United States)

    Sommer, Cesar Adolfo; Silva, Flavio Henrique; Novo, Maria Teresa Marques

    2004-01-01

    Practical classes on protein expression and purification were given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering." The heterologous expression of the green fluorescent protein (GFP)* of "Aequorea victoria" is an interesting system for didactic purposes because it can be viewed easily during…

  20. Construction,expression,purification and identification of prokaryotic expression vector of MART-1 fusion protein

    Directory of Open Access Journals (Sweden)

    Zhao-ting MENG

    2011-09-01

    Full Text Available Objective To construct a prokaryotic expression plasmid containing a fusion gene of MART-1 expressing the His-MART-1 fusion protein in E.coli,and to purify the protein and identify the immunogenicity of His-MART-1.Methods The MART-1 coding sequence was amplified by polymerase chain reaction(PCR,and then cloned into the prokaryotic expression vector(pET-28b containing His tag.The constructed vector,verified by restriction endonuclease digestion,PCR and DNA sequencing,was then transformed into E.coli for expression.The expression of MART-1 recombinant protein was induced by IPTG in E.coli,purified with Ni2+-NTA affinity chromatography method,and identified by SDS-PAGE and Western blotting.ELISA was used to detect the IFN-γ expression secreted by the His-MART-1 specific CD4+ T cells which recognized the His-MART-1 fusion protein presented by dendritic cells(DCs.Results The successful construction of recombinant plasmid was confirmed by restriction digestion,PCR and sequencing.The molecular weight of the purified fusion protein was identified as 13kD by SDS-PAGE,which was identical to the expected value.It was confirmed by western blotting that His-MART-1 fusion protein could be recognized by His monoclonal antibody.ELISA analysis showed that His-MART-1 fusion protein presented by DCs could induce IFN-γ secretion of MART-1 specific CD4+ T cells.Conclusion The recombinant plasmid of pET-28b-MART-1 has been successfully constructed.The expressed His-MART-1 fusion protein has been purified and the immunogenicity of inducing responses between DCs and CD4+ T cells has been determined.

  1. Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli.

    Science.gov (United States)

    Yero, Daniel; Pajón, Rolando; Niebla, Olivia; Sardiñas, Gretel; Vivar, Isbel; Perera, Yasser; García, Darien; Delgado, Maité; Cobas, Karem

    2006-04-01

    In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.

  2. Expression, Purification and Antibacterial Activity of NK-Lysin Mature Peptides from the Channel Catfish (Ictalurus punctatus

    Directory of Open Access Journals (Sweden)

    Shurui Cai

    2016-08-01

    Full Text Available Antimicrobial peptides (AMPs are small peptides and play important roles in host innate immune response against microbial invasion. Aquatic animals secrete different kinds of antimicrobial peptides which have antimicrobial activity towards microorganisms. NK-lysins, mature peptides produced by cytotoxic T lymphocytes and natural killer cells, are comprised of 74–78 amino acid residues, demonstrating broad-spectrum antimicrobial activity against bacteria, fungi, protozoa, and parasites. In this study, three distinct NK-lysin mature peptide (mNKLs, transcripts (76 amino acid residues cloned from the channel catfish (Ictalurus punctatus head kidney were ligated into plasmid vector pET-32a(+ to express the mNKLs fusion protein. The fusion protein was successfully expressed in E. coli Rosetta (DE3 under optimized conditions. After purification by affinity column chromatography, the fusion protein was successfully cleaved by enterokinase and released the peptide mNKLs. Tricine-SDS-PAGE results showed that mNKLs (approximately 8.6 kDa were successfully expressed. The purified peptide mNKLs exhibited antibacterial activity against Staphylococcus aureus and E. coli.

  3. Transformation of Escherichia coli and protein expression using lipoplex mimicry.

    Science.gov (United States)

    Yun, Chul-Ho; Bae, Chun-Sik; Ahn, Taeho

    2016-11-01

    We investigated a "one-step" method for transformation of and protein expression in Escherichia coli (E. coli) using a complex of n-stearylamine, a cationic lipid, and plasmid DNA, which mimics lipoplex-based approaches. When E. coli cells were treated with the cationic lipid-plasmid complex, the transformation efficiencies were in the range of approximately 2-3 × 10(6) colony-forming units. Further increase in the efficiency was obtained by co-treatment with calcium chloride (or rubidium chloride) and the complexes. Moreover, after DNA transfer, E. coli cells successfully expressed plasmid-encoded proteins such as cytochrome P450s and glutathione-S-transferase without overnight incubation of the cells to form colonies, an indispensable step in other bacterial transformation methods. In this study, we provide a simple method for E. coli transformation, which does not require the preparation of competent cells. The present method also shortens the overall procedures for transformation and gene expression in E. coli by omitting the colony-forming step. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Expression, purification, and crystallization of Schizosaccharomyces pombe eIF2B.

    Science.gov (United States)

    Kashiwagi, Kazuhiro; Shigeta, Tomoaki; Imataka, Hiroaki; Ito, Takuhiro; Yokoyama, Shigeyuki

    2016-03-01

    Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution.

  5. Expression, Purification and Characterization of Ricin vectors used for exogenous antigen delivery into the MHC Class I presentation pathway

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    Smith Daniel C.

    2003-01-01

    Full Text Available Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smith et al. J Immunol 2002; 169:99-107]. The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules. Expression in E. coli and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterized in vitro, via an N-glycosidase assay, and in vivo, by a cytotoxicity assay. The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass.

  6. High-level expression of Staphylococcal Protein A in Pichia pastoris and purification and characterization of the recombinant protein.

    Science.gov (United States)

    Hao, Jing; Xu, Li; He, Hongde; Du, Xiaojun; Jia, Lingyun

    2013-08-01

    Staphylococcal Protein A (SPA), a cell wall protein of Staphylococcus aureus, is in high demand because of its ability to bind immunoglobulins. Much of the SPA that we use today is recombinant SPA (rSPA), which is produced in Escherichia coli. As rSPA is obtained by expressing SPA as an intracellular protein, its purification is tedious and time consuming. In order to obtain a large amount of highly purified rSPA with relative ease, we expressed SPA as a secretory form in the yeast Pichia pastoris. To increase the expression level of SPA and repress its proteolysis during fermentation, the cell density (OD600), temperature and pH at which SPA expression was induced as well as the induction time were optimized. The final yield of SPA obtained was about 8.8 g per liter of culture, which under the optimized fermentation condition, accounted for 80% of the total protein in the culture supernatant. The expressed SPA was purified from the culture supernatant by DEAE ion-exchange chromatography (IEC) after the supernatant was subjected to a desalting step. The purified SPA was resolved as a single band by SDS-PAGE and as a single peak by HPLC. Its identity was confirmed by MALDI-TOF MS and western-blot. Moreover, the protein also exhibited excellent affinity for IgG when tested with human IgG. The production and purification of SPA described in this study offers a new method for obtaining high level of SPA in relatively pure form that is suitable for practical application. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Functional expression of spider neurotoxic peptide huwentoxin-I in E. coli.

    Directory of Open Access Journals (Sweden)

    Er Meng

    Full Text Available The coding sequence of huwentoxin-I, a neurotoxic peptide isolated from the venom of the Chinese spider Ornithoctonus huwena, was amplified by PCR using the cDNA library constructed from the spider venom glands. The cloned fragment was inserted into the expression vector pET-40b and transformed into the E. coli strain BL21 (DE3. The expression of a soluble fusion protein, disulfide interchange protein (DsbC-huwentoxin-I, was auto-induced in the periplasm of E. coli in the absence of IPTG. After partial purification using a Ni-NTA column, the expressed fusion protein was digested using enterokinase to release heteroexpressed huwentoxin-I and was further purified using RP-HPLC. The resulting peptide was subjected to gel electrophoresis and mass spectrometry analysis. The molecular weight of the heteroexpressed huwentoxin-I was 3750.69, which is identical to that of the natural form of the peptide isolated from spider venom. The physiological properties of the heteroexpressed huwentoxin-I were further analyzed using a whole-cell patch clamp assay. The heteroexpressed huwentoxin-I was able to block currents generated by human Na(v1.7 at an IC₅₀ of 640 nmole/L, similar to that of the natural huwentoxin-I, which is 630 nmole/L.

  8. Expression, purification and characterization of Oryza sativa L. NAD ...

    African Journals Online (AJOL)

    Jane

    2011-10-17

    Oct 17, 2011 ... Key words: Enzyme activity, GST fusion protein, kinetic properties, NAD-malic enzyme, Oryza sativa L., ... Plant NADP-. MEs were induced by many biotic or abiotic stresses, such as pathogen (Sutherland, 1991), wounding, UV-B radiation (Casati et al., ... E. coli cells containing pGEX-6p-3-OsNAD-ME1.

  9. Expression and affinity purification of recombinant proteins from plants

    Science.gov (United States)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  10. [Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

    Science.gov (United States)

    Ding, Jun-Ying; Meng, Qing-Ling; Guo, Min-Zhuo; Yi, Yao; Su, Qiu-Dong; Lu, Xue-Xin; Qiu, Feng; Bi, Sheng-Li

    2012-10-01

    To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

  11. Synthesis, cloning and expression of a novel pre-miniproinsulin analogue gene in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ahmed A. Abolliel

    2015-09-01

    Full Text Available In the present study, a novel pre-miniproinsulin analogue was designed to have a short 9 residue sequence replacing the 35 residue C-chain, one lysine and one arginine added to the C-terminus of the B-chain in combination with glycine and arginine substitution at A21 and B29, respectively, and a 16-residue fusion partner comprising the pentapeptide sequence (PSDKP of the N-terminus of human tumor necrosis factor-α (TNF-α, 6 histidine residues for Ni2+ chelated affinity purification and a pentapeptide ending with methionine for ease of chemical cleavage fused at the N-terminus. Homology modeling of the designed protein against miniproinsulin (protein databank file 1 efeA as a template showed that the distance between the α-carbons of the C-terminus of the B-chain and the N-terminus of the A-chain did not change; the root-mean-square deviation of the backbone atoms between the structures of modeled miniproinsulin and miniproinsulin template was 0.000 Å. DNA sequencing of the synthesized gene showed 100% identity with theoretical sequence. The gene was constructed taking into account the codon preference of Escherichia coli (CAI value 0.99 in order to increase the expression rate of the DNA in the host strain. The designed gene was synthesized using DNA synthesis technology and then cloned into the expression plasmid pET-24a(+ and propagated in E. coli strain JM109. Gene expression was successful in two E. coli strains: namely JM109(DE3 and BL21(DE3pLysS. SDS–PAGE analysis was carried out to check protein size and to check and optimize expression. Rapid screening and purification of the resulting protein was carried out by Ni–NTA technology. The identity of the expressed protein was verified by immunological detection method of western blot using polyclonal rabbit antibody against insulin.

  12. Recombinant E. coli expressing Vitreoscilla haemoglobin prefers ...

    Indian Academy of Sciences (India)

    Under these conditions, the expression levels of proteins involved in central metabolic pathways, cellular adaptation and cell division were also found to be altered. These results imply that Vitreoscilla haemoglobin expression alters aerobic metabolism specifically, in addition to altering proteins involved in other pathways, ...

  13. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    Science.gov (United States)

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  14. The calcium-binding protein of Entamoeba histolytica as a fusion partner for expression of peptides in Escherichia coli.

    Science.gov (United States)

    Reddi, Honey; Bhattacharya, Alok; Kumar, Vijay

    2002-12-01

    We describe the construction of an Escherichia coli expression vector, CBP that allows the C-terminal fusion of heterologous proteins to the calcium-binding protein (CaBP) of the parasitic protozoan Entamoeba histolytica. The intrinsic nature of this protein to remain soluble on heat treatment has been exploited in its use as a novel fusion partner. The presence of a histidine tag and an enterokinase recognition site, aid in the affinity purification and proteolytic cleavage of the fusion protein. The efficacy of the vector was tested using the preS1 region of the envelope protein of the hepatitis B virus. The CaBP-preS1 fusion protein partitioned in the soluble fraction on heat treatment and this facilitated its rapid purification.

  15. Design parameters to control synthetic gene expression in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Mark Welch

    Full Text Available BACKGROUND: Production of proteins as therapeutic agents, research reagents and molecular tools frequently depends on expression in heterologous hosts. Synthetic genes are increasingly used for protein production because sequence information is easier to obtain than the corresponding physical DNA. Protein-coding sequences are commonly re-designed to enhance expression, but there are no experimentally supported design principles. PRINCIPAL FINDINGS: To identify sequence features that affect protein expression we synthesized and expressed in E. coli two sets of 40 genes encoding two commercially valuable proteins, a DNA polymerase and a single chain antibody. Genes differing only in synonymous codon usage expressed protein at levels ranging from undetectable to 30% of cellular protein. Using partial least squares regression we tested the correlation of protein production levels with parameters that have been reported to affect expression. We found that the amount of protein produced in E. coli was strongly dependent on the codons used to encode a subset of amino acids. Favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. Finally we confirmed the validity of our models by designing, synthesizing and testing new genes using codon biases predicted to perform well. CONCLUSION: The systematic analysis of gene design parameters shown in this study has allowed us to identify codon usage within a gene as a critical determinant of achievable protein expression levels in E. coli. We propose a biochemical basis for this, as well as design algorithms to ensure high protein production from synthetic genes. Replication of this methodology should allow similar design algorithms to be empirically derived for any expression system.

  16. High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Vineet K. Singh

    2014-01-01

    Full Text Available Staphylococcus aureus is a major human and animal pathogen. Autolysins regulate the growth, turnover, cell lysis, biofilm formation, and the pathogenicity of S. aureus. Atl is the major autolysin in S. aureus. The biochemical and structural studies of staphylococcal Atl have been limited due to difficulty in cloning, high level overexpression, and purification of this protein. This study describes successful cloning, high level over-expression, and purification of two forms of fully functional Atl proteins. These pure proteins can be used to study the functional and structural properties of this important protein.

  17. Production and Purification of the Native Saccharomyces cerevisiae Hsp12 in Escherichia coli.

    Science.gov (United States)

    Léger, Antoine; Hocquellet, Agnès; Dieryck, Wilfrid; Moine, Virginie; Marchal, Axel; Marullo, Philippe; Josseaume, Annabelle; Cabanne, Charlotte

    2017-09-20

    Hsp12 is a small heat shock protein produced in many organisms, including the yeast Saccharomyces cerevisiae. It has been described as an indicator of yeast stress rate and has also been linked to the sweetness sensation of wine. To obtain a sufficient amount of protein, we produced and purified Hsp12 without tag in Escherichia coli. A simple fast two-step process was developed using a microplate approach and a design of experiments. A capture step on an anion-exchange salt-tolerant resin was followed by size exclusion chromatography for polishing, leading to a purity of 97%. Thereafter, specific anti-Hsp12 antibodies were obtained by rabbit immunization. An ELISA was developed to quantify Hsp12 in various strains of Saccharomyces cerevisiae. The antibodies showed high specificity and allowed the quantitation of Hsp12 in the yeast. The quantities of Hsp12 measured in the strains differed in direct proportion to the level of expression found in previous studies.

  18. The extended leader peptide of Haemophilus parasuis trimeric autotransporters conditions their protein expression in Escherichia coli.

    Science.gov (United States)

    Pina-Pedrero, Sonia; Olvera, Àlex; Bensaid, Albert

    2017-05-01

    Trimeric autotransporters are surface-exposed proteins of Gram-negative bacteria belonging to the type V secretion system. They are involved in virulence and are targets for vaccine and diagnostic tool development, so optimal systems for their expression and purification are required. In the present study, the impact of the extended leader peptide of the Haemophilus parasuis virulence-associated trimeric autotransporters (VtaA) in its production as recombinant proteins in Escherichia coli was evaluated. The 13 genes encoding the VtaA1 to VtaA13 passenger domains of the strain Nagasaki were cloned in the pASK-IBA33plus plasmid and expressed in E. coli. Recombinant protein production was higher for truncated forms in which the entire leader peptide was deleted, and the recombinant protein accumulated in the cytoplasm of the cells. The yield of protein production of the different VtaAs was size dependent, and reached maximal amount at 2-4 h post -induction. The optimization of these conditions allowed to scale-up the production to obtain enough recombinant protein to immunize large animals. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Nicholas, R.A.; Suzuki, H.; Hirota, Y.; Strominger, J.L.

    1985-07-02

    This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with (/sup 14/C)penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the (/sup 14/C)penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.

  20. Expression, purification and crystallization of Helicobacter pyloril-asparaginase

    Energy Technology Data Exchange (ETDEWEB)

    Dhavala, Prathusha [Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku 20521 (Finland); Krasotkina, Julya [Institute for Biomedical Sciences, Russian Academy of Medical Sciences, Moscow (Russian Federation); Dubreuil, Christine; Papageorgiou, Anastassios C., E-mail: tassos.papageorgiou@btk.fi [Turku Centre for Biotechnology, University of Turku and Åbo Akademi, Turku 20521 (Finland)

    2008-08-01

    l-Asparaginase from H. pylori was overexpressed in E. coli, purified and crystallized. The crystals belonged to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 Å and one molecule in the asymmetric unit. A complete data set to 1.6 Å resolution was collected using synchrotron radiation. The l-asparaginases from Escherichia coli and Erwinia chrysanthemi are effective drugs that have been used in the treatment of acute childhood lymphoblastic leukaemia for over 30 years. However, despite their therapeutic potential, they can cause serious side effects as a consequence of their intrinsic glutaminase activity, which leads to l-glutamine depletion in the blood. Consequently, new asparaginases with low glutaminase activity, fewer side effects and high activity towards l-asparagine are highly desirable as better alternatives in cancer therapy. l-Asparaginase from Helicobacter pylori was overexpressed in E. coli and purified for structural studies. The enzyme was crystallized at pH 7.0 in the presence of 16–19%(w/v) PEG 4000 and 0.1 M magnesium formate. Data were collected to 1.6 Å resolution at 100 K from a single crystal at a synchrotron-radiation source. The crystals belong to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 Å and one molecule of l-asparaginase in the asymmetric unit. Elucidation of the crystal structure will provide insight into the active site of the enzyme and a better understanding of the structure–activity relationship in l-asparaginases.

  1. Codon optimisation is key for pernisine expression in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Marko Šnajder

    Full Text Available Pernisine is an extracellular serine protease from the hyperthermophilic Archaeon Aeropyrum pernix K1. Low yields from the natural host and expression problems in heterologous hosts have limited the potential applications of pernisine in industry.The challenges of pernisine overexpression in Escherichia coli were overcome by codon preference optimisation and de-novo DNA synthesis. The following forms of the pernisine gene were cloned into the pMCSGx series of vectors and expressed in E. coli cells: wild-type (pernisinewt, codon-optimised (pernisineco, and codon-optimised with a S355A mutation of a predicted active site (pernisineS355Aco. The fusion-tagged pernisines were purified using fast protein liquid chromatography equipped with Ni2+ chelate and gel filtration chromatography columns. The identities of the resultant proteins were confirmed with N-terminal sequencing, tandem mass spectrometry analysis, and immunodetection. Pernisinewt was not expressed in E. coli at detectable levels, while pernisineco and pernisineS355Aco were expressed and purified as 55-kDa proforms with yields of around 10 mg per litre E. coli culture. After heat activation of purified pernisine, the proteolytic activity of the mature pernisineco was confirmed using zymography, at a molecular weight of 36 kDa, while the mutant pernisineS355Aco remained inactive. Enzymatic performances of pernisine evaluated under different temperatures and pHs demonstrate that the optimal enzymatic activity of the recombinant pernisine is ca. 100°C and pH 7.0, respectively.These data demonstrate that codon optimisation is crucial for pernisine overexpression in E. coli, and that the proposed catalytic Ser355 has an important role in pernisine activity, but not in its activation process. Pernisine is activated by autoproteolytical cleavage of its N-terminal proregion. We have also confirmed that the recombinant pernisine retains the characteristics of native pernisine, as a calcium

  2. Functional fusion expression of sunflower multicystatin in E. coli and its comparison with a single domain cystatin.

    Science.gov (United States)

    Gholizadeh, Ashraf; Kohnehrouz, B Baghban

    2011-12-01

    Identification of the molecular structure and novel biophysiological functions of plant cystatins or phytocystatins is of great interest in the field of molecular biology. The important requirements for these are the efficient production, purification and correctly folded forms of these proteins. We report here the cloning, easy expression and characterization of a sunflower multicystatin (SMC) as a functional fusion protein in E. coli. For the first time, the amplified cystatin coding region was expressed as a part of maltose-binding fusion protein using pMALc2X over-expression vector in TB1 strain of E. coli without affecting the recombinant bacterial growth. In comparison to the previously prepared recombinant SMC (rSMC), a high amount (-44 mg/L of bacterial cell culture) of purified fused SMC (fSMC) was obtained using single-step purification method. fSMC strongly inhibited papain activity in vitro as compared to Celosia single-domain cystatin. Purified fSMC may be used for basic biochemical, pharmacological or clinical studies without the cleavage of its fusion parts.

  3. Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification.

    Directory of Open Access Journals (Sweden)

    Jayde A Gawthorne

    Full Text Available In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an "effector" protein normally produced by enterohemorrhagic E. coli strains and "injected" into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.

  4. A dual protease approach for expression and affinity purification of recombinant proteins.

    Science.gov (United States)

    Raran-Kurussi, Sreejith; Waugh, David S

    2016-07-01

    We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

  5. Molecular Cloning Expression And Purification Studies With An ORF Of Mycobacterium Tuberculosis

    Directory of Open Access Journals (Sweden)

    Chiranjibi Chaudhary

    2017-08-01

    Full Text Available The study was initiated to develop a recombinant strain for expression and production of large scale protein and to develop its purification protocol. The MRAORF-X was amplified from the genomic DNA of M. tuberculosis H37Ra. The amplicon was successfully cloned in a cloning vector pGEM-T Easy and transformed in cloning host DH5amp945. Recombinant clones were identified by blue-white screening and insert presence was confirmed by restriction digestion of plasmid isolated from white colonies. Expression vector pET32a was used for protein expression. The recombinant plasmid was transformed into expression host BL21 and protein expression was checked by SDS-PAGE. The desired protein was approximately 60 kDa in size including tags. The purification protocol was established for purification from inclusion bodies. The purity of purified protein was assessed by SDS-PAGE gel run and presence of a single band at 60 kDa suggested that the inclusion bodies were a good source of purified protein.

  6. The complex between SOS3 and SOS2 regulatory domain from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis

    Energy Technology Data Exchange (ETDEWEB)

    Sánchez-Barrena, María José; Moreno-Pérez, Sandra; Angulo, Iván; Martínez-Ripoll, Martín; Albert, Armando, E-mail: xalbert@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química Física ‘Rocasolano’, Consejo Superior de Investigaciones Científicas, Serrano 119, E-28006 Madrid (Spain)

    2007-07-01

    Recombinant SOS3 and SOS2 regulatory domain from A. thaliana have been coexpressed in E. coli, purified and crystallized by the hanging-drop vapour-diffusion method. An X-ray data set has been collected at 2.0 Å resolution. The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 Å.

  7. Expression, purification, and characterization of Sss1p, an essential component of the yeast Sec61p protein translocation complex.

    Science.gov (United States)

    Beswick, V; Brodsky, J L; Képès, F; Neumann, J M; Sanson, A; Garrigos, M

    1998-08-01

    Sss1p, a 8.9-kDa membrane protein, is an essential component of the protein translocation complex involved in the transport of secretory proteins across the Saccharomyces cerevisiae endoplasmic reticulum membrane. In order to determine the high resolution structure of Sss1p by NMR, we have undertaken its overexpression and purification. We first inserted the yeast SSS1 gene into the pGEX-2T plasmid expression vector. Sss1p was expressed as fusions with Schistosoma japonica glutathione S-transferase (GST-Sss1p) in MC1061 Escherichia coli cells. Maximum yield of GST-Sss1p was obtained from cells harvested 2 h after induction at 37 degreesC in Luria broth medium. GST-Sss1p was found associated predominantly with the membrane pool and was readily extracted with Triton X-100. Detergent-solubilized GST-Sss1p was isolated by adsorption on glutathione-agarose beads. Sss1p was released from its GST carrier by cleavage with thrombin and its recovery was maximized by addition of dodecyl maltoside. Desorbed Sss1p was loaded on a high-performance liquid chromatography hydroxyapatite column equilibrated in phosphate buffer supplemented with dodecyl maltoside and the fractions containing Sss1p were subsequently purified to homogeneity by reverse-phase chromatography on a C4 column. The entire purification protocol can be completed in 5-6 h and yields about 0.4 mg of Sss1p per gram of transformed cells. CD and preliminary 1H NMR experiments show that purified Sss1p solubilized in SDS micelles is very stable and adopts a helical secondary structure. Copyright 1998 Academic Press.

  8. Floating Escherichia coli by expressing cyanobacterial gas vesicle genes

    Science.gov (United States)

    Wang, Tianhe; Kang, Li; Li, Jiaheng; Wu, Wenjie; Zhang, Peiran; Gong, Minghao; Lai, Weihong; Zhang, Chunyan; Chang, Lei; Peng, Yong; Yang, Zhongzhou; Li, Lian; Bao, Yingying; Xu, Haowen; Zhang, Xiaohua; Sui, Zhenghong; Yang, Guanpin; Wang, Xianghong

    2015-02-01

    Gas vesicles are hollow, air-filled polyprotein structures that provide the buoyancy to cells. They are found in a variety of prokaryotes. In this study, we isolated a partial gas vesicle protein gene cluster containing gvpA and gvpC20Ψ from Planktothrix rubescens, and inserted it into an expression vector and expressed it in E. coli. The gas vesicle was developed in bacterial cells, which made bacterial cells to float on medium surface. We also amplified gvpA and gvpC20Ψ separately and synthesized an artificial operon by fusing these two genes with the standardized gene expression controlling elements of E. coli. The artificial operon was expressed in E. coli, forming gas vesicles and floating bacteria cells. Our findings verified that the whole set of genes and the overall structure of gas vesicle gene cluster are not necessary for developing gas vesicles in bacteria cells. Two genes, gvpA and gvpC20Ψ, of the gas vesicle gene cluster are sufficient for synthesizing an artificial operon that can develop gas vesicles in bacteria cells. Our findings provided a wide range of applications including easing the harvest of cultured microalgae and bacteria, as well as enriching and remediating aquatic pollutants by constructing gas vesicles in their cells.

  9. Cloning, expression and purification of 10 kd culture filtrared protein ...

    African Journals Online (AJOL)

    Recombinant CFP-10 was purified from the soluble supernatant by metal affinity chromatography. SDS-PAGE analysis was performed to confirm expression of CFP-10 as 28 kDa fusion protein. In this study, we cloned, expressed and purified sufficient amounts of CFP-10 that could be usable in sero-diagnostic tests in future ...

  10. Comparison of enzymatic activity of two linoleic acid isomerases expressed in E. coli.

    Science.gov (United States)

    Luo, Xue; Zhang, Lanwei; Li, Hongbo; Zhang, Shuang; Jiao, Yuehua; Wang, Shumei; Xue, Chaohui; Fan, Rongbo

    2013-10-01

    Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of octadecadienoic fatty acid with conjugated double bonds. CLA possesses many important physiological functions and it can be produced from linoleic acid (LA) by LA isomerases. In this report, we first cloned the genes encoding LA isomerases: C12 isomerases and C9 isomerase, then transformed the recombinant plasmids into Escherichia coli TOP10 and induced E. coli with IPTG (isopropylthio-β-D-galactoside) to express the recombinant proteins. Next, we purified the isomerases using a HisTrap™ HP column, followed with the analysis by SDS-PAGE or Western blot. Finally, we compared their enzymatic activity by biotransformation of LA into CLA. Plasmids containing LA isomerase genes were successfully constructed. LA isomerases were found expressed in E. coli, and the molecular weight was 64 KD for C12 LA isomerase and 55 KD for C9 LA isomerase. The enzyme activity (9.93 ± 0.01 U/ml for C12 LA isomerase and 8.12 ± 0.02 U/ml for C9 LA isomerase) of both LA isomerases reached the highest when IPTG concentration is 0.2 mM and the induction time is 18 h. After purification, C9 LA isomerase was enriched in peak 4 and C12 LA isomerase was enriched in peak 3. Optimum pH for C9 LA and C12 LA isomerases were 7.5 and 7.0 separately, and optimum temperatures was 37 °C for highest concentration of CLA. The work may provide theoretical significance for an effective production process of CLA for the medical and nutritional purposes.

  11. Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

    Science.gov (United States)

    El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

    2000-02-15

    We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

  12. Expression and purification of a nanostructure-forming peptide.

    Science.gov (United States)

    Hartmann, B M; Kaar, W; Falconer, R J; Zeng, B; Middelberg, A P J

    2008-05-20

    Peptides have recently attracted interest as building blocks for the assembly of novel functional materials including switchable surfactants, nanocoatings, hydrogels and aqueous vesicles. We expressed a beta-sheet forming peptide that has been widely studied in self-assembly processing, P(11)-2, as a monomer, dimer, tetramer and nonamer fused to an insoluble expression partner, ketosteroid isomerase, using minimal media. Expression was followed by whole cell extraction and isolation of the fusion protein to greater than 90% purity via a single immobilised metal affinity chromatography (IMAC) step. Peptides were chemically cleaved from each other and from the fusion partner, followed by acetone precipitation of the contaminating protein fragments. Pure peptide was recovered by reversed-phase HPLC. The expression level of the fusion protein decreased as the peptide concatamer number increased, as did the efficiency of the chemical cleavage, making the single-peptide process the most efficient overall. Applying this laboratory process to the single-peptide fusion protein nevertheless resulted in a pure peptide yield of greater than 30% of the expressed peptide.

  13. Expression and purification of the cystic fibrosis transmembrane conductance regulator protein in Saccharomyces cerevisiae.

    Science.gov (United States)

    O'Ryan, Liam; Rimington, Tracy; Cant, Natasha; Ford, Robert C

    2012-03-10

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein(1-3). Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed (4-9). Proteins involved in the processing of recombinant CFTR in yeast have been described(6-9) .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)(10). The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for

  14. Purification and light-scattering analysis of penicillin-binding protein 4 from Escherichia coli

    NARCIS (Netherlands)

    Fusetti, F; Dijkstra, BW

    1996-01-01

    Penicillin binding protein 4 (PBP4) from Escherichia coli is a protein involved in the recycling and maturation of the bacterial cell wall and it is inhibited by beta-lactam antibiotics, PBP4 exhibits D-Ala-D-Ala-endopeptidase as well as D-Ala-D-Ala-carboxypeptidase activity. To provide a structural

  15. Expression and purification of the central stalk subunits of Na + ...

    African Journals Online (AJOL)

    , NtpD and NtpG subunits. The aim of the present study was cloning and expression of these central stalk subunits of E. hirae V-type Na+-ATPase. Here we cloned the synthesized DNA fragments, corresponding to ntpC, ntpD and ntpG genes, ...

  16. High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase.

    Science.gov (United States)

    Bordeaux, Mélanie; de Girval, Diane; Rullaud, Robin; Subileau, Maeva; Dubreucq, Eric; Drone, Jullien

    2014-01-01

    We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and moderate operational stability. In this study, we focused our efforts on the production of A13-Red using high-cell-density cultivation (HCDC) of recombinant Escherichia coli. We achieved 455 mg (5,000 nmol) of functional enzyme per liter of culture. Tight control of cultivation parameters rendered the whole process highly reproducible compared with flask cultivations. We optimized the purification of the biocatalyst that can be performed in either two or three steps depending on the application needed to afford A13-Red up to 95 % homogeneous. We investigated different reaction conditions and found that the total turnover numbers of A13-Red during the in vitro hydroxylation of n-octane could reach up to 3,250 to produce 1-octanol (1.6 mM) over a period of 78 h.

  17. Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli

    Directory of Open Access Journals (Sweden)

    Gómez-Lim Miguel A

    2009-02-01

    Full Text Available Abstract Background Gag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB when fused to p24. Results A fusion between ricin toxin B subunit and p24 HIV (RTB/p24 was expressed in E. coli. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin. Conclusion In this work, we report the expression in E. coli of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when

  18. Scalable chromatography-based purification of virus-like particle carrier for epitope based influenza A vaccine produced in Escherichia coli.

    Science.gov (United States)

    Lagoutte, Priscillia; Mignon, Charlotte; Donnat, Stéphanie; Stadthagen, Gustavo; Mast, Jan; Sodoyer, Régis; Lugari, Adrien; Werle, Bettina

    2016-06-01

    Virus-like particles (VLPs) are promising molecular structures for the design and construction of novel vaccines, diagnostic tools, and gene therapy vectors. Size, oligomer assembly and repetitiveness of epitopes are optimal features to induce strong immune responses. Several VLP-based vaccines are currently licensed and commercialized, and many vaccine candidates are now under preclinical and clinical studies. In recent years, the development of genetically engineered recombinant VLPs has accelerated the need for new, improved downstream processes. In particular, a rapid low cost purification process has been identified as a remaining key challenge in manufacturing process development. In the present study we set up a size-exclusion chromatography-based, scalable purification protocol for the purification of a VLP-based influenza A vaccine produced in Escherichia coli. Recombinant VLPs derived from the RNA bacteriophage MS2 displaying an epitope from the ectodomain of Matrix 2 protein from influenza A virus were produced and purified. The 3 steps purification protocol uses a recently developed multimodal size-exclusion chromatography medium (Capto™ Core 700) in combination with detergent extraction and size-exclusion polishing to reach a 89% VLP purity with a 19% yield. The combination of this downstream strategy following production in E. coli would be suited for production of VLP-based veterinary vaccines targeting livestock and companion animals where large amounts of doses must be produced at an affordable price. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Expression, purification and kinase activity analysis of maize ...

    African Journals Online (AJOL)

    Kinase activity is essential for a protein kinase to perform its biological function. In previous study we have cloned a novel plant SnRK2 subfamily gene from maize and named it as ZmSPK1. In this study the cDNA of ZmSPK1 with dHA-His6 tag was amplified by PCR and was subcloned into the yeast expression vector ...

  20. Molecular cloning, sequencing and expression in Escherichia coli cells Thermus thermophilus leucyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Kovalenko O. P.

    2011-12-01

    Full Text Available Aim. Cloning and sequencing of the T. thermophilus leucyl-tRNA synthetase (LeuRSTT followed by the creation of genetically engineered construct for protein expression in E.coli cells and its purification. Methods. Searching for the LeuRSTT gene was performed by Southern blot hybridization with chromosomal DNA, where digoxigenin-labeled PCR fragments of DNA were used as probes. Results. The gene of T. thermophilus HB27 leucyl-tRNA synthetase was cloned and sequenced. The open reading frame encodes a polypeptide chain of 878 amino acid residues in length (molecular mass 101 kDa. Comparison of the amino acid sequence of T. thermophilus LeuRS with that of the enzymes from other organisms showed that LeuRSTT was a part of the group of similar enzymes of prokaryotes, formed by the proteins of protobacteriae, rickettsia and mitochondria of eukaryotes. The resulting phylogenetic tree of LeuRSs reveals dichotomous branching into two lines: prokaryotic/eukaryotic mitochondrial and arhaeal/eukaryotic cytosolic proteins. Differences between prokaryotic and arhaeal branches of the LeuRSs phylogenetic tree are primarily due to the structure of two domains of the enzyme – the editing and the C-terminal. T. thermophilus LeuRS was expressed in E. coli cells by cloning the corresponding gene into pET29b vector. Conclusions. The cloned T. thermophilus leuS gene and expressed recombinant protein will be used for structural and functional studies on LeuRSTT, including X-ray analysis of the enzyme and its mutant forms in complex with different substrates

  1. Heterologous expression and purification of membrane-bound pyrophosphatases

    DEFF Research Database (Denmark)

    Kellosalo, J.; Kajander, T.; Palmgren, Michael Broberg

    2011-01-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low...... and monodisperse active states. To generate M-PPases with an increased hydrophilic surface area, which potentially should facilitate formation of crystal contacts, phage T4 lysozyme was inserted into different extramembraneous loops of one of these M-PPases. Two of these fusion proteins were active and expressed...

  2. Expression, Purification, and Characterization of Interleukin-11 Orthologues

    Directory of Open Access Journals (Sweden)

    Andrei S. Sokolov

    2016-11-01

    Full Text Available Interleukin-11 (IL-11 is a multifunctional cytokine implicated in several normal and pathological processes. The decoding of IL-11 function and development of IL-11-targeted drugs dictate the use of laboratory animals and need of the better understanding of species specificity of IL-11 signaling. Here, we present a method for the recombinant interleukin-11 (rIL-11 production from the important model animals, mouse and macaque. The purified mouse and macaque rIL-11 interact with extracellular domain of human IL-11 receptor subunit α and activate STAT3 signaling in HEK293 cells co-expressing human IL-11 receptors with efficacies resembling those of human rIL-11. Hence, the evolutionary divergence does not impair IL-11 signaling. Furthermore, compared to human rIL-11 its macaque orthologue is 8-fold more effective STAT3 activator, which favors its use for treatment of thrombocytopenia as a potent substitute for human rIL-11. Compared to IL-6, IL-11 signaling exhibits lower species specificity, likely due to less conserved intrinsic disorder propensity within IL-6 orthologues. The developed express method for preparation of functionally active macaque/mouse rIL-11 samples is suited for exploration of the molecular mechanisms underlying IL-11 action and for development of the drug candidates for therapy of oncologic/hematologic/inflammatory diseases related to IL-11 signaling.

  3. Expression, purification, and characterization of recombinant NOD1 (NLRC1): A NLR family member.

    Science.gov (United States)

    Askari, Nadav; Correa, Ricardo G; Zhai, Dayong; Reed, John C

    2012-01-01

    NOD1 (NLRC1) is a member of the NLR family of innate immunity proteins, which are important cellular sensors of various pathogens. Deregulated NOD1 signaling is involved in various autoimmune, inflammatory, and allergic diseases, making it a potential target for drug discovery. However, to date, the successful high-yield purification NOD1 protein has not been reported. Here we describe the large-scale expression of recombinant NOD1 protein in non-adherent mammalian cells. One-step immunoaffinity purification was carried out, yielding highly pure protein with excellent yields. Gel-sieve chromatography studies showed that the purified NOD1 protein eluted almost exclusively as a monomer. Addition of the NOD1 ligand (γ-Tri-DAP) stimulated NOD1 protein oligomerization. Using purified NOD1 protein for nucleotide binding studies by the Fluorescence Polarization Assay (FPA) method, we determined that NOD1 binds preferentially to ATP over ADP and AMP or dATP. We also documented that purified NOD1 protein binds directly to purified pro-apoptotic protein Bid, thus extending recent data that have identified Bid as an enhancer of NOD1 signaling. This expression and purification strategy will enable a wide variety of biochemical studies of mechanisms of NOD1 regulation, as well as laying a foundation for future attempts at drug discovery. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Expression and purification of untagged GlnK proteins from actinobacteria.

    Science.gov (United States)

    Gerhardt, Edileusa C M; Moure, Vivian R; Souza, Andrey W; Pedrosa, Fabio O; Souza, Emanuel M; Diacovich, Lautaro; Gramajo, Hugo; Huergo, Luciano F

    2017-01-01

    The PII protein family constitutes one of the most conserved and well distributed family of signal transduction proteins in nature. These proteins play key roles in nitrogen and carbon metabolism. PII function has been well documented in Gram-negative bacteria. However, there are very few reports describing the in vitro properties and function of PII derived from Gram-positive bacteria. Here we present the heterologous expression and efficient purification protocols for untagged PII from three Actinobacteria of medical and biotechnological interest namely: Mycobacterium tuberculosis, Rhodococcus jostii and Streptomyces coelicolor. Circular dichroism and gel filtration analysis supported that the purified proteins are correctly folded. The purification protocol described here will facilitate biochemical studies and help to uncover the biochemical functions of PII proteins in Actinobacteria.

  5. Membrane chromatography: protein purification from E. coli lysate using newly designed and commercial anion-exchange stationary phases.

    Science.gov (United States)

    Bhut, Bharat V; Christensen, Kenneth A; Husson, Scott M

    2010-07-23

    This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind D membrane adsorber and HiTrap DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind D membrane at equivalent volumetric throughput and higher capacities than the HiTrap DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap DEAE FF resin column and was the same for the new AEX membrane and Sartobind D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  6. Expression, purification and crystallization of pecan (Carya illinoinensis) vicilin.

    Science.gov (United States)

    Lee, BoRam; Zhang, Renhao; Du, Wen-Xian; Grauke, Larry J; McHugh, Tara H; Zhang, Yu-Zhu

    2014-08-01

    Tree nuts are responsible for many cases of severe food allergies. The 7S seed storage protein vicilin has been identified as a food allergen in many kinds of tree nuts. The vicilin protein consists of an N-terminal low-complexity region with antimicrobial activity and a C-terminal domain that forms a trimeric structure that belongs to the cupin superfamily. In this study, vicilin from pecan (Carya illinoinensis) was isolated and was expressed in bacteria for the first time. The cupin structural core of the protein, residues 369-792, was purified by metal-affinity and gel-filtration chromatography to high purity. Vicilin crystals were obtained and the best crystal diffracted to 2.65 Å resolution in space group P212121.

  7. Expression and purification of active, stabilized trimethyllysine hydroxylase.

    Science.gov (United States)

    Kazaks, Andris; Makrecka-Kuka, Marina; Kuka, Janis; Voronkova, Tatyana; Akopjana, Inara; Grinberga, Solveiga; Pugovics, Osvalds; Tars, Kaspars

    2014-12-01

    Trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis - the conversion of N6,N6,N6-trimethyl-l-lysine to 3-hydroxy-N6,N6,N6-trimethyl-l-lysine. By changing carnitine availability it is possible to optimise cardiac energy metabolism, that is beneficial under certain ischemic conditions. Previous efforts have been devoted towards the inhibition of gamma-butyrobetaine dioxygenase, which catalyses the last step in carnitine biosynthesis. However, the effects of TMLH activity regulation are currently unexplored. To facilitate the development of specific ligands of TMLH, large quantities of recombinant protein are necessary for downstream binding and structural studies. Here, we describe an efficient system for expressing and purifying active and stable TMLH as a maltose-binding protein fusion in Escherichiacoli. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Expression, Purification, and Screening of BamE, a Component of the BAM Complex, for Structural Characterization.

    Science.gov (United States)

    Jeeves, Mark; Sridhar, Pooja; Knowles, Timothy J

    2015-01-01

    In Gram-negative bacteria, integral outer membrane β-barrel proteins (OMP) are assembled by the β-barrel assembly machine complex, or BAM complex. This complex includes the essential components BamA, an OMP composed of a carboxyl terminal β-barrel domain and five polypeptide transport-associated domains (POTRA), and the lipoprotein BamD. In Escherichia coli, the complex contains an additional three lipoproteins, BamB, C and E required for efficient delivery of OMPs to the outer membrane. Here we provide methods for production, isotope labeling, purification, and functional screening of BamE for research purposes. Purification strategies of both the soluble and wild-type membrane-tethered forms of BamE are described using techniques including osmotic shock, Ni-NTA purification, and size-exclusion chromatography. Functional screening using a simple plate assay is also described which allows screening for defects in outer membrane permeability.

  9. Expression of recombinant alkaline phosphatase conjugates in Escherichia coli.

    Science.gov (United States)

    Boulain, Jean-Claude; Ducancel, Frédéric

    2004-01-01

    The methods described in this article are relative to the use of a positive cloning/screening recombinant system for the generation in Escherichia coli of foreign proteins fused to a highly active bacterial alkaline phosphatase (PhoA) variant as reporter enzyme. Appropriate insertion of the DNA encoding the foreign peptides, proteic domains, or proteins between codons +6 and +7 of the phoa gene restores the initial frame of the phoa gene in the vector. Consequently, only recombinant clones appear as blue colonies when plating onto an agar medium containing a chromogenic substrate for PhoA. The presence of an intact PhoA signal peptide yields to a systematic secretion of the fusion proteins into the periplasm where the PhoA dimerises to its active form, and disulfides can be formed if necessary. The resultant PhoA-tagged proteins are particularly convenient novel tools that can be used in a wide range of applications, including expression, epitope mapping, histochemistry, immunoblotting, mutant analysis, and competition or sandwich ELISAs. Expression of an scFv antibody fragment derived from an IgG2a/kappa immunoglobulin specific for curaremimetic toxins from snake (named M-alpha2-3), will be used to illustrate the methods utilized for its cloning, expression in E.coli, extraction, and functional characterization.

  10. Secretory expression of active clostripain in Escherichia coli.

    Science.gov (United States)

    Kim, Chang-Kyu; Lee, Sang-Yong; Kwon, Oh-Joong; Lee, Sang-Mok; Nah, Seung-Yeol; Jeong, Sang Min

    2007-09-15

    In this study, the clostripain gene was modified and its signal sequence was replaced with that of penicillin G acylase (PGA). The core clostripain protein fused to the PGA signal peptide was also prepared. With regard to the expression of the clostripain precursors, the majority of clostripain activity was observed in the culture media, thereby indicating that both the clostripain signal peptide and the PGA signal peptide were recognized in the E. coli secretion pathway, and the precursors successfully matured into the active form. Otherwise, the activity was rather low when the core protein was expressed, which indicates that the clostripain pro-peptide is important in the formation of the active enzyme in E. coli. Enzyme activity reached a value of 3200U/L in CGY media for high expression. The recombinant clostripain and porcine carboxypeptidase B were used in the conversion of a proinsulin fusion protein into insulin. The leader peptide (LP) and the proinsulin C-peptide appeared to have been removed simultaneously, and the final cleavage product evidenced an HPLC retention time identical to that of the insulin standard, thereby implying that the clostripain specifically cleaved the arginine residues in the LP and in the C-peptide. We have also demonstrated the possibility that the recombinant clostripain might prove useful in the production of insulin from the proinsulin fusion protein.

  11. Improving E. coli growth performance by manipulating small RNA expression.

    Science.gov (United States)

    Negrete, Alejandro; Shiloach, Joseph

    2017-11-14

    Efficient growth of E. coli, especially for production of recombinant proteins, has been a challenge for the biotechnological industry since the early 1970s. By employing multiple approaches, such as different media composition, various growth strategies and specific genetic manipulations, it is now possible to grow bacteria to concentrations exceeding 100 g/L and to achieve high concentrations of recombinant proteins. Although the growth conditions are carefully monitored and maintained, it is likely that during the growth process cells are exposed to periodic stress conditions, created by fluctuations in pH, dissolved oxygen, temperature, glucose, and salt concentration. These stress circumstances which can occur especially in large volume bioreactors, may affect the growth and production process. In the last several years, it has been recognized that small non-coding RNAs can act as regulators of bacterial gene expression. These molecules are found to be specifically involved in E. coli response to different environmental stress conditions; but so far, have not been used for improving production strains. The review provides summary of small RNAs identified on petri dish or in shake flask culture that can potentially affect growth characteristics of E. coli grown in bioreactor. Among them MicC and MicF that are involved in response to temperature changes, RyhB that responds to iron concentration, Gady which is associated with lower pH, Sgrs that is coupled with glucose transport and OxyS that responds to oxygen concentration. The manipulation of some of these small RNAs for improving growth of E. coli in Bioreactor is described in the last part of the review. Overexpression of SgrS was associated with improved growth and reduced acetate expression, over expression of GadY improved cell growth at acidic conditions and over expression of OxyS reduced the effect of oxidative stress. One of the possible advantages of manipulating sRNAs for improving cell growth is

  12. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of bacterioferritin A from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Vibha [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Gupta, Rakesh K. [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Ram Lal Anand College, University of Delhi, Benito Juarez Road, New Delhi 110021 (India); Khare, Garima [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Salunke, Dinakar M. [National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067 (India); Tyagi, Anil K., E-mail: aniltyagi@south.du.ac.in [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India)

    2008-05-01

    The cloning, purification and crystallization of a bacterioferritin from M. tuberculosis together with preliminary X-ray characterization of its crystals are reported. Bacterioferritins (Bfrs) comprise a subfamily of the ferritin superfamily of proteins that play an important role in bacterial iron storage and homeostasis. Bacterioferritins differ from ferritins in that they have additional noncovalently bound haem groups. To assess the physiological role of this subfamily of ferritins, a greater understanding of the structural details of bacterioferritins from various sources is required. The gene encoding bacterioferritin A (BfrA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein product was purified by affinity chromatography on a Strep-Tactin column and crystallized with sodium chloride as a precipitant at pH 8.0 using the vapour-diffusion technique. The crystals diffracted to 2.1 Å resolution and belonged to space group P4{sub 2}, with unit-cell parameters a = 123.0, b = 123.0, c = 174.6 Å.

  13. Characterization and optimization of ArtinM lectin expression in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Pranchevicius Maria-Cristina S

    2012-08-01

    Full Text Available Abstract Background ArtinM is a d-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM in Escherichia coli system. Results The ArtinM coding region was inserted in pET29a(+ vector and expressed in E. coli BL21(DE3-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C and shaking speeds (130, 200 or 220 rpm during induction, concentrations of the induction agent IPTG (0.01-4 mM and periods of induction (1-19 h. BL21-CodonPlus(DE3-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized d-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM. The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises β-sheet structure. Conclusions Overall, the

  14. Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus

    DEFF Research Database (Denmark)

    Wang, Xiaole; Hald, Helle; Ernst, Heidi Asschenfeldt

    2010-01-01

    The human alanine-serine-cysteine transporter 1 (Asc-1) belongs to the slc7a family of solute carrier transporters. Asc-1 mediates the uptake of D-serine in an exchanger-type fashion, coupling the process to the release of alanine and cysteine. Among the bacterial Asc-1 homologues, one transporter...... by auto-induction, and performed purification and biophysical characterization. In addition, growth studies indicate a preference for alanine as nitrogen source in cells expressing the G. violaceus transporter. It was observed that use of the auto-induction method and subsequent optimization of the length...

  15. Recombinant thermostable AP exonuclease from Thermoanaerobacter tengcongensis: cloning, expression, purification, properties and PCR application

    DEFF Research Database (Denmark)

    Dabrowski, Slawomir; Brillowska-Dabrowska, Anna; Ahring, Birgitte Kiær

    2013-01-01

    and transformed into Escherichia coli. The protein product showed high identity (80%) to human Ape1 nuclease, whereas to E. coli exonuclease III - 78%. This is the first prokaryotic AP nuclease that exhibits such high identity to human Ape1 nuclease. The very high expression level (57% of total soluble proteins......Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis......) of fully active and soluble His6-tagged Tte AP enzyme with His6-tag on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni(2+)-IDA-Sepharose resin. The yield was 90 mg...

  16. Expression and purification of human and Saccharomyces cerevisiae equilibrative nucleoside transporters.

    Science.gov (United States)

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Roe-Žurž, Zygy; Duggan, Kelli D; Schmitz, Hannah; Hays, Franklin A

    2018-02-01

    Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Cloning and Expression of Functional Reteplase in Escherichia coli TOP10

    OpenAIRE

    Khodabakhsh, Fatemeh; Dehghani, Zohreh; Zia, Mohammad Farid; Rabbani, Mohammad; Sadeghi, Hamid Mir Mohammad

    2013-01-01

    Background Production of tissue Plasminogen Activator protein (t-PA) in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. Methods In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli (E. coli) bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBA...

  18. Optimization the expression of human papilloma virus E6 and E7 polytopic construct in E. coli expression system

    Directory of Open Access Journals (Sweden)

    Arian Rahimi

    2015-12-01

    Full Text Available Background: Human papilloma virus is a DNA virus from the papillomavirus family that is most prevalent in human cervical cancers and many studies showed the E6 and E7 proteins are present in the majority of cervical cancer cases. Development of universal HPV peptide-based vaccine with more serotypes coverage has considerable value. The aim of the study was to design a multi-epitope universal vaccine for major HPV based on E6 and E7 proteins and optimization the expression of polytopic construct contains E6 and E7 genes from different genotypes of human papilloma virus as a candid vaccine. Methods: In this experimental study that was carried out in Pasteur Institute of Iran, Virology Department from October 2013 to November 2014. In order to design the polytypic construct, we predicted the most probable immunogenic epitopes of E6 and E7 from common high risk HPV16, 18, 31, 45 along with high prevalent type 6 and 11 using bioinformatics methods. The synthetic pET28a expression vector harboring E6 and E7 protein was transformed into Escherichia coli hosts and its expression was analyzed by SDS-PAGE and western blotting. Finally, in order to expression optimization of recombinant protein, cell density, induction time, growth temperature, IPTG (Isopropyl β-D-1-thiogalactopyranoside concentration and cultures media were studied. Results: In the present study the recombinant fusion protein was expressed successfully and the highest expression of target protein was achieved in super broth medium containing 0.1% glucose and 0.2% L-arabinose. In Super broth medium, the optimum condition for recombinant protein expression was occurred at OD600 of 0.8, 0.1mM IPTG, one hour’s incubation time at 37 °C and BL21 (A1 host. Conclusion: The results of this study show that the optimum expression of E6 and E7 proteins from different genotypes of human papilloma virus can be performed. Moreover, by purification of recombinant protein and evaluation of its

  19. Cloning, expression, and purification of the functional Delta(3)-Delta(2)-enoyl-CoA isomerase fusion protein.

    Science.gov (United States)

    Li, Ding; Wong, Chi-Kin; Yu, Wen-hua; Li, Pingfeng

    2002-10-01

    Delta(3)-Delta(2)-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme for the beta-oxidation of unsaturated fatty acids. The cDNA of the full-length rat liver Delta(3)-Delta(2)-enoyl-CoA isomerase was previously cloned as pAG847. PCR methodologies were used to subclone the gene encoding the functional Delta(3)-Delta(2)-enoyl-CoA isomerase from pAG847 with primers that were designed to add six continuous histidine codon to the 5(') primer. The PCR product was inserted into a pLM1 expression vector and overexpressed in Escherichia coli. The soluble expressed protein was purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity based on Coomassie blue-stained SDS-PAGE and the molecular weight of the protein subunit was 30 kDa. The purified protein had a dimeric structure composed of identical subunits, and the molecular weight of the enzyme determined by gel chromatography was 60 kDa. Kinetic studies have been carried out and K(M) of 81 microM and V(max) of 292 micromol/min/mg were determined. The specific activity of the protein is 201 U/mg, which is significantly higher than that reported before for the same protein isolated from a natural source. The one-step purification of the highly active Delta(3)-Delta(2)-enoyl-CoA isomerase will greatly facilitate the further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogues.

  20. Expression, solubilisation, and purification of a functional CMP-sialic acid transporter in Pichia pastoris.

    Science.gov (United States)

    Maggioni, Andrea; Hadley, Barbara; von Itzstein, Mark; Tiralongo, Joe

    2014-09-01

    Membrane proteins, including solute transporters play crucial roles in cellular function and have been implicated in a variety of important diseases, and as such are considered important targets for drug development. Currently the drug discovery process is heavily reliant on the structural and functional information discerned from high-resolution crystal structures. However, membrane protein structure determination is notoriously difficult, due in part to challenges faced in their expression, solubilisation and purification. The CMP-sialic acid transporter (CST) is considered to be an attractive target for drug discovery. CST inhibition reduces cancer cell sialylation and decreases the metastatic potential of cancer cells and to date, no crystal structure of the CST, or any other nucleotide sugar transporter exists. Here we describe the optimised conditions for expression in Pichia pastoris, solubilisation using n-nonyl β-d-maltopyranoside (NM) and single step purification of a functional CST. Importantly we show that despite being able to solubilise and purify the CST using a number of different detergents, only NM was able to maintain CST functionality. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Purification and characterization of recombinant baculovirus-expressed mouse DNA methyltransferase.

    Science.gov (United States)

    Glickman, J F; Flynn, J; Reich, N O

    1997-01-13

    DNA methylation is essential for normal embryonic development in mice. An understanding of how DNA methylation is controlled is largely dependent upon the isolation and characterization of the cellular components of the DNA methylation system. The enzyme which methylates DNA in eukaryotic cells is a C-5 cytosine DNA methyltransferase. Historically, the characterization of this enzyme has been limited by its availability and purity. Here, we present a single-step purification of 4 mg of baculovirus-expressed mouse DNA methyltransferase containing a nickel-affinity leader peptide. The recombinant DNA methyltransferase copurified with inhibitory RNA which was removed by treatment with ribonuclease A. Like its non-recombinant counterpart, the recombinant enzyme is activated by hemi-methylation. A direct steady-state kinetic comparison between the recombinant baculovirus-expressed enzyme with its MEL cell-derived counterpart is presented.

  2. Barley (Hordeum vulgare L.) cysteine proteases: heterologous expression, purification and characterization

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    2011-01-01

    During germination of barley seeds, mobilization of protein is essential and cysteine proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins. Cysteine proteases exist as pro-enzyme and is activated through reduction of the active...... site cysteines and by removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. A cDNA clone of the barley key cysteine endoprotease...... B2 (HvEPB2) was ligated into the Pichia pastoris expression vector pPICZ Aα and electrotransformed into Pichia pastoris strain KM71H. Heterologous protein production was induced with 2% MeOH and maximum yield were obtained after 4 days where the supernatant was harvested. Purification of HvEPB2 from...

  3. Cloning, expression, purification and crystallographic studies of galectin-11 from domestic sheep (Ovis aries).

    Science.gov (United States)

    Sakthivel, Dhanasekaran; Littler, Dene; Shahine, Adam; Troy, Sally; Johnson, Matthew; Rossjohn, Jamie; Piedrafita, David; Beddoe, Travis

    2015-08-01

    Galectins are an evolutionarily conserved family of proteins that translate glycan recognition into cellular effects. Galectin-11 is a unique member of the galectin family that is only expressed in ruminants such as sheep, goat and cattle and that plays a critical role in several important biological processes, such as reproduction and parasite-mediated innate immune responses. Currently, these two areas are of major importance for the sustainability of ruminant livestock production. Despite the emerging biological significance of galectin-11, no structural information is available. It is expected that structural studies will unravel the functional mechanisms of galectin-11 activity. Here, the expression, purification and crystallization of the ruminant-specific galectin-11 from domestic sheep and the collection of X-ray data to 2.0 Å resolution are reported.

  4. [Gene expression, purification and functional analysis of LiPrmA].

    Science.gov (United States)

    Sun, Yan-Ju; Zhou, Zhong-Wei; Yao, Jian-Xiu; Sun, Ti-Chang; Xu, Yan-Yan; Wen, Ya; Bao, Shi-Lai

    2005-09-01

    Leptospira interrogans (L. interrogans) genomic DNA was used as template to amplify the full-length gene for ribosomal protein L11 methyltransferase (liPrmA) by PCR. The pET22b-/liprmA expression plasmid was successfully constructed in Escherichia coli (E.coli.) strain TOP10 and confirmed by restriction enzyme digest and sequencing. Through optimizing expression of the recombinant liPrmA-6xHis fusion protein in expression host E. coli. BL21, the yield of soluble target protein reached 40 mg (liter culture)-1. The LiPrmA was purified to apparent homogeneity in a single step using Ni-NTA His Bind chromatography. Amino acid homologous analysis showed that liPrmA shared significant identity with other prokaryotic PrmA and eukaryotic putative PrmA in the catalytic region including AdoMet binding domain. Methylation activity experiments showed purified liPrmA was able to catalyze the ribosomal protein L11 of L. interrogans methylated under the presence of S-adenosyl-methionine (AdoMet).

  5. Molecular Cloning, Expression, Purification, and Functional Characterization of Dammarenediol Synthase from Panax ginseng

    Directory of Open Access Journals (Sweden)

    Wei Hu

    2013-01-01

    Full Text Available The objective of this study is to clone and charecterize the expression of dammarenediol synthase gene and then to determine the relationship between the expression of dammarenediol synthase gene that is involved in the ginsenoside biosynthetic pathway and the ginsenoside content. A cDNA phage library was constructed from a five-year-old ginseng root. The cDNA library was screened for the dammarenediol synthase gene by using its specific primers. It was further cloned and expressed in pET-30a vector. The recombinant plasmid pET-30a-DS was expressed in Rosetta E. coli. The recombinant DS protein was purified by affinity chromatography. The production of dammarenediol was detected by liquid chromatography-mass spectrometry (LC-MS. Results showed that dammarenediol synthase gene was cloned from the cDNA library and was expressed in Rosetta E. coli and the SDS-PAGE analysis showed the presence of purified DS protein. LS-MS showed the activity of DS protein, as the protein content increases the dammarenediol increases. Our results indicate that the recombinant dammarenediol synthase protein could increase the production of dammarenediol and the expression of DS played a vital role in the biosynthesis of ginsenosides in P. ginseng.

  6. High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    Directory of Open Access Journals (Sweden)

    You Bang-Jau

    2011-07-01

    Full Text Available Abstract Background Chicken anemia virus (CAV, the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Results Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. Conclusions Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

  7. High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development.

    Science.gov (United States)

    Lee, Meng-Shiou; Hseu, You-Cheng; Lai, Guan-Hua; Chang, Wen-Te; Chen, Hsi-Jien; Huang, Chi-Hung; Lee, Meng-Shiunn; Wang, Min-Ying; Kao, Jung-Yie; You, Bang-Jau; Lin, Wen- Hsin; Lien, Yi-Yang; Lin, Ming-Kuem

    2011-07-23

    Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

  8. Expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath

    Directory of Open Access Journals (Sweden)

    Kursula Petri

    2010-01-01

    Full Text Available Abstract Background 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase is an enigmatic enzyme specifically expressed at high levels in the vertebrate myelin sheath, whose function and physiological substrates are unknown. The protein consists of two domains: an uncharacterized N-terminal domain with little homology to other proteins, and a C-terminal phosphodiesterase domain. Findings In order to be able to fully characterize CNPase structurally and functionally, we have set up expression systems for different domains of CNPase, using a total of 18 different expression constructs. CNPase was expressed in E. coli with a TEV-cleavable His-tag. Enzymatic activity assays indicated that the purified proteins were active and correctly folded. The folding of both the full-length protein, as well as the N- and C-terminal domains, was also studied by synchrotron CD spectroscopy. A thermal shift assay was used to optimize buffer compositions to be used during purification and storage. The assay also indicated that CNPase was most stable at a pH of 5.5, and could be significantly stabilized by high salt concentrations. Conclusions We have been able to express and purify recombinantly several different domains of CNPase, including the isolated N-terminal domain, which is folded mainly into a β-sheet structure. The expression system can be used as an efficient tool to elucidate the role of CNPase in the myelin sheath.

  9. An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Gaurav Chhetri

    2015-01-01

    • In addition to being inexpensive, easy to manage, universal, and quick to perform, the proposed method does not require any commercial kits and, can be used for various recombinant proteins expressed in the E. coli expression system.

  10. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Siti Aisyah Mualif

    Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  11. Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

    Science.gov (United States)

    Meiyalaghan, Sathiyamoorthy; Latimer, Julie M; Kralicek, Andrew V; Shaw, Martin L; Lewis, John G; Conner, Anthony J; Barrell, Philippa J

    2014-11-04

    The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts. To overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system. We present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.

  12. Molecular Cloning, Expression and Purification of Protein TB10.4 Secreted by Mycobacterium Tuberculosis

    Directory of Open Access Journals (Sweden)

    Aida Gholoobi

    2010-09-01

    Full Text Available Objective(sTuberculosis (TB is the leading cause of mortality among the infectious diseases, especially in developing countries. One of the main goals in tuberculosis research is to identify antigens which have the ability of inducing cellular and/or humoral immunity in order to use them in diagnostic reagents or vaccine design. The aim of this study was to clone and express the TB10.4 protein in Escherichia coli expression system.Materials and MethodsDNA was extracted from Mycobacterium tuberculosis H37Rv. Gene specific primers were designed using Gene Runner software according to sanger sequence database. Gene tb10.4 fragment was amplified by PCR method and purified tb10.4 gene was cloned into pET 102/D vector. Plasmid containing pET102/D-10.4 was transformed into competence E. coli TOP10. A positive transformant was chosen and plasmids DNA was isolated and subsequently transformed into competence E. coli BL21(DE3. The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. Purified recombinant protein was achieved using metal affinity chromatography (Ni-nitrilotriacetic acid.ResultsTB10.4 molecule was successfully cloned, expressed, and purified. An approximately 26.4 kDa exogenous protein was observed on the SDS-PAGE. The recombinant protein was confirmed by DNA sequencing of correct insert.ConclusionThe success of expressing the TB10.4 protein could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and diagnostic method.

  13. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

    Science.gov (United States)

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia

    2015-12-01

    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

    Science.gov (United States)

    Bruni, Renato

    2014-01-01

    Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

  15. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    Science.gov (United States)

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.

  16. Genotypic Characterization of Egypt Enterotoxigenic Escherichia coli Isolates Expressing Coli Surface Antigen 6

    Science.gov (United States)

    2013-02-01

    USA Abstract Introduction: One approach to control enterotoxigenic Escherichia coli (ETEC) infections has been to develop vaccines focused on...results show a lack of clonality among Egypt CS6 E. coli isolates and supports the use and the further research on vaccines targeting this cell surface...organisms must colonize the mucosal epithelium; this process utilizes fimbrial and non-fimbrial colonization factors, also referred to as coli surface

  17. Expression, purification and biochemical characterization of Schizosaccharomyces pombe Mcm4, 6 and 7.

    Science.gov (United States)

    Xu, Meng; Chang, Y Paul; Chen, Xiaojiang S

    2013-02-27

    The hetero-hexamer of the eukaryotic minichromosome maintenance (MCM) proteins plays an essential role in replication of genomic DNA. The ring-shaped Mcm2-7 hexamers comprising one of each subunit show helicase activity in vitro, and form double-hexamers on DNA. The Mcm4/6/7 also forms a hexameric complex with helicase activity in vitro. We used an Escherichiai coli expression system to express various domains of Schizosaccharomyces pombe Mcm4, 6 and 7 in order to characterize their domain structure, oligomeric states, and possible inter-/intra-subunit interactions. We also successfully employed a co-expression system to express Mcm4/6/7 at the same time in Escherichiai coli, and have purified functional Mcm4/6/7 complex in a hexameric state in high yield and purity, providing a means for generating large quantity of proteins for future structural and biochemical studies. Based on our results and those of others, models were proposed for the subunit arrangement and architecture of both the Mcm4/6/7 hexamer and the Mcm2-7 double-hexamer.

  18. Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells

    Directory of Open Access Journals (Sweden)

    Baschieri Selene

    2007-02-01

    Full Text Available Abstract Background Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa and a truncated form (p25, 25 kDa. Here we report the expression and purification of HIV Nef from transgenic tobacco. Results We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. Conclusion We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

  19. Expression and Purification of Haemophilus influenzae Rhomboid Intramembrane Protease GlpG for Structural Studies.

    Science.gov (United States)

    Panwar, Pankaj; Lemieux, M Joanne

    2014-04-01

    Rhomboid proteases are membrane-embedded proteases that cleave peptide bonds of transmembrane proteins. They play a variety of roles in cell signaling events. The rhomboid protease GlpG from Haemophilus influenzae (hiGlpG) is a canonical form of rhomboid protease having six transmembrane segments. In this unit, detailed protocols are presented for optimization of hiGlpG expression using the araBAD promotor system in the pBAD vector. The parameters for optimization include concentration of inducing agent, induction temperature, and time. Optimization of these key factors led to the development of a protocol yielding 1.6 to 2.5 mg/liter protein purified after ion metal affinity chromatography (IMAC). Further purification can include size exclusion chromatography (SEC). Copyright © 2014 John Wiley & Sons, Inc.

  20. Cloning, Expression, Purification, Crystallization and Preliminary X-ray Analysis of Mycoplasma Genitalium Protein MG289

    Energy Technology Data Exchange (ETDEWEB)

    Sippel, K.; Boehlein, S; Sakai, Y; Quirit, J; Agbandje-McKenna, M; Rosser, C; McKenna, R

    2009-01-01

    Mycoplasma genitalium is a human pathogen that is associated with nongonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 {angstrom} resolution. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 {angstrom}. The diffraction data after processing had an overall R{sub merge} of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.

  1. Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract

    DEFF Research Database (Denmark)

    Connell, Hugh; Agace, William; Klemm, Per

    1996-01-01

    of Escherichia coli for the urinary tract by promoting bacterial persistence and enhancing the inflammatory responce to infection. In a clinical study, we observed that disease severity was greater in children infected with E. coli O1:K1:H7 isolates expressing type 1 fimbriae than in those infected with type 1...... negative isolates of the same serotype. The E. coli O1:K1:H7 isolates had the same electrophoretic type, were hemolysin-negative, expressed P fimbriae, and carried the fim DNA sequences. When tested in a mouse urinary tract infection model, the type 1-positive E. coli O1:K1:H7 isolates survived inhigher...... urinary tract infection model. E. coli CN1016 reconstituted with type 1 fimbriae had restored virulence similar to that of the wild-type parent strain. These results show that type 1 fimbriae in the genetic background of a uropathogenic strain contribute to the pathogenesis of E. coli in the urinary tract....

  2. Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences.

    Science.gov (United States)

    Carvalho, Rimenys Junior; Cabrera-Crespo, Joaquin; Tanizaki, Martha Massako; Gonçalves, Viviane Maimoni

    2012-05-01

    Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.

  3. [Expression of pyruvate oxidase gene sopox from Streptococcus sanguis in E. coli].

    Science.gov (United States)

    Hou, B; Zhang, J; Zhang, R; Zhang, Y

    2001-03-01

    To reconstruct the expression plasmid of Sopox gene for further understanding the regulation of its expression. Sopox was recombined with expression vector pBV220 and the expression of Sopox in E. coli JM105 was observed after transformation. pBV220/Sopox/JM105 expressed a protein with molecular weight of 65 kd on SDS-PAGE after induction, and the expression reached the maximal amount with induction at 42 degrees C for 4 hours. Sopox was successfully cloned into pBV220 and expressed in E. coli JM105.

  4. Co-expression and co-purification of archaeal and eukaryal box C/D RNPs.

    Directory of Open Access Journals (Sweden)

    Yu Peng

    Full Text Available Box C/D ribonucleoprotein particles (RNPs are 2'-O-methylation enzymes required for maturation of ribosomal and small nuclear RNA. Previous biochemical and structural studies of the box C/D RNPs were limited by the unavailability of purified intact RNPs. We developed a bacterial co-expression strategy based on the combined use of a multi-gene expression system and a tRNA-scaffold construct that allowed the expression and purification of homogeneous archaeal and human box C/D RNPs. While the co-expressed and co-purified archaeal box C/D RNP was found to be fully active in a 2'-O-methylation assay, the intact human U14 box C/D RNP showed no detectable catalytic activity, consistent with the earlier findings that assembly of eukaryotic box C/D RNPs is nonspontaneous and requires additional protein factors. Our systems provide a means for further biochemical and structural characterization of box C/D RNPs and their assembly factors.

  5. Impact of intramammary treatment on gene expression profiles in bovine Escherichia coli mastitis

    OpenAIRE

    Anja Sipka; Suzanne Klaessig; Duhamel, Gerald E.; Jantijn Swinkels; Pascal Rainard; Ynte Schukken

    2014-01-01

    Clinical mastitis caused by E. coli accounts for significant production losses and animal welfare concerns on dairy farms worldwide. The benefits of therapeutic intervention in mild to moderate cases are incompletely understood. We investigated the effect of intramammary treatment with cefapirin alone or in combination with prednisolone on gene expression profiles in experimentally-induced E. coli mastitis in six mid-lactating Holstein Friesian cows. Cows were challenged with E. coli in 3 qua...

  6. Application to Photocatalytic H2 Production of a Whole-Cell Reaction by Recombinant Escherichia coli Cells Expressing [FeFe]-Hydrogenase and Maturases Genes.

    Science.gov (United States)

    Honda, Yuki; Hagiwara, Hidehisa; Ida, Shintaro; Ishihara, Tatsumi

    2016-07-04

    A photocatalytic H2 production system using an inorganic-bio hybrid photocatalyst could contribute to the efficient utilization of solar energy, but would require the development of a new approach for preparing a H2 -forming biocatalyst. In the present study, we constructed a recombinant strain of Escherichia coli expressing the genes encoding the [FeFe]-hydrogenase and relevant maturases from Clostridium acetobutylicum NBRC 13948 for use as a biocatalyst. We investigated the direct application of a whole-cell of the recombinant E. coli. The combination of TiO2 , methylviologen, and the recombinant E. coli formed H2 under light irradiation, demonstrating that whole cells of the recombinant E. coli could be employed for photocatalytic H2 production without any time-consuming and costly manipulations (for example, enzyme purification). This is the first report of the direct application of a whole-cell reaction of recombinant E. coli to photocatalytic H2 production. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. [Expression, purification and protective antigen analysis of cell wall protein MRP of Streptococcus suis type 2].

    Science.gov (United States)

    Wang, Ping-ping; Pian, Ya-ya; Yuan, Yuan; Zheng, Yu-ling; Jiang, Yong-qiang; Xiong, Zheng-ying

    2012-02-01

    To amplify the mrp gene of Streptococcus suis type 2 05ZYH33, express it in E.coli BL21 in order to acquire high purity recombinant protein MRP, then evaluate the protective antigen of recombinant protein MRP. Using PCR technology to obtain the product of mrp gene of 05ZYH33, and then cloned it into the expression vector pET28a(+). The recombinant protein was purified by affinity chromatography, later immunized New Zealand rabbit to gain anti-serum, then test the anti-serum titer by ELISA. The opsonophagocytic killing test demonstrated the abilities of protective antigen of MRP. The truncated of MRP recombinant protein in E.coli BL21 expressed by inclusion bodies, and purified it in high purity. After immunoprotection, the survival condition of CD-1 was significantly elevated. The survival rate of wild-type strain 05ZYH33 in blood was apparently decreased after anti-serum opsonophagocyticed, but the mutant delta; MRP showed no differences. MRP represent an important protective antigen activity.

  8. Chromatin architecture and gene expression in Escherichia coli

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2004-01-01

    Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli.......Two recent genome-scale analyses underscore the importance of DNA topology and chromatin structure in regulating transcription in Escherichia coli....

  9. Expression, purification, crystallization and preliminary X-ray studies of Lactobacillus jensenii enolase

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Paul T.; Raghunathan, Kannan; Spurbeck, Rachel R.; Arvidson, Cindy G.; Arvidson, Dennis N. (MSU)

    2010-09-02

    Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25 {angstrom} resolution. The crystals belonged to space group I4, with unit-cell parameters a = b = 145.31, c = 99.79 {angstrom}. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient V{sub M} of 2.8 {angstrom}{sup 3} Da{sup -1}, corresponding to 55.2% solvent content.

  10. Construction, Expression and Purification of Wild and Mutant Type of nm23-H1 in Prokaryotic Expression System

    Directory of Open Access Journals (Sweden)

    Xueqin YANG

    2009-01-01

    Full Text Available Background and objective Nm23-H1 is a metastasis-suppressor gene. However, its molecular mechanism of suppressing metastasis is unknown until now. The aim of this study is to construct prokaryotic expression vector of wild and mutant type of nm23-H1 (WT, P96S, H118F, and then express and purify the proteins. Methods wild and mutant type of nm23-H1 fragments were amplified by PCR. The prokaryotic expression vectors of pET28anm23-H1 were constructed by gene recombination technique and verified by restriction enzyme analysis and sequencing. The positive clones were transformed into E. coli BL21 (DE3 and soluble analysis of the expression was conducted in this system. The proteins were purified by nickel column chromatography and identified by Western blot. Results Thesequences and open read frames of all the pET28a-nm23-H1 plasmids were completely correct. After transforming, these plasmids can express the target proteins. The protein production was very high, and all the proteins were soluble expression. The molecular weight of wild and mutant type of nm23-H1 was 20 kDa detected by Western blot, which was as the same as the objective protein. Conclusion We have succeeded in constructing the prokaryotic expression vectors ofpET28a-nm23-H1 (WT, P96S, H118F and the proteins which expressed can be used in following studies.

  11. Optimization of expression and purification of human mortalin (Hsp70): Folding/unfolding analysis

    Science.gov (United States)

    Khan, Mohd Shahnawaz; Ahmed, Anwar; Tabrez, Shams; Islam, Badar ul; Rabbani, Nayyar; Malik, Ajamaluddin; Ismael, Mohamad A.; Alsenaidy, Mohammad A.; Alsenaidy, Abdulrahman M.

    2017-12-01

    Human mortalin is a Hsp70 mitochondrial protein that plays an essential role in the biogenesis of mitochondria. The deregulation of mortalin expression and its functions could lead to several age-associated disorders and some types of cancers. In the present study, we optimized the expression and purification of recombinant human mortalin by the use of two-step chromatography. Low temperature (18 °C) and 0.5 mM (IPTG) was required for optimum mortalin expression. Chaperone activity of mortalin was assessed by the citrate synthase and insulin protection assay, which suggested their protective role in mitochondria. Folding and unfolding assessments of mortalin were carried out in the presence of guanidine hydrochloride (GdnHCl) by intrinsic fluorescence measurement, ANS (8-analino 1-nephthlene sulfonic acid) binding and CD (circular dichroism) analysis. Under denaturing conditions, mortalin showed decrease in tryptophan fluorescence intensity along with a red shift of 11 nm. Moreover, ANS binding studies illustrated decrease in hydrophobicity. CD measurement of mortalin showed a predominant helical structure. However, the secondary structure was lost at low concentration of GdnHCl (1 M). We present a simple and robust method to produce soluble mortalin and warranted that chaperones are also susceptible to unfolding and futile to maintain protein homeostasis.

  12. Injury and mechanism of recombinant E. coli expressing STa on piglets colon.

    Science.gov (United States)

    Lv, Yang; Li, Xueni; Zhang, Lin; Shi, Yutao; DU, Linxiao; Ding, Binying; Hou, Yongqing; Gong, Joshua; Wu, Tao

    2018-02-09

    Enterotoxigenic Escherichia coli (ETEC) is primary pathogenic bacteria of piglet diarrhea, over two thirds of piglets diarrhea caused by ETEC are resulted from STa-producing ETEC strains. This experiment was conducted to construct the recombinant E. coli expressing STa and study the injury and mechanism of recombinant E. coli expressing STa on 7 days old piglets colon. Twenty-four 7 days old piglets were allotted to four treatments: control group, STa group (2 × 10 9 CFU E. coli LMG194-STa), LMG194 group (2 × 10 9 CFU E. coli LMG194) and K88 group (2 × 10 9 CFU E. coli K88). The result showed that E. coli infection significantly increased diarrhea rates; changed DAO activity in plasma and colon; damaged colonic mucosal morphology including crypt depth, number of globet cells, density of lymphocytes and lamina propria cell density; substantially reduced antioxidant capacity by altering activities of GSH-Px, SOD, and TNOS and productions of MDA and H 2 O 2 ; obviously decreased AQP3, AQP4 and KCNJ13 protein expression levels; substantially altered the gene expression levels of inflammatory cytokines. Conclusively, STa group had the biggest effect on these indices in four treatment groups. These results suggested that the recombinant strain expressed STa can induce piglets diarrhea and colonic morphological and funtional damage by altering expression of proteins connect to transportation function and genes associated with intestinal injury and inflammatory cytokines.

  13. Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis

    NARCIS (Netherlands)

    van Dijl, J M; Jong, de Anne; Smith, H; Bron, Sierd; Venema, G

    The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half

  14. Construction of an Expression and Site-Directed Mutagenesis System of Haloalkane Dehalogenase in Escherichia coli

    NARCIS (Netherlands)

    Schanstra, J.P.; Rink, R.; Pries, F.; Janssen, D.B.

    1993-01-01

    Haloalkane dehalogenase from Xanthobacter autotrophicus was efficiently expressed in Escherichia coli BL21(DE3) and E. coli JM101. After introduction of restriction sites hy PCR the haloalkane dehalogenase gene (dhlA) was translationally fused behind the T7 (φ10), trc, and tac promoters. This

  15. Expression, purification and biochemical characterization of a family 6 carboxylesterase from Methylococcus capsulatus (bath).

    Science.gov (United States)

    Soni, Surabhi; Odaneth, Annamma A; Lali, Arvind M; Chandrayan, Sanjeev K

    2016-06-01

    The genome of Methylococcus capsulatus (bath) encodes a protein R-est6 that is annotated as a lipase family 3 protein. The phylogenetic and the sequence analyses linked this protein to the family 6 carboxylesterase. The gene encoding R-est6 was cloned and overexpressed in Escherichia coli and the recombinant 6x-His tagged protein was purified by Ni-NTA affinity chromatography. The buffers used in the purification were modified by adding 1% glycerol instead of the salt to prevent the protein aggregation. Far UV-CD spectrum and gel filtration chromatography of the purified R-est6 confirmed that the protein was well folded like a typical α/β hydrolase and had the quaternary structure of a tetramer, in addition to a compact monomer. The optimum pH was in the range of 7.0-9.0 and the optimum temperature was at 55 °C for the hydrolysis of pNP-butyrate. As expected, being a member of the family 6 carboxylesterase, R-est6 hydrolyzed triglycerides, pNP esters of the small and the medium fatty acid chain esters and an aryl ester-phenyl acetate. However, R-est6 was also found to hydrolyze the long-chain fatty acid ester which had never been reported for the family 6 carboxylesterase. Additionally, R-est6 was stable and active in the different water-miscible organic solvents. Therefore, the broad substrate range and the structural stability of R-est6 would be advantageous for its application in industrial processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. [Cloning and expression of novel swine gene BCL-G(L) in E.coli and preparation of its polyclonal antibody in guinea pigs].

    Science.gov (United States)

    Jiang, Peng-Fei; Cao, Jin-Suo; Liu, Chao; Zhao, Hai-Ping; Zhang, De-Li

    2010-12-01

    In order to express a novel gene named as BCL-G(L); of swine in E.coli and prepare its polyclonal antibody. The contig sequence of the gene was predicted and in silicon cloned by blasting the human BCL-G(L); in swine ESTs database in NCBI. The cloning sequence was obtained by RT-PCR from swine spleen. The cloning sequence was identified by sequencing and compared with the contig sequence. Then the gene was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET32a-BCL-G(L);. The fusion protein pET32a-BCL-G(L); was expressed in E.coli BL21 and purified using a His-tag fusion protein purification kit. Then guinea pigs were immunized with the purified protein to get the specific polyclonal antibody. The titer of the antibody was 1:800 detected by ELISA. The protein BCL-G(L); can be specifically detected by western blot assay using the polyclonal antibody. The novel swine gene BCL-G(L); was cloned and expressed in E.coli and its polyclonal antibody was prepared successfully.

  17. Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene.

    Science.gov (United States)

    Wang, C L; Lum, A M; Ozuna, S C; Clark, D S; Keasling, J D

    2001-08-01

    The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.

  18. Global gene expression profiling of asymptomatic bacteriuria Escherichia coli during biofilm growth in human urine

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Klemm, Per

    2007-01-01

    Urinary tract infection (UTI) is an important health problem worldwide, with many millions of cases each year, and Escherichia coli is the most common organism causing UTI in humans. Also, E. coli is responsible for most infections in patients with chronic indwelling bladder catheter. The two...... asymptomatic bacteriuria (ABU) E. coli strains 83972 and VR50 are significantly better biofilm formers in their natural growth medium, human urine, than the two uropathogenic E. coli isolates CFT073 and 536. We used DNA microarrays to monitor the expression profile during biofilm growth in urine of the two ABU...

  19. A generic system for the expression and purification of soluble and stable influenza neuraminidase.

    Directory of Open Access Journals (Sweden)

    Peter M Schmidt

    Full Text Available The influenza surface glycoprotein neuraminidase (NA is essential for the efficient spread of the virus. Antiviral drugs such as Tamiflu (oseltamivir and Relenza (zanamivir that inhibit NA enzyme activity have been shown to be effective in the treatment of influenza infections. The recent 'swine flu' pandemic and world-wide emergence of Tamiflu-resistant seasonal human influenza A(H1N1 H(274Y have highlighted the need for the ongoing development of new anti-virals, efficient production of vaccine proteins and novel diagnostic tools. Each of these goals could benefit from the production of large quantities of highly pure and stable NA. This publication describes a generic expression system for NAs in a baculovirus Expression Vector System (BEVS that is capable of expressing milligram amounts of recombinant NA. To construct NAs with increased stability, the natural influenza NA stalk was replaced by two different artificial tetramerization domains that drive the formation of catalytically active NA homotetramers: GCN4-pLI from yeast or the Tetrabrachion tetramerization domain from Staphylothermus marinus. Both recombinant NAs are secreted as FLAG-tagged proteins to allow for rapid and simple purification. The Tetrabrachion-based NA showed good solubility, increased stability and biochemical properties closer to the original viral NA than the GCN4-pLI based construct. The expressed quantities and high quality of the purified recombinant NA suggest that this expression system is capable of producing recombinant NA for a broad range of applications including high-throughput drug screening, protein crystallisation, or vaccine development.

  20. Expression, purification and immunochemical characterization of recombinant OMP28 protein of Brucella species

    Directory of Open Access Journals (Sweden)

    Y. Manat

    2016-05-01

    Full Text Available Brucellosis is the lion’s share of infectious disease of animals and it has a particular socio-economic importance for the Republic of Kazakhstan. Sixty percent of epizootic outbreaks of brucellosis identified in the Commonwealth of Independent States (CIS originated from Kazakhstan in recent years. Definitive diagnosis of brucellosis remains a difficult task. Precisely for this reason, we evaluated a purified recombinant out membrane protein 28 (rOMP28 of Brucella species (Brucella spp. produced in Escherichia coli (E. coli as a diagnostic antigen in an Indirect ELISA (I-ELISA for bovine brucellosis. The gene encoding OMP28 was synthesized using a two-round PCR procedure. In order to produce the rOMP28, the de novo synthesized DNA was cloned into the expression vector pET-22b(+. Then, the rOMP28 was expressed in E. coli system and characterized in the present study. We further estimated the diagnostic potential of purified rOMP28 of Brucella spp. for screening bovine sera. To determine if rOMP28 has a valuable benefit for use in the serodiagnosis of bovine brucellosis, rOMP28-based I-ELISA was performed. Brucella spp. positive (n=62 and Brucella spp. negative (n=28 samples from tube agglutination test (TAT were positive (n=59 and negative (n=27 by I-ELISA, respectively. These findings show that the rOMP28 of Brucella spp. could be a good candidate for improving serological diagnostic methods for bovine brucellosis.

  1. Expression and purification of the active soluble form of Bacillus sphaericus binary toxin for structural analysis.

    Science.gov (United States)

    Srisucharitpanit, Kanokporn; Inchana, Pattarapong; Rungrod, Amporn; Promdonkoy, Boonhiang; Boonserm, Panadda

    2012-04-01

    The binary toxin produced from Bacillus sphaericus is highly toxic against larvae of Culex and Anopheles mosquitoes. The two major components of the binary toxin are 42-kDa BinA and 51-kDa BinB, which are produced as crystalline inclusions during sporulation. Currently, there is no detailed knowledge of the molecular mechanism of the binary toxin, mainly due to the lack of structural information. Herein, we describe an expression protocol with modified conditions allowing production of soluble, biologically active BinA and BinB for further structural analysis. The binA and binB genes from B. sphaericus 2297 strain were independently cloned and fused with a polyhistidine tag at their N-termini. Both (His)(6)-tagged BinA and (His)(6)-tagged BinB were expressed as soluble forms at low temperature. Highly pure proteins were obtained after two-step purification by Ni-NTA affinity and size exclusion chromatography. In vitro activation by trypsin digestion generated a resistant fragment, of 40kDa for BinA, and of 45kDa for BinB, and an oligomeric complex of BinA and BinB in solution was observed after proteolytic activation. Their functional and structural properties were confirmed by a biological assay and far-UV circular dichroism, respectively. The mixture of BinA and BinB, either as a protoxin or as a trypsin-activated form, exhibited high mosquito-larvicidal activity against Culex quinquefasciatus larvae with LC(50) of about 10ng/ml, while no toxicity was observed from the single binary toxin component. Results from far-UV circular dichroism of BinA and BinB suggest the presence of mainly β-structure. The expression and purification protocols reported here will be useful for the production of the active and homogeneous binary toxin to allow further detailed structural investigation. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L..

    Directory of Open Access Journals (Sweden)

    Osama Elhag

    Full Text Available Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L, and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a, sarcotoxin (2b, sarcotoxin3, stomoxynZH1, and stomoxynZH1(a. Among these genes, a 189-basepair gene (stomoxynZH1 was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice-10, and fungus Sclerotinia sclerotiorum (Lib. de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L. could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens.

  3. Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L.).

    Science.gov (United States)

    Elhag, Osama; Zhou, Dingzhong; Song, Qi; Soomro, Abdul Aziz; Cai, Minmin; Zheng, Longyu; Yu, Ziniu; Zhang, Jibin

    2017-01-01

    Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens.

  4. Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase.

    Science.gov (United States)

    Berwal, Ritu; Gopalan, Natarajan; Chandel, Kshitij; Prasad, G B K S; Prakash, Shri

    2008-10-01

    Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 degrees C with 0.5mM isopropyl beta-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 degrees C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 micromol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k(cat) [(3.2+/-0.02)x10(4)] with 3-acetylpyridine adenine dinucleotide (APAD+). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.

  5. High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression.

    Science.gov (United States)

    Lai, Guan-Hua; Lin, Yen-Chang; Tsai, Yi-Lun; Lien, Yi-Yang; Lin, Ming-Kuem; Chen, Hsi-Jien; Chang, Wen-Te; Tzen, Jason T C; Lee, Meng-Shiou

    2014-05-22

    Pigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap (or C1) gene of PiCV, has been shown to be responsible for not only capsid assembly, but also has been used as antigen for detecting antibody when the host is infected with PiCV. The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system. The PiCV cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to have good antigenic

  6. Article Expression, Purification, and Characterization of Cu/ZnSOD from Panax Ginseng

    Directory of Open Access Journals (Sweden)

    Dayong Ding

    2014-06-01

    Full Text Available Superoxide dismutase (SOD has a strong antioxidant effect, but the traditional SOD extraction method is not the most efficient method of SOD amplification. In this study, we report the cloning of the Cu/ZnSOD gene from Panax ginseng into a temperature-regulated expression plasmid, pBV220. Cu/ZnSOD inclusion bodies were expressed in E. coli at a high level. Then, the inclusion bodies were purified by ion-exchange chromatography and molecular sieve chromatography. Finally, we obtained stable SOD in the bacterial broth, with a protein content of 965 mg/L and enzyme specific activity of 9389.96 U/mg. These results provide a foundation for future studies on the antioxidant mechanisms of ginseng and the development and application of ginseng Cu/ZnSOD.

  7. Optimized expression and purification of NavAb provide the structural insight into the voltage dependence.

    Science.gov (United States)

    Irie, Katsumasa; Haga, Yukari; Shimomura, Takushi; Fujiyoshi, Yoshinori

    2018-01-01

    Voltage-gated sodium channels are crucial for electro-signalling in living systems. Analysis of the molecular mechanism requires both fine electrophysiological evaluation and high-resolution channel structures. Here, we optimized a dual expression system of NavAb, which is a well-established standard of prokaryotic voltage-gated sodium channels, for E. coli and insect cells using a single plasmid vector to analyse high-resolution protein structures and measure large ionic currents. Using this expression system, we evaluated the voltage dependence and determined the crystal structures of NavAb wild-type and two mutants, E32Q and N49K, whose voltage dependence were positively shifted and essential interactions were lost in voltage sensor domain. The structural and functional comparison elucidated the molecular mechanisms of the voltage dependence of prokaryotic voltage-gated sodium channels. © 2017 Federation of European Biochemical Societies.

  8. Expression of highly toxic genes in E. coli: special strategies and genetic tools.

    Science.gov (United States)

    Saïda, F; Uzan, M; Odaert, B; Bontems, F

    2006-02-01

    Escherichia coli (E. coli) remains the most efficient widely-used host for recombinant protein production. Well-known genetics, high transformation efficiency, cultivation simplicity, rapidity and inexpensiveness are the main factors that contribute to the selection of this host. With the advent of the post-genomic era has come the need to express in this bacterium a growing number of genes originating from different organisms. Unfortunately, many of these genes severely interfere with the survival of E. coli cells. They lead to bacteria death or cause significant defects in bacteria growth that dramatically decrease expression capabilities. In this paper, we review special strategies and genetics tools successfully used to express, in E. coli, highly toxic genes. Suppression of basal expression from leaky inducible promoters, suppression of read-through transcription from cryptic promoters, tight control of plasmids copy numbers and proteins production as inactive (but reversible) forms are among the solutions presented and discussed. Special expression vectors and modified E. coli strains are listed and their effectiveness illustrated with key examples, some of which are related to our study of the highly toxic phage T4 restriction endoribonuclease RegB. We mainly selected those strategies and tools that permit E. coli normal growth until the very moment of highly toxic gene induction. Expression then occurs efficiently before cells die. Because they do not target a particular toxic effect, these strategies and tools can be used to express a wide variety of highly toxic genes.

  9. Large-Scale Recombinant Expression and Purification of Human Tyrosinase Suitable for Structural Studies.

    Directory of Open Access Journals (Sweden)

    Xuelei Lai

    Full Text Available Human tyrosinase (TYR is a glycoprotein that initiates the first two reactions in the melanin biosynthesis pathway. Mutations in its encoding gene cause Oculocutaneous Albinism type I (OCA1, the most severe form of albinism, which is a group of autosomal recessive disorders characterized by reduced or absent production of melanin in skin, hair and eyes. Despite extensive structural and characterization studies of its homologues in lower eukaryotic organisms, the catalytic mechanism of human TYR and the molecular basis of OCA1 are largely unknown. In this work, we have carried out a large-scale recombinant expression of TYR that has enabled us to obtain high yields of pure and active protein, required for crystallization trials and screening of skin whitening agents, which is highly demanded in the cosmetic industry. Addition of an N-terminal honeybee melittin signal peptide for secretion of the produced protein into the (protein-free medium, as well as a cleavable His-tag at the C-terminus, was crucial for increasing the yield of pure protein. We have successfully crystallized two TYR variants, in both glycosylated and deglycosylated forms, showing preliminary X-ray diffraction patterns at 3.5 Å resolution. Hence, we have established an expression and purification protocol suitable for the crystal structure determination of human TYR, which will give unique atomic insight into the nature and conformation of the residues that shape the substrate binding pocket that will ultimately lead to efficient compound design.

  10. Cloning, expression and purification of cindoxin, an unusual Fmn-containing cytochrome p450 redox partner.

    Science.gov (United States)

    Hawkes, David B; Slessor, Kate E; Bernhardt, Paul V; De Voss, James J

    2010-05-17

    Cytochromes P450 (P450s) belong to a superfamily of haemoproteins that catalyse a remarkable variety of oxidative transformations. P450 catalysis generally requires that cognate redox proteins transfer electrons, derived ultimately from NAD(P)H, to the P450 for oxygen activation. P450(cin) (CYP176A1) is a bacterial P450 that is postulated to allow Citrobacter braakii to live on cineole as its sole carbon source by initiating cineole biodegradation. Here we report the cloning, expression, purification and characterisation of one of its postulated redox partners, cindoxin (Cdx), which has strong similarity to the FMN domain of cytochrome P450 reductase. Cindoxin reductase (CdR), which displays strong similarity to NADPH-dependent ferredoxin reductases, was unable to be expressed in a functional form. Mass spectrometric and HPLC analyses confirmed that the flavin cofactor of cindoxin was FMN. Redox potentiometric titrations were performed with cindoxin within the range 6reductase (Fpr) acting as the terminal redox partner in the absence of CdR. Our results show that Cdx and Fpr support regio- and stereoselective P450(cin)-catalysed cineole oxidation to (1R)-6beta-hydroxycineole with turnover rates up to 1500 min(-1). This system is tightly coupled with 80 % of NADPH reducing equivalents funnelled into substrate oxidation.

  11. An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures.

    Science.gov (United States)

    Grant, A J; Haigh, R; Williams, P; O'Connor, C D

    2001-12-12

    Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of Tn Omega P(BAD) to place the highly regulable, arabinose inducible P(BAD) promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P(BAD) promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.

  12. High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

    Directory of Open Access Journals (Sweden)

    Craveiro R.B.

    2006-01-01

    Full Text Available Carboxypeptidase M (CPM is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1. This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa, suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

  13. Expression of the mouse metallothionein-I gene in Escherichia coli: increased tolerance to heavy metals.

    Science.gov (United States)

    Hou, Y M; Kim, R; Kim, S H

    1988-11-10

    The cDNA of mouse metallothionein, a small metal-binding protein rich in cysteine, has been cloned downstream from a bacterial inducible promoter and expressed in Escherichia coli. Upon induction, E. coli harboring this cDNA clone contained a protein species readily labelled by [35S]cysteine in vivo and incorporated 10-times as much 109Cd from the medium than would otherwise be the case. We show that expression of metallothionein endows resistance in E. coli to heavy metal ions such as mercury, silver, copper, cadmium and zinc by sequestering rather than exclusion or conversion, common mechanisms of metal resistance in bacteria.

  14. Design and Construction of ctxB-gfp-stxB Gene Cassette and Investigation of Its Expression in E. coli Bl21 (DE3

    Directory of Open Access Journals (Sweden)

    Aghil Esmaeili

    2013-06-01

    Full Text Available Background & Objective: In order to enhance the expression of soluble proteins and facilitate their purification and development of multi-functional polypeptide , chimerical recombinant proteins have been invented . The purpose of this study was to construct ctxB-gfp-stxB gene cassette to measure the uptake and excretion of chimerical antigen in future studies.   Materials & Methods: After preparation of primers for gfp gene as a reporter gene , ctxB and stxB, attempts were made to amplify the genes via the PCR techniques . The amplified genes were clone d in the pGEM vector; and after confirmation of the gene fragments, they were fused as ctxB-gfp-stxB. The gene cassette was thereafter sub-cloned in the pET28a(+ expression vector. E. coli Bl21 (DE3 was transformed by the recombinant vector pET28a(+, and the expression of the recombinant protein was investigated by IPTG induction and SDS-PAGEelectrophoresis.   Results: The amplified genes were cloned in the pGEM vector, and were confirmed via PCR, restriction enzymes, and sequence analyzing system. The confirmed gene fragments were mixed together as ctxB-gfp-stxB . The existence of the gene cassette was confirmed after sub-cloning. The expression was not observed for this gene cassette in E . coli.   Conclusion: The presence of a large number of E. coli rare codons in ctxB and stxB gene sequences precluded the expression of the gene cassette in E. coli; it, therefore, requires the discovery of a suitable host cell for its expression and optimization. Given the gene cassette structure and position of restriction enzymes on the constructed fragment, this gene can be replaced with different genes and can produce a variety of gene fragments.

  15. Pyranose 2-oxidase from Phanerochaete chrysosporium : expression in E. coli and biochemical characterization

    Science.gov (United States)

    Ines Pisanelli; Magdalena Kujawa; Oliver Spadiut; Roman Kittl; Petr Halada; Jindrich Volc; Michael D. Mozuch; Philip Kersten; Dietmar Haltrich; Clemens Peterbauer

    2009-01-01

    The presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in...

  16. Expression, purification, and characterization of the protein repair l-isoaspartyl methyltransferase from Arabidopsis thaliana.

    Science.gov (United States)

    Thapar, N; Clarke, S

    2000-11-01

    Protein l-isoaspartate (d-aspartate) O-methyltransferase (EC 2.1.1. 77) is a repair enzyme that methylates abnormal l-isoaspartate residues in proteins which arise spontaneously as a result of aging. This enzyme initiates their conversion back into the normal l-aspartate form by a methyl esterification reaction. Previously, partial cDNAs of this enzyme were isolated from the higher plant Arabidopsis thaliana. In this study, we report the cloning and expression of a full-length cDNA of l-isoaspartyl methyltransferase from A. thaliana into Escherichia coli under the P(BAD) promoter, which offers a high level of expression under a tight regulatory control. The enzyme is found largely in the soluble fraction. We purified this recombinant enzyme to homogeneity using a series of steps involving DEAE-cellulose, gel filtration, and hydrophobic interaction chromatographies. The homogeneous enzyme was found to have maximum activity at 45 degrees C and a pH optimum from 7 to 8. The enzyme was found to have a wide range of affinities for l-isoaspartate-containing peptides and displayed relatively poor reactivity toward protein substrates. The best methyl-accepting substrates were KASA-l-isoAsp-LAKY (K(m) = 80 microM) and VYP-l-isoAsp-HA (K(m) = 310 microM). We also expressed the full-length form and a truncated version of this enzyme (lacking the N-terminal 26 amino acid residues) in E. coli under the T7 promoter. Both the full-length and the truncated forms were active, though overexpression of the truncated enzyme led to a complete loss of activity. Copyright 2000 Academic Press.

  17. Purification, crystallization and preliminary X-ray analysis of the HsdR subunit of the EcoR124I endonuclease from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Lapkouski, Mikalai [Institute of Physical Biology, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady (Czech Republic); Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady (Czech Republic); Panjikar, Santosh [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany); Kuta Smatanova, Ivana; Csefalvay, Eva, E-mail: jindrova@greentech.cz [Institute of Physical Biology, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady (Czech Republic); Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady (Czech Republic)

    2007-07-01

    The purification, crystallization and preliminary diffraction analysis of the HsdR subunit of the EcoR124I endonuclease are described. EcoR124I is a multicomplex enzyme belonging to the type I restriction-modification system from Escherichia coli. Although EcoR124I has been extensively characterized biochemically, there is no direct structural information available about particular subunits. HsdR is a motor subunit that is responsible for ATP hydrolysis, DNA translocation and cleavage of the DNA substrate recognized by the complex. Recombinant HsdR subunit was crystallized using the sitting-drop vapour-diffusion method. Crystals belong to the primitive monoclinic space group, with unit-cell parameters a = 85.75, b = 124.71, c = 128.37 Å, β = 108.14°. Native data were collected to 2.6 Å resolution at the X12 beamline of EMBL Hamburg.

  18. Expression and purification of two Family GH31 α-glucosidases from Bacteroides thetaiotaomicron.

    Science.gov (United States)

    Chaudet, Marcia M; Allen, Jenny-Lyn; Rose, David R

    2012-12-01

    Microorganisms in the human gut outnumber human cells by a factor of 10. These microbes have been shown to have relevance to the human immune, nutrition and metabolic systems. A dominant symbiont of this environment is Bacteroides thetaiotaomicron which is characterized as being involved in degrading non-digestible plant polysaccharides. This organism's genome is highly enriched in genes predicted to be involved in the hydrolysis of various glycans. Presented here is a comparative functional analysis of two α-glucosidases (designated BT_0339 and BT_3299), Family 31 Glycoside Hydrolases from B. thetaiotaomicron. The purpose of this research is to explore the contributions these enzymes may have to human nutrition and specifically starch digestion. Expression of both α-glucosidases in pET-29a expression vector resulted in high levels of expressed protein in the soluble fraction. Two-step purification allowed for the isolation of the enzymes of interest in significant yield and fractions were observed to be homogenous. Both enzymes demonstrated activity on maltose, isomaltose and malto-oligosaccharide substrates and low level of activity on lactose and sucrose. Enzymatic kinetics revealed these enzymes both preferentially cleave the α1-6 linkage in comparison to the expected α1-4 and specifically favor maltose-derived substrates of longer length. The flexible hydrolytic capabilities of BT_0339 and BT_3299 reveal the ability of this bacterium to maintain its dominant position in its environment by utilizing an array of substrates. Specifically, these enzymes demonstrate an important aspect of this organism's contribution to starch digestion in the distal gut and the overall energy intake of humans. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

    Science.gov (United States)

    Maggi, Maristella; Scotti, Claudia

    2017-08-01

    Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae.

    Science.gov (United States)

    Verba, Kliment A; Agard, David A

    2017-10-05

    Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of Hsp90-Cdc37-Cdk4 kinase protein complex from Saccharomyces cerevisiae utilizing the viral 2A sequences to titrate the three proteins at similar levels.

  1. Recombinant expression, purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae.

    Science.gov (United States)

    Hao, Qing; Liu, Xiaoguang; Zhao, Guozhong; Jiang, Lu; Li, Ming; Zeng, Bin

    2016-03-01

    To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae. A full-length cDNA encoding a candidate ACBP from A. oryzae (AoACBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoACBP protein was purified by an amylose resin chromatography column. SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa. Microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for palmitoyl-CoA (K d = 80 nM) than for myristoyl-CoA (K d = 510 nM), thus demonstrating the preference of AoACBP for long-chain acyl-CoA. The data support the identification of AoACBP as a long-chain ACBP in A. oryzae.

  2. Recombinant expression and purification of an Oxysterol Binding Protein from Aspergillus oryzae 3.042

    Directory of Open Access Journals (Sweden)

    Zhang Xian

    2017-01-01

    Full Text Available A full-length cDNA encoding a candidate Oxysterol-binding protein(OSBP from Aspergillus oryzae (AoOSBP was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP fusion protein. The MBP-AoOSBP protein from the importantly industrial fungus A. oryzae was purified by amylose resin and chromatography column. SDS-PAGE showed that MBP-AoOSBP has an estimated molecular weight of 182 kDa. OSBP and its homologues (ORPs own the affinity for oxysterols, cholesterol and glycerophospholipids. According to the superiority of A. oryzae in the fermented foods and also in food-grade productions pharmaceutical enzyme manufacture, it is meaningful to identify the biochemical properties of OSBP in A. oryzae.

  3. Expression, Purification, Crystallization And Preliminary X-Ray Studies of Histamine Dehydrogenase From Nocardioides Simplex

    Energy Technology Data Exchange (ETDEWEB)

    Reed, T.M.; Hirakawa, H.; Mure, M.; Scott, E.E.; Limburg, J.

    2009-05-21

    Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 {angstrom} resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 {angstrom}.

  4. Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase from Corynebacterium glutamicum

    Energy Technology Data Exchange (ETDEWEB)

    Schoepe, Jan, E-mail: jschoepe@smail.uni-koeln.de; Niefind, Karsten; Chatterjee, Shivani; Schomburg, Dietmar [Institute for Biochemistry, University of Köln, Zülpicher Strasse 47, Köln, NRW 50974 (Germany)

    2006-07-01

    The crystallization and preliminary X-ray characterization of a shikimate dehydrogenase from C. glutamicum is presented. The shikimate dehydrogenase from Corynebacterium glutamicum has been cloned into an Escherichia coli expression vector, overexpressed and purified. Native crystals were obtained by the vapour-diffusion technique using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the centred monoclinic space group C2, with unit-cell parameters a = 118.77, b = 63.17, c = 35.67 Å, β = 92.26° (at 100 K), and diffract to 1.64 Å on a synchrotron X-ray source. The asymmetric unit is likely to contain one molecule, corresponding to a packing density of 2.08 Å{sup 3} Da{sup −1} and a solvent content of about 41%.

  5. Expression and Purification of the Individual Bam Components BamB-E.

    Science.gov (United States)

    Aulakh, Suraaj; Kim, Kelly H; Paetzel, Mark

    2015-01-01

    BamB, BamC, BamD, and BamE are lipoproteins that, along with the integral membrane protein BamA, form the β-barrel assembly machinery (BAM) complex in the outer-membrane of Gram-negative bacteria. Elucidating the roles that these lipoproteins play in the β-barrel assembly process requires both structural and functional studies that rely on milligram quantities of pure protein. Here, we describe a simple protocol for expressing individual BamB-BamE proteins in Escherichia coli and purifying them by nickel affinity and size-exclusion chromatography. This protocol yields pure proteins in amounts that are sufficient for crystallization trials, in vitro protein-protein interaction studies, NMR, and other biochemical experiments.

  6. Effective ethanol production from whey powder through immobilized E. coli expressing Vitreoscilla hemoglobin.

    Science.gov (United States)

    Sar, Taner; Stark, Benjamin C; Yesilcimen Akbas, Meltem

    2017-03-04

    Ethanol production from whey powder was investigated by using free as well as alginate immobilized E. coli and E. coli expressing Vitreoscilla hemoglobin (VHb) in both shake flask and fermenter cultures. Media with varying levels of whey (lactose contents of 3%, 5%, 8% or 15%) and yeast extract (0.3% or 0.5%) were evaluated with fermentation times of 48-96 h. Immobilization and VHb expression resulted in higher ethanol production with all media; the increases ranged from 2% to 89% for immobilization and from 2% to 182% for VHb expression. It was determined that growth medium containing 8% lactose with 0.5% yeast extract yielded the highest ethanol production for free or immobilized strains, with or without VHb expression, in both shake flask and fermenter cultures. Immobilization with alginate was found to be a promising process for ethanol production by VHb-expressing ethanologenic E. coli.

  7. Cloning, expression, purification and preliminary crystallographic analysis of the short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica

    Energy Technology Data Exchange (ETDEWEB)

    Harmer, Nicholas J., E-mail: nic@cryst.bioc.cam.ac.uk [Department of Biochemistry, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); King, Jerry D. [Department of Biochemistry, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Department of Veterinary Medicine, Cambridge CB3 0ES (United Kingdom); Palmer, Colin M. [Department of Biochemistry, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Preston, Andrew [Department of Veterinary Medicine, Cambridge CB3 0ES (United Kingdom); Department of Molecular and Cellular Biology, University of Guelph, Ontario N1G 2W1 (Canada); Maskell, Duncan J. [Department of Veterinary Medicine, Cambridge CB3 0ES (United Kingdom); Blundell, Tom L. [Department of Biochemistry, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom)

    2007-08-01

    The expression, purification, and crystallisation of the short-chain dehydrogenases WbmF, WbmG and WbmH from B. bronchiseptica are described. Native diffraction data to 1.5, 2.0, and 2.2 Å were obtained for the three proteins, together with complexes with nucleotides. The short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica were cloned into Escherichia coli expression vectors, overexpressed and purified to homogeneity. Crystals of all three wild-type enzymes were obtained using vapour-diffusion crystallization with high-molecular-weight PEGs as a primary precipitant at alkaline pH. Some of the crystallization conditions permitted the soaking of crystals with cofactors and nucleotides or nucleotide sugars, which are possible substrate compounds, and further conditions provided co-complexes of two of the proteins with these compounds. The crystals diffracted to resolutions of between 1.50 and 2.40 Å at synchrotron X-ray sources. The synchrotron data obtained were sufficient to determine eight structures of the three enzymes in complex with a variety of cofactors and substrate molecules.

  8. Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli.

    Science.gov (United States)

    Torres, Leticia L; Ferreras, Eloy R; Cantero, Angel; Hidalgo, Aurelio; Berenguer, José

    2012-08-09

    Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 °C. The half-life of

  9. Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Torres Leticia L

    2012-08-01

    Full Text Available Abstract Background Penicillin acylases (PACs are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. Results Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1. The optimum pH was aprox. 4 and the optimum

  10. Identification, cloning and heterologous expression of active [NiFe]-hydrogenase 2 from Citrobacter sp. SG in Escherichia coli.

    Science.gov (United States)

    Maier, Johannes A H; Ragozin, Sergey; Jeltsch, Albert

    2015-04-10

    Hydrogen (H2) is a potential alternative energy carrier which only produces water and heat upon combustion. Today, industrial hydrogen production mainly uses thermochemical processes based on fossil fuels or electrolysis of water. Therefore, biotechnological approaches to produce H2 from biomass are an interesting alternative. We introduce here a novel direct hydrogen measurement system using a semiconducting device specific for hydrogen detection. Using this device, a bacterium producing considerable amounts of hydrogen under aerobic cultivation was isolated and identified by 16S ribosomal DNA sequencing as Citrobacter sp. The enzyme responsible for the observed hydrogenase activity was partially purified by 3 chromatographic purification steps and could be identified by peptide mass fingerprinting to be a type 2 [NiFe]-hydrogenase. Expression of the [NiFe]-hydrogenase 2 containing operon from Citrobacter sp. SG in Escherichia coli allowed recombinant hydrogen production. The [NiFe]-hydrogenase 2 identified here may be useful for biotechnological hydrogen production. We speculate that the expression of the hydrogenase in Citrobacter may be an adaptation to growth in acidic conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Expression, purification, and functional characterisation of Flagellin, a TLR5-ligand

    Directory of Open Access Journals (Sweden)

    Irshad Ahmed Hajam

    2013-06-01

    Full Text Available Flagellin, a Toll-like receptor 5 (TLR5-ligand, is known for its activities like adjuvant, induction of pro-inflammatory cytokines and innate immunity. In this context, fliC gene of Salmonella Typhimurium was cloned into pET32a expression plasmid using in-house designed gene specific primers. The frame and orientation of the inserted fliC gene was confirmed upon colony PCR, restriction enzyme analysis and sequencing. Sequence analysis of fliC revealed proper orientation of the gene and had 1,485 nucleotides. Following transformation of pET-fliC plasmid into Escherichia coli BL21 (DE3 cells, the gene was expressed after inducing with IPTG (Isopropylβ-D-1-thiogalactopyranoside. The polyHis-tag-fliC was ~70kDa as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE. The identity/authenticity of the recombinant-fliC was confirmed by its specific reactivity with commercial anti-fliC MAb of S. Typhimurium. Further, the antigenic and functional properties of recombinant-fliC were determined espousing its ability to induce antigen specific antibodies in G pigs and increased m-RNA expression of certain pro-inflammatory mediators like TNF-α and GM-CSF in vitro.

  12. [hHO-1 structure prediction and its mutant construct, expression, purification and activity analysis].

    Science.gov (United States)

    Xia, Zhen Wei; Cui, Wen Jun; Zhou, Wen Pu; Zhang, Xue Hong; Shen, Qing Xiang; Li, Yun Zhu; Yu, Shan Chang

    2004-10-01

    Human Heme Oxygenase-1 (hHO-1) is the rate-limiting enzyme in the catabolism reaction of heme, which directly regulates the concentration of bilirubin in human body. The mutant structure was simulated by Swiss-pdbviewer procedure, which showed that the structure of active pocket was changed distinctly after Ala25 substituted for His25 in active domain, but the mutated enzyme still binded with heme. On the basis of the results, the expression vectors, pBHO-1 and pBHO-1(M), were constructed, induced by IPTG and expressed in E. coli DH5alpha strain. The expression products were purified with 30%-60% saturation (NH4)2SO4 and Q-Sepharose Fast Flow column chromatography. The concentration of hHO-1 in 30%-60% saturation (NH4)2SO4 components and in fractions through twice column chromatography was 3.6-fold and 30-fold higher than that in initial product, respectively. The activity of wild hHO-1 (whHO-1) and mutant hHO-1 (deltahHO-1) showed that the activity of deltahHO-1 was reduced 91.21% compared with that of whHO-1. The study shows that His25 is of importance for the mechanism of hHO-1, and provides the possibility for effectively regulating the activity to exert biological function.

  13. Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens.

    Science.gov (United States)

    Mahmoudi, Shima; Mamishi, Setareh; Ghazi, Mona; Hosseinpour Sadeghi, Reihaneh; Pourakbari, Babak

    2013-12-01

    ESAT-6 (6-kDaearly secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) have been described as dominant antigens recognized by T-cells and considered as virulence factors in Mycobacterium tuberculosis. The aim of this study was to clone, express and purify recombinant ESAT-6 andCFP-10 proteins of M. tuberculosis in soluble form. ESAT-6 andCFP-10 genes were amplified by PCR, cloned into pET32a (+) vector, and overexpress-ed using isopropyl-beta-D-thiogalactopyranoside in E. coli BL21 (DE3). ESAT-6 andCFP-10 proteins were purified by Ni-NTA affinity chromatography and were detected by anti- ESAT-6 and anti -CFP-10 antibodies. ESAT-6 andCFP-10 genes were successfully expressed and purified. Anti- ESAT-6 and anti-CFP-10 antibodies were produced after induction of immunization against purified ESAT-6 andCFP-10 proteins in rabbit. In this study, we cloned, expressed and purified sufficient amounts of ESAT-6 andCFP-10 and it would be tested for the development of diagnostic kit for M. tuberculosis in future.

  14. Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Kalimuthu Karuppanan

    2017-01-01

    Full Text Available Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2 protein fused via a linker to the fragment crystallizable (Fc domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50% N-glycan structure was GlcNAc2(XylMan3(FucGlcNAc2 in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting.

  15. Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins.

    Science.gov (United States)

    Yeh, Hung-Yueh; Hiett, Kelli L; Line, John E; Oakley, Brian B; Seal, Bruce S

    2013-05-06

    Campylobacter jejuni, a flagellated, spiral-rod Gram-negative bacterium, is the leading etiologic agent of human acute bacterial gastroenteritis worldwide. The source of this microorganism for human infection has been implicated as consumption and handling of poultry meat where this microorganism is a commensal in the gut. Because the genomes of many C. jejuni isolates have been sequenced, our ultimate goal is to develop protein arrays for exploring this microorganism and host interactions. In this communication, we report cloning, expression and purification of C. jejuni flagellar proteins in a bacterial expression system. Twelve recombinant proteins were purified, which were confirmed by SDS-PAGE analysis and a His tag detection kit. The FlgE1, FlgG, FlgK, FliE, FlgH/FliH and FlaA recombinant proteins were further confirmed by LC-ESI-MS/MS. The purified recombinant proteins were tested whether they were immunogenic using antibodies from several sources. BacTrace anti-Campylobacter species antibody reacted to the FlaA recombinant protein, but not others. Rabbit anti-MOMP1 peptide antibody reacted strongly to FliE and weakly to FlaA, but not others. Rabbit anti-MOMP2 peptide antibody reacted strongly to the FlaA, FliG, FliE, FlhF, FlgG, FlgE1 and FliD recombinant proteins, less to FlgK and FlgH/FliH, and did not react to the FliY, FliS and FliH recombinant proteins. These antibody studies suggest that these recombinant flagellar proteins have potential for novel targets for vaccine development. It is also anticipated that these recombinant proteins provide us a very useful tool for investigating host immune response to C. jejuni. Published by Elsevier GmbH.

  16. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E. (UIC)

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  17. The TGV transgenic vectors for single-copy gene expression from the Escherichia coli chromosome.

    Science.gov (United States)

    Gumbiner-Russo, L M; Lombardo, M J; Ponder, R G; Rosenberg, S M

    2001-07-25

    Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inability to achieve complete repression of gene expression; non-physiological overexpression of the cloned gene; titration of regulatory proteins; and the requirement for antibiotic selection. We describe a simple system for cloning and expression of genes in single copy in the E. coli chromosome, using a non-antibiotic selection for transgene insertion. The transgene is inserted into a vector containing homology to the chromosomal region flanking the attachment site for phage lambda. This vector is then linearized and introduced into a recombination-proficient E. coli strain carrying a temperature-sensitive lambda prophage. Selection for replacement of the prophage with the transgene is performed at high temperature. Once in the chromosome, transgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident prophage. Additional vector constructs provide an arabinose-inducible promoter (P(BAD)), P(BAD) plus a translation-initiation sequence, and optional chloramphenicol-, tetracycline-, or kanamycin-resistance cassettes. These Transgenic E. coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function.

  18. Identification and validation of novel chromosomal integration and expression loci in Escherichia coli flagellar region 1.

    Directory of Open Access Journals (Sweden)

    Mario Juhas

    Full Text Available Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci.

  19. Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Nagendra Suryanarayana

    2016-01-01

    Full Text Available Bacillus anthracis secretory protein protective antigen (PA is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1 compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein’s functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform.

  20. Inactivation and Gene Expression of a Virulent WastewaterEscherichia coliStrain and the Nonvirulent CommensalEscherichia coliDSM1103 Strain upon Solar Irradiation

    KAUST Repository

    Aljassim, Nada I.

    2017-03-06

    This study examined the decay kinetics and molecular responses of two Escherichia coli strains upon solar irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E. coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs 1.33 ± 0.52 h and 2.04 ± 0.36 min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar irradiation remained effective in reducing the numbers of both strains by more than 5-log10 in <24 h, comparative genomics and transcriptomics revealed differences in the genomes and overall regulation of genes between the two E. coli strains. A wider arsenal of genes related to oxidative stress, cellular repair and protective mechanisms were upregulated in E. coli PI-7. Subpopulations of E. coli PI-7 expressed genes related to dormancy and persister cell formation during the late decay phase, which may have accounted for its prolonged persistence. Upon prolonged solar irradiation, both E. coli strains displayed upregulation of genes related to horizontal gene transfer and antibiotic resistance. Virulence functions unique to E. coli PI-7 were also upregulated. Our findings collectively indicated that, whereas solar irradiation is able to reduce total cell numbers, viable E. coli remained and expressed genes that enable survival despite solar treatment. There remains a need for heightened levels of concern regarding risks arising from the dissemination of E. coli that may remain viable in wastewater after solar irradiation.

  1. Molecular cloning, expression and purification of lactoferrin from Tibetan sheep mammary gland using a yeast expression system.

    Science.gov (United States)

    Li, Jianbo; Zhu, Wuzheng; Luo, Meirong; Ren, Honghui; Tang, Lu; Liao, Honghai; Wang, Yong

    2015-05-01

    This paper reports the successful expression of a lactoferrin gene-obtained from the mammary gland tissue of Tibetan sheep-in the yeast Pichia pastoris GS115 using pPICZαA as the recombinant plasmid and α-factor signal sequence for secretion. The recombinant lactoferrin was purified by ammonium sulfate precipitation, ion-exchange column chromatography and gel-filtration chromatography, and it had a molecular mass of 76kDa. We obtained an expression yield of >60mgL(-1) and specific activity of 2533.33Umg(-1). The antimicrobial activities and iron-binding behaviors of recombinant lactoferrin indicated that it was correctly folded and functional. Additionally, recombinant lactoferrin inhibited the growth of Escherichia coli JM109 and Staphylococcus aureus. These findings indicate that recombinant lactoferrin is a potential antibiotic for use on humans. This study also demonstrates the successful expression of recombinant lactoferrin using the eukaryotic host organism P. pastoris, paving the way for protein engineering using this gene. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Recombinant protein expression and solubility screening in Escherichia coli: a comparative study

    NARCIS (Netherlands)

    Berrow, N.S.; Folkers, G.E.

    2006-01-01

    Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. Avariety of expression-screening

  3. [The expression, purification and characterization of human His-ATG4B protein].

    Science.gov (United States)

    Huang, Rong; Xu, Xiaojie; Liang, Yingchun; Zhang, Li; Wang, Tao; Zhou, Liying; DU, Nan; Ye, Qinong

    2015-02-01

    To construct the prokaryotic expression vector of human autophagy-related protein 4 homolog B (ATG4B) labeled with His-tag, obtain the purified His-ATG4B protein, and identify its activity preliminarily. ATG4B coding region was amplified from human mammary gland cDNA library by PCR, and was cloned into the prokaryotic expression vector pET-28a⁺. After verification by enzyme digestion, the correct recombinant plasmid His-ATG4B was introduced into E.coli Rossate. The expressed recombinant plasmid was purified by Ni-NTA beads and identified by SDS-PAGE and Western blot analysis. The function of the purified protein His-ATG4B was detected by GST pull-down assay. The DNA fragment of about 1100 bp was successfully amplified from human mammary gland cDNA by PCR, and inserted into pET-28a vector correctly. The results of double digestion and sequencing suggested that the His-ATG4B recombinant plasmid was successfully obtained. His-ATG4B protein of about Mr 47 000 was induced and identified by SDS-PAGE analysis. GST pull-down assay showed that His-ATG4B could interact with LC3B in vitro, suggesting that it has a good biological function. The prokaryotic expression protein of His-ATG4B has been obtained successfully, which lays a foundation for further research on the function of ATG4B in autophagy.

  4. Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

    Directory of Open Access Journals (Sweden)

    Ren Wang

    2008-06-01

    Full Text Available Lipoxygenase (LOX, EC1.13.11.12 is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a(+ and transformed into Escherichia coli BL21 (DE3. Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside (IPTG and the purified recombinant protein was obtained by His·Bind® Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 (acetate buffer and the optimum temperature was 30°C for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.

  5. Expression, purification and crystallization of the SARS-CoV macro domain

    Energy Technology Data Exchange (ETDEWEB)

    Malet, Hélène; Dalle, Karen; Brémond, Nicolas; Tocque, Fabienne; Blangy, Stéphanie; Campanacci, Valérie; Coutard, Bruno; Grisel, Sacha; Lichière, Julie; Lantez, Violaine; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre, E-mail: marie-pierre.egloff@afmb.univ-mrs.fr [Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, UMR 6098-Case 932, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France)

    2006-04-01

    The SARS-CoV macro domain was expressed, purified and crystallized. Selenomethionine-labelled crystals diffracted to 1.8 Å resolution. Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1′′-phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macro domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182–355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 37.5, b = 55.6, c = 108.9 Å, β = 91.4°, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8 Å.

  6. [Integration and expression of sdh gene in Escherichia coli].

    Science.gov (United States)

    Gao, Shu-ying; Zhang, Wei-cai; Wang, Jian-hua; Guo, Ai-guang

    2005-02-01

    The chloramphenicol-resistant cassette with short shared sequences of ptsG gene on both ends was PCR-generated from plasmid pKF3 and ligated to pMD18-T to construct pMD18-PC. The sdh gene for sorbose dehydrogenase was generated from plasmid pQE60-SDH and inserted into pMD18-PC, then pMD18-PC-SDH was constructed. It was digested with Pvu II and the target fragment ptsG1-cat-sdh-ptsG2 was recovered and electroporated into Escherichia coli JM109/pKD46. Homologous-recombination between linear DNA cassettes and Escherichia coli chromosomes took place by Red recombination. The detection result showed that the integron JM109s was of sorbose dehydrogenase activity. The PCR products assay using the upstream and downstream sequences of ptsG gene as primers and JM109s genomic DNA as template, indicated that sdh gene had been integrated at the ptsG gene site in Escherichia coli.

  7. Heterologous expression of Homo sapiens alpha-folate receptors in E. coli by fusion with a trigger factor for enhanced solubilization.

    Science.gov (United States)

    Miranda, Beatriz Nogueira Messias; Fotoran, Wesley Luzetti; Canduri, Fernanda; Souza, Dulce Helena Ferreira; Wunderlich, Gerhard; Carrilho, Emanuel

    2018-02-01

    The role of Alpha folate receptors (FRα) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FRα complexed with different ligands were described relying on an expensive and time-consuming production process. Here, we constructed an efficient system for the expression and purification of a human FRα in E. coli. Unlike a conventional expression method we used a specific protein fusion expressing the target protein together with a trigger factor (TF). This factor is a chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains. The activity of rTFFRα was comparable to glycosylphosphatidylinositol (GPI) anchored proteins extracted from HeLa tumor cells. Our work demonstrates a straightforward and versatile approach for the production of active human FRα by heterologous expression; this approach further enhances the development of inhibition studies and biotechnological applications. The purified product was then conjugated to liposomes, obtaining a 35% higher signal from densitometry measurement on the immunoblotting assay in the contruct containing the Ni-NTA tag, as a mimesis of an exosome, which is of vital importance to nanotherapeutic techniques associated to treatment and diagnosis of tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis.

    Science.gov (United States)

    Shih, Kuo-Shun; Lin, Chih-Chang; Hung, Hsiao-Fang; Yang, Yu-Chi; Wang, Chung-An; Jeng, Kee-Ching; Fu, Hua-Wen

    2013-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.

  9. One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Kuo-Shun Shih

    Full Text Available Helicobacter pylori neutrophil-activating protein (HP-NAP, a major virulence factor of Helicobacter pylori (H. pylori, is capable of activating human neutrophils to produce reactive oxygen species (ROS and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis. This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.

  10. One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

    Science.gov (United States)

    Shih, Kuo-Shun; Lin, Chih-Chang; Hung, Hsiao-Fang; Yang, Yu-Chi; Wang, Chung-An; Jeng, Kee-Ching; Fu, Hua-Wen

    2013-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection. PMID:23577158

  11. Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli.

    Science.gov (United States)

    Sequeira, Ana Filipa; Turchetto, Jeremy; Saez, Natalie J; Peysson, Fanny; Ramond, Laurie; Duhoo, Yoan; Blémont, Marilyne; Fernandes, Vânia O; Gama, Luís T; Ferreira, Luís M A; Guerreiro, Catarina I P I; Gilles, Nicolas; Darbon, Hervé; Fontes, Carlos M G A; Vincentelli, Renaud

    2017-01-17

    Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli. The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal. This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.

  12. Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli

    OpenAIRE

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Jafari, Anis; Irani, Shiva

    2015-01-01

    Background: Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. Protein D (PD) belongs to the minor outer-membrane proteins of H. influenza. Moreover, it has been shown that this protein is one of the most potent vaccine candidates against the NTHi strain. Objectives: In the present study, a new truncated form of PD was designed based on conserved areas, and recombina...

  13. Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli

    DEFF Research Database (Denmark)

    Bommarius, B.; Jenssen, Håvard; Elliott, M.

    2010-01-01

    Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibioticresistant bacteria, viruses and even parasites, but there are substantial roadblocks to the...... in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans....... to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial...

  14. Expression, Purification, and Analysis of Unknown Translation Factors from "Escherichia Coli": A Synthesis Approach

    Science.gov (United States)

    Walter, Justin D.; Littlefield, Peter; Delbecq, Scott; Prody, Gerry; Spiegel, P. Clint

    2010-01-01

    New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described…

  15. Cd(II) and As(III) bioaccumulation by recombinant Escherichia coli expressing oligomeric human metallothioneins.

    Science.gov (United States)

    Ma, Yao; Lin, Jianqun; Zhang, Chengjia; Ren, Yilin; Lin, Jianqiang

    2011-01-30

    Metallothioneins (MTs) are a family of metal binding proteins. Recombinant Escherichia coli expressing the human MT (hMT-1A) gene was constructed for bioaccumulation of heavy metals. In order to increase protein stability, the glutathione S-transferase (GST) gene was fused with the hMT-1A gene and coexpressed. In order to increase MT expression efficiency and metal binding capacity, two, three or four hMT-1A genes were integrated in series and overexpressed in E. coli. The recombinant E. coli expressing the GST fused trimeric hMT-1A protein exhibited the highest Cd(II) and As(III) bioaccumulation ability, 6.36 mg Cd/g dry cells and 7.59 mg As/g dry cells, respectively. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. Relation between tetR and tetA expression in tetracycline resistant Escherichia coli

    DEFF Research Database (Denmark)

    Møller, Thea S. B.; Overgaard, Martin; Nielsen, Søren S.

    2016-01-01

    Background: Tetracyclines are among the most used antibiotics in livestock worldwide. Resistance is widely disseminated in Escherichia coli, where it is generally mediated by tetracycline efflux pumps, such as TetA. Expression of tetracycline efflux pumps is tightly controlled by the repressor Tet......R, which has been shown to be tetracycline-responsive at sub-MIC tetracycline concentrations. The objective of this study was to investigate the effects of increasing tetracycline concentrations on the growth of TetA-producing E. coli, and to determine how expression of tetA and tetR related to each other...... in different growth phases in the presence of tetracycline. Results: A tetracycline resistant E. coli strain containing tetA and tetR on the chromosome was constructed and cultured in the presence of increasing concentrations of tetracycline. Expression of tetR and tetA was measured at four time points...

  17. Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli.

    Science.gov (United States)

    De Luca, G; Asso, M; Belaich, J P; Dermoun, Z

    1998-02-24

    Based on the DNA sequence of its structural genes, clustered in the hnd operon, the NADP-reducing hydrogenase of Desulfovibrio fructosovorans is thought to be a heterotetrameric complex in which HndA and HndC constitute the NADP-reducing unit and HndD constitutes the hydrogenase unit, respectively. The weak representativity of the enzyme among cell proteins has prevented its purification. This paper discusses the purification and characterization of the HndA subunit of this unique tetrameric iron hydrogenase overproduced in Escherichia coli. The purified subunit contains 1.7 mol of non-heme iron and 1.7 mol of acid-labile sulfide/mol. EPR analysis of the reduced form of HndA indicates that it contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with values of gx, gy, and gz equal to 1.915, 1.950, and 2. 000, respectively, and a midpoint redox potential of -395 mV. The UV-visible and EPR spectra of the [2Fe-2S] cluster indicate that HndA belongs to the [2Fe-2S] family typified by the Clostridium pasteurianum [2Fe-2S] ferredoxin. The C-terminal sequence of HndA shows 27% identity with the C-terminal sequence of the 25-kDa subunit of NADH: quinone oxidoreductase from Paracoccus denitrificans, 33% identity with the C-terminal sequence of the 24-kDa subunit from Bos taurus complex I, and 32% identity with the entire sequence of C. pasteurianum [2Fe-2S] ferredoxin. The four cysteine residues involved in HndA cluster binding have been tentatively identified on the basis of sequence identity considerations. Evidence of a HndA organization based on two independent structural domains is discussed.

  18. Switchable gene expression in Escherichia coli using a miniaturized photobioreactor

    National Research Council Canada - National Science Library

    Lee, Jae Myung; Lee, Junhyeong; Kim, Taesung; Lee, Sung Kuk

    2013-01-01

    .... This system also ensures homogenous expression across the entire cell population. We also report the design of a miniaturized photobioreactor to be used in combination with the light-switchable gene expression system...

  19. Expression, purification and low-resolution structure of human vitamin C transporter SVCT1 (SLC23A1.

    Directory of Open Access Journals (Sweden)

    Rajendra Boggavarapu

    Full Text Available Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1 in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.

  20. High-level expression, purification and production of the fungal immunomodulatory protein-gts in baculovirus-infected insect larva.

    Science.gov (United States)

    Wu, Tzong-Yuan; Chen, Hsin-An; Li, Feng-Yin; Lin, Ching-Ting; Wu, Chi-Ming; Hsieh, Feng-Chia; Tzen, Jason Tze-Cheng; Hsieh, Sheng-Kuo; Ko, Jiunn-Liang; Jinn, Tzyy-Rong

    2013-02-01

    Fip-gts, a fungal immunomodulatory protein (Fip) isolated from Ganoderma tsugae (gts), has been reported to possess therapeutic effects in the treatment of cancer and autoimmune disease. To cost-effectively produce Fip-gts and bypass the bottleneck involved in its time-consuming purification from G. tsugae, in this study, we incorporated the SP(bbx) secretion signal into recombinant baculovirus for expressing glycosylated and bioactive rFip-gts in baculovirus-infected insect cells and Trichoplusia ni larva. This is the first study to employ the aerosol infecting T. ni larva with recombinant baculovirus for economical and high-level production of foreign proteins. In this study, one purification could yield 10 mg of rFip-gts protein merely from ∼100 infected T. ni larvae by aerosol inoculation, corresponding to 5 L (5 × 10⁹ cells) of the infected Sf21 culture. In addition, the rFip-gts purified from T. ni larvae could induce the expression of interleukin-2 in murine splenocytes with an immunoresponsive level similar to that induced by LZ-8 (a known potent immunomodulatory protein purified from Ling zhi, Ganoderma lucidum). Thus, our results demonstrated that the larva-based baculovirus expression system can successfully express rFip-gts with the assembling capability required for maintaining immunomodulatory and anticancer activity. Our approach will open a new avenue for the production of rFip-gts and facilitate the immunoregulatory activity of rFip-gts available in the future.

  1. Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression

    DEFF Research Database (Denmark)

    Beloin, C.; Valle, J.; Latour-Lambert, P.

    2004-01-01

    The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K-12 biofilm. We show that, when biofilm is compared...... that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological...

  2. Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate

    Energy Technology Data Exchange (ETDEWEB)

    Inaoka, Daniel Ken; Takashima, Eizo; Osanai, Arihiro; Shimizu, Hironari [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Nara, Takeshi; Aoki, Takashi [Department of Parasitology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Harada, Shigeharu [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Kita, Kiyoshi, E-mail: kitak@m.u-tokyo.ac.jp [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2005-10-01

    The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate. Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R{sub merge} of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V{sub M}) of 2.2 Å{sup 3} Da{sup −1} and a solvent content of 44%.

  3. Expression, purification and functional characterization of a novel 3α-hydroxysteroid dehydrogenase from Pseudomonas aeruginosa.

    Science.gov (United States)

    Chen, Jianmin; Gao, Xiufeng; Hong, Lin; Ma, Liting; Li, Yongsheng

    2015-11-01

    3α-Hydroxysteroid dehydrogenase (3α-HSD) catalyzes the oxidation of the 3-hydroxyl group of steroids. The enzymatic conversion is a critical step in the enzymatic assay of urinary sulfated bile acids (SBAs), which is a valuable diagnosis index of hepatobiliary diseases. However, the source of 3α-HSD for clinical applications is limited. In this study, an open reading frame (ORF) encoding a novel 3α-HSD was successfully cloned from Pseudomonas aeruginosa and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified by immobilized metal ion affinity chromatography. Enzyme characterization studies revealed that the protein has 3α-HSD activity and the Km value for sodium cholate is 1.06 mmol L(-1). More than 60% relative enzyme activity was observed in a wide range of pH and temperature, with an optimum pH at 8.0 and an optimum temperature at 30°C. The enzyme's good thermostability under 40°C would be favorable in clinical applications. Ion interference experiments indicated that Zn(2+) was an activating cofactor which increased the enzyme activity 1.75-fold. With the favorable characteristics mentioned above, the new 3α-HSD is a promising enzyme for clinical applications. More importantly, the present work is the first report on a 3α-HSD from P. aeruginosa. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Expression, purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe.

    Science.gov (United States)

    Hu, Lulu; Gao, Chen-Hui; Hong, Chao; Zhong, Qiao; Dong, Hong-Liang; Gao, Xiao-Ming

    2016-01-01

    Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343-682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.

  5. Production of Protein Kinases in E. coli.

    Science.gov (United States)

    Dodson, Charlotte A

    2017-01-01

    Recombinant protein expression is widely used to generate milligram quantities of protein kinases for crystallographic, enzymatic, or other biophysical assays in vitro. Expression in E. coli is fast, cheap, and reliable. Here I present a detailed protocol for the production of human Aurora-A kinase. I begin with transformation of a suitable plasmid into an expression strain of E. coli, followed by growth and harvesting of bacterial cell cultures. Finally, I describe the purification of Aurora-A to homogeneity using immobilized metal affinity and size exclusion chromatographies.

  6. Comparison of Bacteroides thetaiotaomicron and Escherichia coli 16S rRNA gene expression signals.

    Science.gov (United States)

    Mastropaolo, Matthew D; Thorson, Mary L; Stevens, Ann M

    2009-08-01

    There are barriers to cross-expression of genes between Bacteroides spp. and Escherichia coli. In this study, a lux-based reporter system was developed for Bacteroides and used to compare the promoter structure and function of a Bacteroides thetaiotaomicron 4001 (BT4001) 16S rRNA promoter with those of E. coli in vivo. Analysis of the BT4001 sequences upstream of the 16S rRNA gene revealed the same overall structure known for E. coli 16S rRNA promoters in that there were two promoters separated by approximately 150 bp. However, the BT4001 16S rRNA promoter contains the proposed Bacteroides -7 and -33 consensus sequences instead of the E. coli -10 and -35 consensus sequences. The biological activity of various configurations of the BT4001 16S rRNA promoter was analysed. Experiments pairing the BT4001 16S rRNA promoter with an E. coli RBS, and vice-versa, confirmed that gene expression between the two species is restricted at the level of transcription. In Bacteroides, a difference in translation initiation also appears to limit expression of foreign genes.

  7. Expression and processing of Vibrio anguillarum zinc-metalloprotease in Escherichia coli.

    Science.gov (United States)

    Zhang, Fengli; Chen, Jixiang; Chi, Zhenming; Wu, Long-Fei

    2006-07-01

    The extracellular zinc-metalloprotease of Vibrio anguillarum is a secreted virulence factor. It is synthesized from the empA gene as a 611-residue preproprotease and processed to the active mature protease (EmpA) with concomitant secretion via the type II secretion pathway. Active EmpA has been found only in the V. anguillarum culture supernatant and the process of the activation seems to vary depending on strains analyzed. To better understand the mechanism of EmpA export and processing, the empA gene was cloned and expressed in Escherichia coli strains. Expression of empA did not have toxic effect on bacterial growth. Rupturing E. coli TOP10 cells by heating in gel-loading buffer resulted in activation of EmpA and severe proteolysis of the samples. In contrast, the same treatment of the E. coli MC4100A strain did not lead to the general proteolysis. In this strain, EmpA was exported into the periplasm via the Sec pathway. The periplasmic EmpA was detected in two active conformations. Therefore, in E. coli processing of EmpA precursor to an active enzyme did not require secretion to the media and the help of other V. anguillarum protein. Like in V. anguillarum, heterologous expression of empA in E. coli showed strain-specific activation process.

  8. Expression, purification, and characterization of a carbohydrate-active enzyme: A research-inspired methods optimization experiment for the biochemistry laboratory.

    Science.gov (United States)

    Willbur, Jaime F; Vail, Justin D; Mitchell, Lindsey N; Jakeman, David L; Timmons, Shannon C

    2016-01-01

    The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern techniques and instrumentation commonly found in a research laboratory. Unlike in a traditional cookbook-style experiment, students generate their own hypotheses regarding expression conditions and quantify the amount of protein isolated using their selected variables. Over the course of three 3-hour laboratory periods, students learn to use sterile technique to express a protein using recombinant DNA in E. coli, purify the resulting enzyme via affinity chromatography and dialysis, analyze the success of their purification scheme via SDS-PAGE, assess the activity of the enzyme via an HPLC-based assay, and quantify the amount of protein isolated via a Bradford assay. Following the completion of this experiment, students were asked to evaluate their experience via an optional survey. All students strongly agreed that this laboratory module was more interesting to them than traditional experiments because of its lack of a pre-determined outcome and desired additional opportunities to participate in the experimental design process. This experiment serves as an example of how research-inspired, discovery-based experiences can benefit both the students and instructor; students learned important skills necessary for real-world biochemistry research and a more concrete understanding of the research process, while generating new knowledge to enhance the scholarly endeavors of the instructor. © 2015 The International Union of Biochemistry and Molecular Biology.

  9. Recombinant expression and purification of 'virus-like' bacterial encapsulin protein cages

    NARCIS (Netherlands)

    Rurup, W.F.; Cornelissen, Jeroen Johannes Lambertus Maria; Koay, M.S.T.; Orner, Brendan P.

    2014-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules

  10. Recombinant expression and purification of 'virus-like' bacterial encapsulin protein cages

    NARCIS (Netherlands)

    Rurup, W.F.; Cornelissen, Jeroen Johannes Lambertus Maria; Koay, M.S.T.; Orner, Brendan P.

    2015-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules

  11. Expression of a gene encoding chitinase (pCA 8 ORF) from Aeromonas sp. no. 10S-24 in Escherichia coli and enzyme characterization.

    Science.gov (United States)

    Sutrisno, A; Ueda, M; Inui, H; Kawaguchi, T; Nakano, Y; Arai, M; Miyatake, K

    2001-01-01

    A gene encoding chitinase from Aeromonas sp. no. 10S-24 was expressed using pTrc99A in Escherichia coli JM 105 which yielded a 5-fold higher activity than when pUC19 was used. Three different truncated enzymes (SA-1, SA-2 and SA-3) were obtained after purification. Their isoelectric points were 7.0, 6.9, and 6.7, respectively. The enzymes showed two optimum pHs, 4.0 and 7.0, when incubated with ethylene glycol chitin as the substrate, and were stable over a wide pH range (3.0-9.0). The optimum temperature was 60 degrees C and the enzymes were stable up to 50 degrees C. The chitinases exhibited wide substrate specificities for chitin-related compounds.

  12. Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags.

    Science.gov (United States)

    Gillies, Alison R; Hsii, Judy F; Oak, Seachol; Wood, David W

    2008-10-01

    We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.

  13. Cloning and expression of Brucella outer membrane protein 36kDa (OMP2b in E. coli

    Directory of Open Access Journals (Sweden)

    Nazanin Behshti

    2011-09-01

    Full Text Available Background & Objective: Brucellosis is an important zoonotic disease of economic significance. Brucella species are gram-negative, facultative intracellular bacteria, and are capable of replicating in the phagosomes of macrophages. They cause infection in several animal species and humans. Prevention of new diseases and diagnosis of cases infected with the organism are both essential for eradication of the disease. Characterization and evaluation of different antigens of Brucella cells has a key role in progression of prevention and diagnosis programs. Here, we report the production and purification of recombinant 31kDa outer membrane protein Brucella abortus (Omp2b. Materials & Methods: Brucella abortus 36kDa outer membrane protein gene was amplified with PrimeSTAR® HS DNA polymerase, cloned in pJET1.2. The target gene was subcloned in pET28a (+. Recombinant pET28a vectors were transformed into E coli BL21 (DE3. Expression of recombinant protein was induced with 1mM IPTG. Proteins were absorbed to Ni-NTA agarose resins and Recombinant proteins were eluted with 250mM imidazol. Imidazol removed by dialysis. Proteins were assayed by Western-blotting and rOmp2b was probed by Brucella rabbit anti serum. Result: Appearance of a golden brown band at the site of reaction, in Western blotting confirmed successfully clone and expression. We purified Omp2b by affinity chromatography and this method prepared refolds proteins on the column. Conclusion: Omp2b were successfully cloned, expressed and purified. The recombinant proteins were recognized by polyclonal antiserum which suggests the accuracy of procedure.

  14. Bacterial expression, purification and preliminary X-ray crystallographic characterization of the invertase inhibitor Nt-CIF from tobacco.

    Science.gov (United States)

    Hothorn, Michael; Bonneau, Fabien; Stier, Gunter; Greiner, Steffen; Scheffzek, Klaus

    2003-12-01

    Acid invertases catalyzing the breakdown of sucrose are regulated at the post-translational level by extracellular inhibitory proteins of 16-20 kDa molecular weight in a pH-dependent manner. Little is known about the characteristics of the underlying protein-protein interaction. Here, the expression, purification, characterization, crystallization and initial X-ray analysis of a biologically active invertase inhibitor Nt-CIF from tobacco is reported. Four crystal forms covering a wide pH range have been obtained and data sets at resolutions higher than 2.5 A have been collected.

  15. Prevalence and molecular characterization of clinical isolates of Escherichia coli expressing an AmpC phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Rikke Lind; Nielsen, Jesper Boye; Friis-Møller, Alice

    2010-01-01

    OBJECTIVES: To establish the prevalence of the AmpC beta-lactamase phenotype in clinical isolates of Escherichia coli and characterize the genetic resistance mechanisms causing the observed phenotype. METHODS: Clinical E. coli (n = 74) with reduced susceptibility to third-generation cephalosporins...... and resistance to cefoxitin were collected from the Department of Clinical Microbiology at Hvidovre Hospital, Denmark, in 2006. The AmpC disc test was used to confirm expression of AmpC, and test-positive strains were selected for further antimicrobial susceptibility testing and molecular characterization....... Sequencing of ampC showed that most isolates were not clonally related. CONCLUSIONS: E. coli expressing an AmpC phenotype occur sporadically and cause significant resistance to cephalosporins. The majority of these are hyperproducing chromosomal ampC although some isolates have acquired pAmpC....

  16. Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse

    Science.gov (United States)

    Volpert, Marianna; Mangum, Jonathan E.; Jamsai, Duangporn; D'Sylva, Rebecca; O'Bryan, Moira K.; McIntyre, Peter

    2014-02-01

    While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

  17. Co-expression and characterization of enterocin CRL35 and its mutant in Escherichia coli Rosetta

    Directory of Open Access Journals (Sweden)

    Masías Emilse

    2014-01-01

    Full Text Available Even though many sequences and structures of bacteriocins from lactic acid bacteria have been fully characterized so far, little information is currently available about bacteriocins heterologously produced by Escherichia coli. For this purpose, the structural gene of enterocin CRL35, munA, was PCR-amplified using specific primers and cloned downstream of PelB sequence in the pET22b (+ expression vector. E. coli Rosetta (DE3 pLysS was chosen as the host for production and enterocin was purified by an easy two-step protocol. The bacteriocin was correctly expressed with the expected intramolecular disulfide bond. Nevertheless, it was found that a variant of the enterocin, differing by 12 Da from the native polypeptide, was co-expressed by E. coli Rosetta in comparable amount. Indeed, the mutant bacteriocin contained two amino acid substitutions that were characterized by matrix assisted laser desorption ionization-time of flight (MALDI-TOF and HPLCelectrospray (ESI-Q-TOF tandem mass spectrometry (MS/ MS sequencing. This is the first report regarding the production of mutants of pediocin-like bacteriocins in the E. coli expression system.

  18. Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

    NARCIS (Netherlands)

    Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

    2005-01-01

    The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC

  19. Induction studies with Escherichia coli expressing recombinant interleukin-13 using multi-parameter flow cytometry

    DEFF Research Database (Denmark)

    Shitu, J. O.; Woodley, John; Wnek, R.

    2009-01-01

    The expression of interleukin-13 (IL13) following induction with IPTG in Escherichia coli results in metabolic changes as indicated by multi-parameter flow cytometry and traditional methods of fermentation profiling (O-2 uptake rate, CO2 evolution rate and optical density measurements). Induction...

  20. Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escheria coli

    NARCIS (Netherlands)

    Godde, C.; Sahm, K.; Brouns, S.J.J.; Kluskens, L.D.; Oost, van der J.; Vos, de W.M.; Antranikian, G.

    2005-01-01

    A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two

  1. Artificial regulation of gene expression in Escherichia coli by RNase P.

    Science.gov (United States)

    Guerrier-Takada, C; Li, Y; Altman, S

    1995-11-21

    Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo. A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants.

  2. Comparison of cellular stress levels and green-fluorescent-protein expression in several Escherichia coli strains.

    Science.gov (United States)

    Seo, Jeong Hyun; Kang, Dong Gyun; Cha, Hyung Joon

    2003-04-01

    Constructs comprising stress-gene promoter elements from rpoH (Sigma 32), clpB or dnaK linked to a green-fluorescent-protein (GFP) expression vector were previously used as non-invasive "stress probes" in Escherichia coli. We compared cellular stress responses in four E. coli strains: production hosts JM105 and BL21, and cloning hosts HB101 and TOP10. When GFP was also used as a model for foreign protein production, we generally observed that the level of expression was inversely proportional to the level of cellular stress. JM105 showed the highest cellular stress level and very low GFP expression, while BL21 exhibited the lowest cellular stress level and the highest GFP expression, in both normal and heat-shock stress environments.

  3. Cloning, expression, purification and characterization of triosephosphate isomerase from Trypanosoma cruzi.

    Science.gov (United States)

    Ostoa-Saloma, P; Garza-Ramos, G; Ramírez, J; Becker, I; Berzunza, M; Landa, A; Gómez-Puyou, A; Tuena de Gómez-Puyou, M; Pérez-Montfort, R

    1997-03-15

    The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L. mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to

  4. Purification, characterization, and expression of multiple glutamine synthetases from Prevotella ruminicola 23.

    Science.gov (United States)

    Kim, Jong Nam; Cann, Isaac K O; Mackie, Roderick I

    2012-01-01

    The Prevotella ruminicola 23 genome encodes three different glutamine synthetase (GS) enzymes: glutamine synthetase I (GSI) (ORF02151), GSIII-1 (ORF01459), and GSIII-2 (ORF02034). GSI, GSIII-1, and GSIII-2 have each been heterologously expressed in and purified from Escherichia coli. The subunit molecular mass of GSI was 56 kDa, while GSIII-1 and GSIII-2 were both 83 kDa. Optimal conditions for γ-glutamyl transferase activity were found to be 35°C at pH 5.6 with 0.25 mM Mn(2+) ions (GSI) or 37°C at pH 6.0 (GSIII-1 and GSIII-2) with 0.50 to 1.00 mM Mn(2+) ions. GSIII biosynthetic activity was found to be optimal at 50 to 60°C and pH 6.8 to 7.0 with 10 mM Mn(2+) ions, while GSI displayed no GS biosynthetic activity. Kinetic analysis revealed K(m) values for glutamate and ammonium as well as for hydrolysis of ATP to be 8.58, 0.48, and 1.91 mM, respectively, for GSIII-1 and 1.72, 0.43, and 2.65 mM, respectively, for GSIII-2. A quantitative reverse transcriptase PCR assay (qRT-PCR) revealed GSIII-2 to be significantly induced by high concentrations of ammonia, and this corresponded with increases in measured GS activity. Collectively, these results show that both GSIII enzymes in P. ruminicola 23 are functional and indicate that GSIII-2, flanked by GOGAT (gltB and gltD genes), plays an important role in the acquisition and metabolism of ammonia, particularly under nonlimiting ammonia growth conditions.

  5. Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis.

    Science.gov (United States)

    Mishra, Saurabh; Bhagavat, Raghu; Chandra, Nagasuma; Vijayarangan, Namperumalsamy; Rajeswari, Haryadi; Ajitkumar, Parthasarathi

    2012-11-01

    The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, remains unknown. In this regard, M. tuberculosis fic gene (Mtfic) was cloned, overexpressed, and purified to homogeneity for its biochemical characterisation. It has the characteristic FIC motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Neither the His-tagged nor the GST-tagged MtFic protein, overexpressed in Escherichia coli, nor expression of Mtfic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtFic (MBP-MtFic) could be obtained partly in the soluble fraction. The cloned, overexpressed, and purified recombinant MBP-MtFic showed conversion of ATP, GTP, CTP, and UTP into AMP, GMP, CMP, and UMP, respectively. Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The design of NMP formation assay using the recombinant, soluble MtFic would enable identification of its target substrate for NMPylation. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Identification of succinic semialdehyde reductases from Geobacter: expression, purification, crystallization, preliminary functional, and crystallographic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Gao, Xiaoli; Zheng, Yi; Garavito, R. Michael (MSU)

    2012-04-30

    Succinic semialdehyde reductase (SSAR) is an important enzyme involved in {gamma}-aminobutyrate (GABA) metabolism. By converting succinic semialdehyde (SSA) to {gamma}-hydroxybutyrate (GHB), the SSAR facilitates an alternative pathway for GABA degradation. In this study, we identified SSARs from Geobacter sulfurreducens and Geobacter metallireducens (GsSSAR and GmSSAR, respectively). The enzymes were over-expressed in Escherichia coli and purified to near homogeneity. Both GsSSAR and GmSSAR showed the activity of reducing SSA using nicotinamide adenine dinucleotide phosphate as a co-factor. The oligomeric sizes of GsSSAR and GmSSAR, as determined by analytical size exclusion chromatography, suggest that the enzymes presumably exist as tetramers in solution. The recombinant GsSSAR and GmSSAR crystallized in the presence of NADP{sup +}, and the resulting crystals diffracted to 1.89 {angstrom} (GsSSAR) and 2.25 {angstrom} (GmSSAR) resolution. The GsSSAR and GmSSAR crystals belong to the space groups P2{sub 1}22{sub 1} (a = 99.61 {angstrom}, b = 147.49 {angstrom}, c = 182.47 {angstrom}) and P1 (a = 75.97 {angstrom}, b = 79.14 {angstrom}, c = 95.47 {angstrom}, {alpha} = 82.15{sup o}, {beta} = 88.80{sup o}, {gamma} = 87.66{sup o}), respectively. Preliminary crystallographic data analysis suggests the presence of eight protein monomers in the asymmetric units for both GsSSAR and GmSSAR.

  7. Look and See if it is Time to Induce Protein Expression in Eschericia coli Cultures†

    Science.gov (United States)

    Kelley, K. Danielle; Olive, Lorenzo Q.; Hadziselimovic, Arina; Sanders, Charles R.

    2010-01-01

    It is shown that Methyl Red can be used as an indicator dye that changes color in E. coli culture as a result of time- and cell density-dependent bleaching by azoreductase produced by the bacteria. For cell cultures that are being used to express a recombinant protein this phenomenon can be exploited to provide a simple visual cue that cell cultures have reached an appropriate growth phase for addition of an agent to induce protein expression, such as isopropylthiogalactoside. PMID:20540494

  8. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  9. [Fed-batch fermentation of Escherichia coli that express fab fragment of anti-HBsAg].

    Science.gov (United States)

    An, Feng; Chen, Yu-Chuan; Fan, Lie-Ying; Han, Huan-Xing

    2003-01-01

    To develop a fed-batch fementation process of E. coli TOP10 containing a recombinant plasmid pBAD/HBs Fab. Cells were grown in semi-defined medium at 37 degrees C, and the feed operation using glycerol as carbon source was performed when dissolved oxygen increased. When the target cell concentration reached to 64g/L, arabinose was added to a final concentration of 0.02%. Cells were grown for another 5h with the culture temperature decreased from 37 degrees C to 30 degrees C. In the whole process, cell growth was monitored by measuring OD600 of samples taken at 1/2h intervals and the dissolved oxygen was kept above 30%. After the fementation, E. coli pellets were collected for purification of Fab protein. The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot. Cell concentration we got is 96g wet bacteria per liter, the Fab protein is about 6% of total protein of the host, that is 80mg per liter. Stable fermentation parameters were obtained for fermentation to improve productivity of the Fab protein. The Fab protein was produced in the form of soluble biologically active protein, it's better than inclusion bodies from which biologically active protein can only be recovered by complicated and costly denaturation and refolding process.

  10. Cloning, Expression, Isotope Labeling, and Purification of Transmembrane Protein MerF from Mercury Resistant Enterobacter sp. AZ-15 for NMR Studies

    Directory of Open Access Journals (Sweden)

    Aatif Amin

    2017-07-01

    Full Text Available Mercury resistant (HgR Enterobacter sp. AZ-15 was isolated from heavy metal polluted industrial wastewater samples near to districts Kasur and Sheikhupura, Pakistan. 16S rDNA ribotyping and phylogentic analysis showed 98% homology with already reported Enterobacter species. The merF gene encoding transmembrane protein-MerF was amplified from genomic DNA and ligated into pET31b+ vector using restriction endonucleases, SphI and XhoI. The genetic codons of merF gene encoding cysteine residues were mutated into codons, translating into serine residues by site-directed mutagenesis. Ketosteroid isomerase (KSI, a fusion tag which is present in pET31b+ vector, was used in the expression of merFm gene. KSI was used to drive the target peptide (MerFm into inclusion bodies so that the peptide yield and purity were increased. The stable plasmid pET31b+:merFm was transformed into C43(DE3 E.coli cells. The high expression of uniformly 15N isotopically labeled-MerFm protein was induced with 1 mM IPTG. The purification of 15N-MerFm recombinant protein by Ni-NTA and size exclusion chromatography involved an unfolding/refolding procedure. The two-dimensional HSQC NMR spectra of MerFm protein showed the purity and correct number of resonances for each amide. 1H–15N HSQC NMR experiment also confirmed that no modification of the tryptophan residue occurred during cyanogen bromide cleavage. A small scale reservoir of Luria Bertani (LB medium supplemented with 20 μg/ml of HgCl2 showed 90% detoxification of Hg by Enterobacter sp. AZ-15. The accumulation of Hg on the cell surface of this strain was visualized by scanning electron microscopy (SEM which confirmed its potential use in Hg-bioremediation.

  11. Molecular cloning of Reteplase and its expression in E. coli using tac promoter

    Directory of Open Access Journals (Sweden)

    Safieh Aghaabdollahian

    2014-01-01

    Full Text Available Background and Aims: This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression. Materials and Methods: Reteplase cDNA was amplified by polymerase chain reaction (PCR with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG, expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE. Results: Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG. Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli.

  12. Sortase A-aided Escherichia coli expression system for functional osteoprotegerin cysteine-rich domain.

    Science.gov (United States)

    Jin, Mengmeng; Chen, Yuan; Zhao, Yunfeng; Che, Luyang; Ma, Yanyan; Li, Jingzhe; Wang, Yi; Tao, Hua; Ma, Juan; Pan, Bing; Liu, Changzhen; Huang, Peng

    2017-06-01

    As a natural inhibitor of the receptor activator of nuclear factor-кB ligand (RANKL), osteprotegerin (OPG) is considered a promising treatment for metabolic bone diseases. Typical approaches for preparing recombinant OPG or its derivatives employ eukaryotic expression systems. Due to the advantages of a prokaryotic expression system, which include its convenience, low cost, and abundant production, in this study, we establish a strategy for preparing functional OPG using the Escherichia coli expression system. After initial failures in preparation of OPG and its truncation, OPG cysteine-rich domain (OPG-CRD/OPGT) by using pET and pGEX vectors, we constructed a sortase A (SrtA)-aided E. coli expression system, in which the expressed protein was a self-cleaving SrtA fusion protein. Using this system, we successfully prepared the recombinant OPGT protein. The BIAcore analyses indicated that the prepared OPGT had high affinities in binding with RANKL and TRAIL. Cell experiments confirmed the inhibitory effects of the prepared OPGT on RANKL-induced osteoclast differentiation and TRAIL-induced tumor cell apoptosis. The sortase A-aided E. coli expression system for OPGT established in this study may contribute to further studies and commercial applications of OPG.

  13. Molecular cloning of Reteplase and its expression in E. coli using tac promoter.

    Science.gov (United States)

    Aghaabdollahian, Safieh; Rabbani, Mohammad; Ghaedi, Kamran; Sadeghi, Hamid Mir Mohammad

    2014-01-01

    This study aimed to clone and express the reteplase cDNA, a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in E. coli, utilizing tac promoter for its expression. Reteplase cDNA was amplified by polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57R plasmid. The cloned cDNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion. By using 0.2, 0.5 and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), expression of reteplase was induced in E. coli TOP10 cells and analyzed by SDS-PAGE. Electrophoresis of PCR product and also double digested recombinant pTZ57R plasmid, also, pGEX-5x-1 vector, showed a 1068bp band of reteplase. SDS-PAGE analysis showed a 60 KDa band of protein product induced with different concentrations of IPTG. In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector will be used for the optimization of the expression of reteplase in E. coli.

  14. [Handling G-protein-coupled receptors: expression, purification and in vitro stabilization].

    Science.gov (United States)

    Banères, Jean-Louis; Mouillac, Bernard

    2012-10-01

    Among the different classes of integral membrane proteins, G protein-coupled receptors (GPCR) constitute the largest family. They are involved in most essential physiological functions and particularly play a key role in cell-to-cell communication and sensory signal transduction. They represent targets for approximately 30% of currently marketed drugs. In order to better understand their functioning, define their tridimensional structure and develop novel selective and efficient therapeutic compounds, it is crucial to purify these proteins for a full characterization. However, this biochemical step is not trivial since GPCR are present in membranes at very low levels and they require detergents to be extracted from their natural lipid environment and be handled as functional proteins. No universal strategy for GPCR production, purification and stabilization is currently available; each single GPCR possesses a unique set of physicochemical characteristics, preference for some detergents upon solubilization and specific conditions for purification. During the last decade, major breakthroughs regarding overexpression, purification and above all GPCR stabilization, thanks to amphipols and nanodiscs, opened very exciting perspectives for structural and dynamic investigations of these membrane proteins. The aim of this chapter is to provide an overview of the different aspects of GPCR handling. © 2012 médecine/sciences – Inserm / SRMS.

  15. Regulation and expression of human Fabs under the control of the Escherichia coli arabinose promoter, PBAD.

    Science.gov (United States)

    Clark, M A; Hammond, F R; Papaioannou, A; Hawkins, N J; Ward, R L

    1997-10-01

    The L-arabinose operon from E. coli contains an inducible promoter PBAD which has been extensively studied for the control of gene expression. PBAD has a number of potential advantages over Plac, and has been used successfully to promote high level expression of recombinant proteins. The aim of this study was to investigate PBAD as an alternative system to Plac for the bacterial expression of recombinant Fabs. The promoter PBAD from the E. coli arabinose operon araBAD and the gene encoding the regulator of this promoter, were cloned into the phagemid expression vector MCO1. Expression of human recombinant tetanus toxoid (TT) and c-erbB2 Fabs under the control of PBAD was compared at two induction temperatures with the same Fabs produced under the control of Plac. Expression of TT and c-erbB2 Fabs under the control of PBAD was comparable to Fab expression from Plac. However, highly expressed TT Fab under the control of PBAD was localised to the soluble periplasmic fraction whereas under the control of Plac, there was greater leakage of Fab into the culture supernatant. In addition, Fab expression from PBAD could be more tightly repressed than from Plac. PBAD is a useful and cheaply inducible alternative to the more commonly used Plac for the rapid expression of soluble recombinant human antibody fragments.

  16. Global transcriptional regulatory network for Escherichia coli robustly connects gene expression to transcription factor activities

    DEFF Research Database (Denmark)

    Fang, Xin; Sastry, Anand; Mih, Nathan

    2017-01-01

    Transcriptional regulatory networks (TRNs) have been studied intensely for >25 y. Yet, even for the Escherichia coli TRN-probably the best characterized TRN-several questions remain. Here, we address three questions: (i) How complete is our knowledge of the E. coli TRN; (ii) how well can we predict...... were collected from published, validated chromatin immunoprecipitation (ChIP) data and RegulonDB. For 21 different TF knockouts, up to 63% of the differentially expressed genes in the hiTRN were traced to the knocked-out TF through regulatory cascades. Second, we trained supervised machine learning...

  17. Evaluation of a QIAamp DNA stool purification kit for Shiga-toxigenic Escherichia coli detection in bovine fecal swabs by PCR Evaluación del kit QIAamp DNA stool purification para la detección de Escherichia coli productor de toxina Shiga en hisopados de materia fecal bovina por PCR[

    Directory of Open Access Journals (Sweden)

    A. Gioffré

    2004-03-01

    Full Text Available A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC by polymersase chain reaction (PCR directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol, and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73 % were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64% than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.Un kit comercial diseñado para la eliminación de inhibidores de la polimerasa Taq fue ensayado para la detección de STEC por PCR en muestras fecales de bovinos. Cuarenta y cinco muestras fueron evaluadas por la presencia de genes stx. Los resultados fueron comparados con aquéllos obtenidos por otros dos métodos: amplificación de ADN purificado por un procedimiento no comercial (protocolo de lisis por calor, y amplificación de ADN de muestras cultivadas en medio sólido, comúnmente usado en nuestro laboratorio. El mismo número de muestras positivas (33/45, 73 %, fueron obtenidas con el QIAamp DNA stool purification kit y el procedimiento de cultivo, sugiriendo una eliminación adecuada de inhibidores que interfieren con la amplificación en materia fecal. Por otro lado, el número de muestras positivas detectadas usando ADN purificado por el protocolo no comercial fue menor, 25/39 (64%. En conclusión, el uso del kit QIAamp DNA stool

  18. High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery.

    Science.gov (United States)

    Turchetto, Jeremy; Sequeira, Ana Filipa; Ramond, Laurie; Peysson, Fanny; Brás, Joana L A; Saez, Natalie J; Duhoo, Yoan; Blémont, Marilyne; Guerreiro, Catarina I P D; Quinton, Loic; De Pauw, Edwin; Gilles, Nicolas; Darbon, Hervé; Fontes, Carlos M G A; Vincentelli, Renaud

    2017-01-17

    Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics. In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active. Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large

  19. Expression of Treponema pallidum Antigens in Escherichia coli

    Science.gov (United States)

    Walfield, Alan M.; Hanff, Philip A.; Lovett, Michael A.

    1982-04-01

    Treponema pallidum DNA was cloned in a bacteriophage. Clones were screened for expression of Treponema pallidum antigens by an in situ radio-immunoassay on nitrocellulose, with the use of subsequent reactions with syphilitic serum and radioiodinated Staphylococcus aureus protein A. One clone, which gave a strong signal, codes for at least seven antigens that react specifically with human antibodies to Treponema pallidum.

  20. Identification and purification of human induced pluripotent stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression.

    Directory of Open Access Journals (Sweden)

    Rebecca Josowitz

    Full Text Available The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN. When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.

  1. High-level soluble expression of Serratia marcescens H30 lipase in Escherichia coli.

    Science.gov (United States)

    Su, Erzheng; Xu, Jingjing; Wu, Xiangping

    2015-01-01

    Serratia marcescens lipase (SmL) is an important biocatalyst used to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphynyl) glycidic acid methyl ester. However, the economically justified level recombinant soluble expression of SmL in Escherichia coli has not been established. Thus, fusion genes of lipase from S. marcescens H30 with different fusion tags were constructed and expressed in E. coli. The effects of fusion tags were revealed. A significant increase in recombinant lipase solubility showed that E. coli BL21 (DE3)/pET32a-SmL was a suitable choice for SmL production. To optimize the performance of recombinant SmL production, changes in culture medium compositions and induction conditions were systematically tested. Finally, the recombinant SmL activity and productivity reached approximately 23,000 U/L and 1,278 U/L/H in shake flasks, respectively. This value is the highest SmL activity attained by heterogeneous recombinant expression in E. coli. Lipase activity and productivity reached 19,650 U/L and 1,228 U/L/H, respectively, by scaling up SmL production in a 7.0 L fermenter. The existence of the Trx tag did not influence the chiral selectivity of recombinant SmL. These findings indicate a possibility for soluble and economical SmL expression in E. coli to meet industrial needs. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  2. Expression of mastoparan B, a venom peptide, via Escherichia coli C43 (DE3) coupled with an artificial oil body-cyanogen bromide technology platform.

    Science.gov (United States)

    Jinn, Tzyy-Rong; Hsieh, Sheng-Kuo; Yu, Yu-Jen; Tang, Nou-Ying; Lin, Jhao-Ren

    2017-07-24

    Here, we successfully use the oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB peptide which, in a manner identical to that of its native counterpart. In this study, the MPB peptide was interlinked with recombinant oleosin (rOle(127M→L)) by a methionine residue, a CNBr cleavage site. The recombinant rOle(127M→L)-MPB fusion gene was cloned into the pET30a(+) vector and was expressed in E. coli (DE3) after IPTG induction under optimized conditions. Furthermore, the expressed MPB was released from this AOB-CNBr platform, since AOB purification system provides a useful machinery to purify target protein. The purified MPB was identified by MALDI-MS analysis, and its bioactivity was examined by antimicrobial test. It is important to note that this study not only provides a new insight into the expression of active MPB, but also provides an alternative and reliable model to express other valuable venom peptides, such as mastoparan-II, and matoparan A, B, C, J, L, M, T, X, and melittin, which possesses important functional peptides in medical applications. Take together, in this study provides a new avenue for the production of active MPB and facilitate the studies and applications of the peptide in the future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. A novel expression system for production of soluble prion proteins in E. coli

    Directory of Open Access Journals (Sweden)

    Abskharon Romany NN

    2012-01-01

    Full Text Available Abstract Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX, a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.

  4. A Comparative Analysis of Recombinant Expression and Solubility Screening of Two Phytases in E. coli

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    Ashok Pandey

    2011-01-01

    Full Text Available Microbial phytases, especially from fungal and bacterial sources, have received much attention as food additives in human nutrition and as feed supplements for monogastric animals. An effective expression screening method for recombinant production of this enzyme on a small scale is industrially desirable. An effort has been made in this work to clone and express phytase genes from Aspergillus sp. and Escherichia coli with the selected host, vector and inducer combination. Albeit the formation of insoluble inclusion bodies by fungal phytase, recombinant E. coli appA was effectively expressed in a cost-effective manner in the periplasm of BL21plysS using an inducer concentration of 0.01 mM in 4 h of growth. Enzyme was purified in three consecutive steps and functional studies were carried out.

  5. A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Pearl Laurence H

    2006-02-01

    Full Text Available Abstract Background The antiphospholipid syndrome (APS, characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI of human beta 2 glycoprotein I (β2GPI is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL, which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. Methods Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b and expressed in BL21(DE3 Escherichia coli (E. coli. By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. Results Purified, soluble his-tagged DI in yields of 750 μg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of β2GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. Conclusion By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the

  6. A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein.

    Science.gov (United States)

    Dar, Saira; Shuja, Rukhsana N; Shakoori, Abdul Rauf

    2013-02-01

    Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10-30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd(2+)-ions.

  7. Alternative purification method for recombinant measles viral nucleoprotein expressed in insect cells by ion-exchange chromatography.

    Science.gov (United States)

    Lee, Han Saem; Kim, You-Jin; Yang, Jeongsun; Yoon, Hee Sook; Kim, Seung Tae; Kim, Kisoon

    2014-03-01

    Recombinant measles virus nucleoproteins (rMeV N) and fusion (F) proteins were characterized as major antigenic proteins expressed in insect cells mediated by recombinant baculoviruses (rBVs). Band intensities were analyzed by Western blotting to recognize IgG and IgM antibodies against the rMeV N and F proteins in human sera and cerebrospinal fluids (CSFs) from patients with measles infections. Positive results from the blots using the rMeV N were consistent with the results of enzyme-linked immunosorbent assays (ELISAs) in which whole viral proteins were used as antigens. Human sera and CSFs reacted more strongly with the rMeV N than with the rMeV F proteins prepared in an identical expression system. For efficient and reliable purification, ion-exchange chromatography using Source Q anion resin was applied, and high-purity rMeV N protein was harvested. To characterize the similarity with the native viral protein to purified N protein, structural mimicry of purified recombinant proteins with intact rMeV N was shown through transmission electron microscopy, and the truncation and the phosphorylation status of the expressed protein were analyzed. These results suggest that the rMeV N purified by ion-exchange chromatography has features similar to those of naïve N including a self-assembled structure, phosphorylation and antigenic function. Thus, these expression and purification methods can be applied to the large-scale production of the rMeV N, which is essential for the development of new diagnostic tools and vaccines for acute and chronic MeV infections. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12.

    Science.gov (United States)

    Scarabel, M; Penin, F; Bonod-Bidaud, C; Nègre, D; Cozzone, A J; Cortay, J C

    1995-02-03

    A DNA fragment encoding the DNA-binding domain (amino acids 1-60) of the Escherichia coli fru transcriptional regulator was cloned into the pGEX-KT vector and expressed in frame with the fused gene encoding glutathione S-transferase. The fusion protein was purified to homogeneity by affinity chromatography on immobilized glutathione, and then cleaved with thrombin. After separation by a cation-exchange chromatography step, the DNA-binding domain exhibited proper folding, as shown by proton NMR analysis. Furthermore, it showed specific interaction with the operator region of the ace operon, as checked by gel retardation and DNA methylation-protection experiments.

  9. Purification and crystallization of the yeast translation elongation factor eEF3

    DEFF Research Database (Denmark)

    Andersen, Christian Folsted; Anand, Monika; Boesen, Thomas

    2004-01-01

    A Saccharomyces cerevisiae strain expressing full-length histidine-tagged translation elongation factor 3 (eEF3) as the only form of the protein facilitated purification of the factor for both structural and functional studies. Additionally, an identical full-length form has been successfully...... expressed in Escherichia coli and a C-terminally truncated form of histidine-tagged eEF3 has been successfully expressed in E. coli and S. cerevisiae. Both forms have been crystallized and crystals of the truncated protein expressed in yeast diffract synchrotron radiation to a maximum resolution of 2.3 A...

  10. Gene Expression during Survival of Escherichia coli O157:H7 in Soil and Water

    Directory of Open Access Journals (Sweden)

    Ashley D. Duffitt

    2011-01-01

    Full Text Available The in vitro survival of Escherichia coli O157:H7 at 15∘C under two experimental conditions (sterile soil and sterile natural water was examined. DNA microarrays of the entire set of E. coli O157:H7 genes were used to measure the genomic expression patterns after 14 days. Although the populations declined, some E. coli O157:H7 cells survived in sterile stream water up to 234 days and in sterile soil for up to 179 days. Cells incubated in soil microcosms for 14 days expressed genes for antibiotic resistance, biosynthesis, DNA replication and modification, metabolism, phages, transposons, plasmids, pathogenesis and virulence, antibiotic resistance, ribosomal proteins, the stress response, transcription, translation, and transport and binding proteins at significantly higher levels than cells grown in Luria broth. These results suggest that E. coli O157:H7 may develop a different phenotype during transport through the environment. Furthermore, this pathogen may become more resistant to antibiotics making subsequent infections more difficult to treat.

  11. Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides.

    Science.gov (United States)

    Nikel, P I; Ramirez, M C; Pettinari, M J; Méndez, B S; Galvagno, M A

    2010-08-01

    Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen-restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1.4-fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro-oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6.5 +/- 0.3 g l(-1) ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l(-1) ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl-coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two-stage bioreactor cultures were conducted in a minimal medium containing 100 microg l(-1) calcium d-pantothenate to evaluate oxic acetyl-CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15.4 +/- 0.9 g l(-1) with a volumetric productivity of 0.34 +/- 0.02 g l(-1) h(-1). Escherichia coli responded to adhE over-expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl-CoA played a key role in micro-oxic ethanol synthesis and growth. Insight into the micro-oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.

  12. Secretory expression of nattokinase from Bacillus subtilis YF38 in Escherichia coli.

    Science.gov (United States)

    Liang, Xiaobo; Jia, Shifang; Sun, Yufang; Chen, Meiling; Chen, Xiuzhu; Zhong, Jin; Huan, Liandong

    2007-11-01

    Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l(-1) isopropyl-beta-D- thiogalactopyranoside (IPTG) at 20 degrees C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.

  13. Stability, oviposition attraction, and larvicidal activity of binary toxin from Bacillus sphaericus expressed in Escherichia coli.

    Science.gov (United States)

    da Silva Pinto, Luciano; Gonçales, Relber Aguiar; Conceição, Fabricio Rochedo; Knabah, Paula Ferreira; Borsuk, Sibele; Campos, Vinicius Farias; Arruda, Francisco Vassiliepe; Leite, Fabio Pereira Leivas

    2012-09-01

    Bacillus sphaericus produces a two-chain binary toxin composed of BinA (42 kDa) and BinB (51 kDa), which are deposited as parasporal crystals during sporulation. The toxin is highly active against Culex larvae and Aedes and Anopheles mosquitoes, which are the principal vectors for the transmission of malaria, yellow fever, encephalitis, and dengue. The use of B. sphaericus and Bacillus thuringiensis in mosquito control programs is limited by their sedimentation in still water. In this study, the binA and binB genes were cloned and the recombinant BinAB protein was expressed in three strains of Escherichia coli. These recombinant strains were used in a toxicity assay against Culex quinquefasciatus larvae. The highest expression level was achieved when both proteins were expressed in a single operon construct. The BinAB protein expressed in the E. coli Arctic strain showed higher larvicidal activity than either of the recombinant proteins from the E. coli Ril or pLysS strains. Furthermore, it had the highest oviposition attraction (49.1%, P < 0.05). These data suggest that biologically active recombinant BinA and BinB toxins might be useful in mosquito control programs, delivered by inactivated bacterial cells or in traps.

  14. Solubility of recombinant Src homology 2 domains expressed in E. coli can be predicted by TANGO.

    Science.gov (United States)

    Andersen, Thorny Cecilie Bie; Lindsjø, Kjersti; Hem, Cecilie Dahl; Koll, Lise; Kristiansen, Per Eugen; Skjeldal, Lars; Andreotti, Amy H; Spurkland, Anne

    2014-01-14

    Signalling proteins often contain several well defined and conserved protein domains. Structural analyses of such domains by nuclear magnetic spectroscopy or X-ray crystallography may greatly inform the function of proteins. A limiting step is often the production of sufficient amounts of the recombinant protein. However, there is no particular way to predict whether a protein will be soluble when expressed in E.coli. Here we report our experience with expression of a Src homology 2 (SH2) domain. The SH2 domain of the SH2D2A protein (or T cell specific adapter protein, TSAd) forms insoluble aggregates when expressed as various GST-fusion proteins in Escherichia coli (E. coli). Alteration of the flanking sequences, or growth temperature influenced expression and solubility of TSAd-SH2, however overall yield of soluble protein remained low. The algorithm TANGO, which predicts amyloid fibril formation in eukaryotic cells, identified a hydrophobic sequence within the TSAd-SH2 domain with high propensity for beta-aggregation. Mutation to the corresponding amino acids of the related HSH2- (or ALX) SH2 domain increased the yield of soluble TSAd-SH2 domains. High beta-aggregation values predicted by TANGO correlated with low solubility of recombinant SH2 domains as reported in the literature. Solubility of recombinant proteins expressed in E.coli can be predicted by TANGO, an algorithm developed to determine the aggregation propensity of peptides. Targeted mutations representing corresponding amino acids in similar protein domains may increase solubility of recombinant proteins.

  15. Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter.

    Science.gov (United States)

    Shafiee, Fatemeh; Moazen, Fatemeh; Rabbani, Mahammad; Mir Mohammad Sadeghi, Hamid

    2015-02-01

    Reteplase is a mutant version of t-PA (tissue plasminogen activator) with prolonged half-life. In the present study, E. coli Top 10 bacteria were utilized in the production of reteplase, which is the nonglycosylated active domain of t-PA. Reteplase gene was ligated into pBAD/gIII plasmid which, allows secretion of this protein in periplasmic space. It would allow the correct formation of disulfide bonds in protein structure. This study aimed at expression of reteplase in optimum condition. In this study, the reteplase gene was cloned and expressed in Escherichia coli top 10 as a suitable host cell and its expression was optimized. The recombinant plasmid, pET15b/reteplase was digested by NcoI and BamHI restriction enzymes; while pBAD/gIIIA vector was digested by NcoI and BglII. Then the insert and vector were ligated and used for transformation of E. coli Top10 cells by heat shock method. Overnight culture of transformed bacteria was induced by L-arabinose in various concentrations (0.2, 0.02, 0.002, and 0.0002%) and at various temperatures. The obtained recombinant plasmid was sequenced to confirm the presence and correct framing of reteplase gene regarding the expression of reteplase. Maximum production of this enzyme was obtained under the following condition: 0.0002% L-arabinose at 37°C for 2 hours incubation. The purified protein was detected on SDS-PAGE (sodium dodecyl sulfate Polyacrylamide gel electrophoresis) as a 66 kDa band. The concentration of t-PA standard was 1 unit which is equal to 12 µg/mL. The enzymatic activity of samples was measured as 0.8 units compared to the standards. Reteplase was expressed in E. coli Top 10 after activation of pBAD/gIIIA promoter region by arabinose and optimized.

  16. Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10

    Directory of Open Access Journals (Sweden)

    Felsberg Jürgen

    2011-01-01

    Full Text Available Abstract Background Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. Results A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+ and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec expressed in Escherichia coli BL21-Gold(DE3(pOK101/pTf16 was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT, indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn356 was present in Nit-ANigRec but missing in Nit-ANigWT and Asp298-Val313 peptide was shortened to Asp298-Arg310 in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an

  17. Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

    DEFF Research Database (Denmark)

    Guerra, B; Niefind, K; Pinna, L A

    1998-01-01

    The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameter...

  18. In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E. coli

    Directory of Open Access Journals (Sweden)

    Wu Xiangping

    2012-09-01

    Full Text Available Abstract Background Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA and its chaperone (LipB from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E. coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. Results In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp and lipase specific foldase gene lipB (1023 bp. One single expression plasmid system E. coli BL21/pET28a-lipAB and two dual expression plasmid systems E. coli BL21/pETDuet-lipA-lipB and E. coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E. coli BL21/pET28a-lipAB and E. coli BL21/pETDuet-lipA-lipB were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by E. coli BL21/pACYCDuet-lipA-lipB was up to 8500 U/L. The lipase LipA had an optimal temperature of 30°C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol

  19. Cloning and expression of the Legionella micdadei "common antigen" in Escherichia coli

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Collins, M T; Høiby, N

    1989-01-01

    To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15-25 kilobase pairs (kb] were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand...... ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli-absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly...... will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function....

  20. The Genomic Pattern of tDNA Operon Expression in E. coli.

    Directory of Open Access Journals (Sweden)

    2005-06-01

    Full Text Available In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates.

  1. The Modulation of Polymorphonuclear Neutrophil Function by Cytotoxic Necrotizing Factor Type 1 - Expressing Uropathogenic Escherichia coli

    Science.gov (United States)

    2005-01-01

    Ph.D. Professor, Department of Microbiology and Immunology Uropathogenic Escherichia coli ( UPEC ) cause more than 85% of all urinary tract...models of UTI pathogenesis have provided some insight into the role of various UPEC virulence factors. In these animal studies, the toxin Cytotoxic...expressing UPEC infection in the in vivo models was magnitude of the acute iii inflammatory response. Compared to a cnf1 isogenic mutant, CNF1

  2. Expression of human β-defensin-1 in recombinant Escherichia coli ...

    African Journals Online (AJOL)

    Escherichia coli BL21(DE3) was transformed with a pHCD1 plasmid harboring the human β-defensin-1 (hBD1) gene fused in frame behind a disulfide bond isomerase (DsbC), a His-tag, and an enterokinase cleavage site. After induction, the DsbC-hBD1 was expressed as a ~36 kDa soluble fusion protein in recombinant E.

  3. Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells.

    Science.gov (United States)

    Lawrence, D; Strandberg, L; Grundström, T; Ny, T

    1989-12-22

    Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1. In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E. coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks

  4. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    Directory of Open Access Journals (Sweden)

    Safar Farajnia

    2013-02-01

    Full Text Available Purpose: Recombinant tumor necrosis factor-alpha (TNF-α has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.

  5. Prokaryotic expression, purification, and production of glutathione S-transferase-tagged neural stem cell specific peptides from phage display screening.

    Science.gov (United States)

    Jin, Lei; Zhao, Wenxiu; Ma, Lan

    2013-02-01

    Combining stem cell with phage display to discover novel biomarkers is a new field in stem cell studies. Novel peptides obtained through phage display panning have been functionally identified and involved into initial applications as cell culture support or cell label. In the present study, we designed a glutathione S-transferase (GST)-tagged peptide complex to construct an easy and efficient prokaryotic expression and purification platform for peptides obtained from phage display panning. The purified GST-peptide protein could specifically bind to neural stem (NS) cells and could be flexibly used for GST pull down assay or NS cell labeling in future studies. The results of the present study would be useful for cell labeling and future investigations of special receptors on stem cell surface.

  6. Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme.

    Science.gov (United States)

    Pan, Yuan T; Koroth Edavana, Vineetha; Jourdian, William J; Edmondson, Rick; Carroll, J David; Pastuszak, Irena; Elbein, Alan D

    2004-11-01

    Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess

  7. Molecular studies on selected colicin types (Expression, purification and characterization of colicin U)

    OpenAIRE

    Bhusal, Keshav Raj

    2016-01-01

    Colicins are ribosomally synthesized polypeptides produced by most E.coli strains. They exhibit a modular structure comprised of a receptor, translocator and cytotoxic domain, each with distinct function. Colicins exert their effect on the susceptible cell by first binding to a specific receptor, followed by translocation through the outer membrane via the Tol or Ton system and finally killing the cell by pore formation in the membrane or nuclease activity. The study of the colicin structure ...

  8. Expression and characterization of streptococcal rgp genes required for rhamnan synthesis in Escherichia coli.

    Science.gov (United States)

    Shibata, Yukie; Yamashita, Yoshihisa; Ozaki, Kazuhisa; Nakano, Yoshio; Koga, Toshihiko

    2002-06-01

    Six genes (rgpA through rgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans were previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol. 180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen of Streptococcus sobrinus have the same rhamnan backbone as the RGP of S. mutans. Escherichia coli harboring plasmid pRGP1 containing all six rgp genes did not synthesize complete RGP. However, E. coli carrying a plasmid with all of the rgp genes except for rgpE synthesized the rhamnan backbone of RGP without glucose side chains, suggesting that in addition to rgpE, another gene is required for glucose side-chain formation. Synthesis of the rhamnan backbone in E. coli required the initiation of transfer of N-acetylglucosamine to a lipid carrier and the expression of the rgpC and rgpD genes encoding the putative ABC transporter specific for RGP. The similarities in RGP synthesis between E. coli and S. mutans suggest common pathways for rhamnan synthesis. Therefore, we evaluated the rhamnosyl polymerization process in E. coli by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lipooligosaccharide (LOS). An E. coli transformant harboring rgpA produced the LOS modified by the addition of a single rhamnose residue. Furthermore, the rgpA, rgpB, and rgpF genes of pRGP1 were independently mutated by an internal deletion, and the LOS chemotypes of their transformants were examined. The transformant with an rgpA deletion showed the same LOS profile as E. coli without a plasmid. The transformant with an rgpB deletion showed the same LOS profile as E. coli harboring rgpA alone. The transformant with an rgpF deletion showed the LOS band with the most retarded migration. On the basis of these results, we speculated that RgpA, RgpB, and RgpF, in that order, function in rhamnan polymerization.

  9. Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum

    Science.gov (United States)

    Wu, Yong-Qing; Jiang, Pei-Hong; Fan, Chang-Sheng; Wang, Jian-Gang; Shang, Liang; Huang, Wei-Da

    2003-01-01

    AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine. METHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation. RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly. CONCLUSION: Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production. PMID:12532463

  10. Toxicity Biosensor for Sodium Dodecyl Sulfate Using Immobilized Green Fluorescent Protein Expressing Escherichia coli

    Directory of Open Access Journals (Sweden)

    Lia Ooi

    2015-01-01

    Full Text Available Green fluorescent protein (GFP is suitable as a toxicity sensor due to its ability to work alone without cofactors or substrates. Its reaction with toxicants can be determined with fluorometric approaches. GFP mutant gene (C48S/S147C/Q204C/S65T/Q80R is used because it has higher sensitivity compared to others GFP variants. A novel sodium dodecyl sulfate (SDS toxicity detection biosensor was built by immobilizing GFP expressing Escherichia coli in k-Carrageenan matrix. Cytotoxicity effect took place in the toxicity biosensor which leads to the decrease in the fluorescence intensity. The fabricated E. coli GFP toxicity biosensor has a wide dynamic range of 4–100 ppm, with LOD of 1.7 ppm. Besides, it possesses short response time (0.98, and long-term stability (46 days. E. coli GFP toxicity biosensor has been applied to detect toxicity induced by SDS in tap water, river water, and drinking water. High recovery levels of SDS indicated the applicability of E. coli GFP toxicity biosensor in real water samples toxicity evaluation.

  11. Human Interleukine-1 receptor antagonist:Cloning, Expression and Optimization in E.coli Host

    Directory of Open Access Journals (Sweden)

    Gh. Barati

    2014-07-01

    Full Text Available Introduction & Objective: Interleukine-1 receptor antagonist (IL-1RA is a powerful anti-inflammatory cytokine which limits the biological effects of IL-1. Due to structural similarity between IL-1 and its antagonist, IL-1RA competitively binds to IL-1 receptor which leads to no signal transduction. Therefore , it is applied in the treatment of patients with inflammatory diseases such as Rheumatoid Arthritis. The aim of this study is cloning, expression and op-timization of IL-1RA in E. coli. Materials & Methods: In this experimental study synthetically prepared cDNA was amplified by PCR. After double digestion with NdeI and XhoI restriction enzymes, this gene was cloned in pET28a expression vector. Expression of desired gene was analyzed at RNA level by RT-PCR and at protein level by SDS-PAGE and followed by western blot to confirm SDS-PAGE results. Optimization of recombinant protein expression was performed in dif-ferent IPTG concentrations and harvesting times after induction. Results: The presence of gene in pET28a was determined by colony-PCR and confirmed by restriction digestion. Transcription of cloned gene and expression of high yield recombinant protein were shown by RT-PCR and SDS-PAGE, respectively. The result of SDS-PAGE was confirmed by western blot. Expression was optimized in different induction time and IPTG concentrations Conclusion: The result of this study demonstrated expression of this recombinant protein at high level in E.coli system by pET28a expression vector. This study also showed a direct as-sociation between the increased level of expression and time of induction . Therefore, an overnight induction time with 0.1 mM IPTG concentration is recommended for a high level expression. (Sci J Hamadan Univ Med Sci 2014; 21 (2:145-151

  12. Development of expression vectors for Escherichia coli based on the pCR2 replicon

    Directory of Open Access Journals (Sweden)

    Deb J K

    2007-05-01

    Full Text Available Abstract Background Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility. Results We utilized the pCR2 replicon of Corynebacterium renale, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different Escherichia coli plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these E.coli plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the lac promoter in an expression plasmid pCR2GFP. Also high level production of human IFNγ was obtained by cloning the h-IFNγ under a T7 promoter in the expression plasmid pCR2-IFNγ and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNγ, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility. Conclusion We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression.

  13. Heterologous expression of mycobacterial Esx complexes in Escherichia coli for structural studies is facilitated by the use of maltose binding protein fusions.

    Directory of Open Access Journals (Sweden)

    Mark A Arbing

    Full Text Available The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.

  14. Secretory expression and enzymatic characterization of recombinant Agarivorans albus β-agarase in Escherichia coli.

    Science.gov (United States)

    Yoon, Sug-Young; Lee, Hyung-Min; Kong, Ji-Na; Kong, Kwang-Hoon

    2017-11-26

    Agarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding β-agarase from Agarivorans albus (Aaβ-agarase) in Escherichia coli. The synthetic β-agarase gene was designed based on the biased codons of E. coli to optimize its expression and extracellular secretion in an active, soluble form. The synthesized agarase gene, including its signal sequence, was cloned into the pET-26 expression vector, and the pET-Aaβ-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aaβ-agarase in Luria-Bertani broth containing 0.6 mM isopropyl β-D-1-thiogalactopyranoside for 9 h at 37°C. The expressed recombinant Aaβ-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding ∼10 mg/L Aaβ-agarase. The purified recombinant Aaβ-agarase exhibited optimal activity at pH 7 and 40°C, and its activity was strongly inhibited by Cu 2+ , Mn 2+ , Zn 2+ , and Al 3+ ions. Furthermore, the K M and k cat values for purified Aaβ-agarase were ∼0.02 mM and ∼45/s, respectively. These kinetic values were up to approximately 15-100-fold lower than the K M values reported for other agarases and approximately 7-30-fold higher than the k cat /K M values reported for other agarases, indicating that recombinant Aaβ-agarase exhibited good substrate-binding ability and high catalytic efficiency. These results demonstrated that the E. coli expression system was capable of producing recombinant Aaβ-agarase in an active form, at a high yield, and with attributes useful in the relevant industries.

  15. Inhibition of expression of virulence genes of Yersinia pestis in Escherichia coli by external guide sequences and RNase P.

    Science.gov (United States)

    Ko, Jae-hyeong; Izadjoo, Mina; Altman, Sidney

    2008-08-01

    External guide sequences (EGSs) targeting virulence genes from Yersinia pestis were designed and tested in vitro and in vivo in Escherichia coli. Linear EGSs and M1 RNA-linked EGSs were designed for the yscN and yscS genes that are involved in type III secretion in Y. pestis. RNase P from E. coli cleaves the messages of yscN and yscS in vitro with the cognate EGSs, and the expression of the EGSs resulted in the reduction of the levels of these messages of the virulence genes when those genes were expressed in E. coli.

  16. Functional expression of a human GDP-L-fucose transporter in Escherichia coli.

    Science.gov (United States)

    Förster-Fromme, Karin; Schneider, Sarah; Sprenger, Georg A; Albermann, Christoph

    2017-02-01

    To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes. The heterologous expression of the recombinant and codon-adapted human GDP-L-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-L-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of 3 H-GDP-L-fucose with a V max of 8 pmol/min mg with a K m of 4 µM. The functional expression of SLC35C1 in GDP-L-fucose overproducing E. coli led to the export of GDP-L-fucose to the culture supernatant. The export of GDP-L-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

  17. Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli.

    Science.gov (United States)

    Yang, Yu; Zhang, Dongxu; Liu, Song; Jia, Dongxu; Du, Guocheng; Chen, Jian

    2012-01-01

    Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.

  18. Development of Escherichia coli and Mycobacterium smegmatis recombinants expressing major Mycobacterium tuberculosis-specific antigenic proteins.

    Science.gov (United States)

    Amoudy, Hanady A; Safar, Hussain A; Mustafa, Abu S

    2016-12-01

    Mycobacterium tuberculosis is an obligate pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most tuberculosis (TB) cases. Until today, the only approved TB vaccine is Bacille Calmette Guerin (BCG), which has been used since 1921. While BCG provides fairly effective protection for infants and young children, its efficacy in adults is variable around the world. This could be due to several parameters including strains of the vaccine and exposure of individuals to different environmental bacterial infections. The situation is complicated by the emergence of multidrug resistant strains of M. tuberculosis. This urged the demand to develop new improved vaccines and immunotherapies against TB. Development of nonpathogenic recombinant constructs delivering M. tuberculosis-specific antigenic proteins provides the chance to evaluate candidates to be included in diagnostic tools and preventive vaccines. In our study, we are introducing some of the major M. tuberculosis genes in Escherichia coli and Mycobacterium smegmatis. DNA corresponding to the genes Rv3891, Rv3020, Rv0287, Rv3875, Rv3874, Rv3872, Rv2346c, and Rv3619 were PCR-amplified from M. tuberculosis genomic DNA and visualized on gel electrophoresis at the expected DNA size. Products were subsequently ligated to the plasmid pGEMTeasy and used to transform TOP10 E. coli. Transformed colonies were selected on appropriate media. At the second stage, genes-DNA were subcultured in expression vectors pDE22 and pGESTH1; the recombinant plasmids were finally used to transform. M. smegmatis and E. coli, respectively. Expression of proteins in E. coli was confirmed by Western blotting and in M. smegmatis by reverse transcriptase polymerase chain reaction (RT-PCR). Amplified genes were successfully cloned and transformed in E. coli and M. smegmatis. Colonies of recombinant bacteria were detected on appropriate media. Western blotting and RT-PCR confirmed the expression of our corresponding

  19. Screening of genetic parameters for soluble protein expression in Escherichia coli

    DEFF Research Database (Denmark)

    Vernet, Erik; Kotzsch, Alexander; Voldborg, Bjørn

    2011-01-01

    Soluble expression of proteins in a relevant form for functional and structural investigations still often remains a challenge. Although many biochemical factors are known to affect solubility, a thorough investigation of yield-limiting factors is normally not feasible in high-throughput efforts....... Here we present a screening strategy for expression of biomedically relevant proteins in Escherichia coli using a panel of six different genetic variations. These include engineered strains for rare codon supplementation, increased disulfide bond formation in the cytoplasm and novel vectors...... for secretion to the periplasm or culture medium. Combining these variants with expression construct truncations design, we report on parallel cloning and expression of more than 300 constructs representing 24 selected proteins; including full-length variants of human growth factors, interleukins and growth...

  20. Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Troii; Emmons, Thomas L.; Chrencik, Jill E.; Gormley, Jennifer A.; Weinberg, Robin A.; Leone, Joseph W.; Hirsch, Jeffrey L.; Saabye, Matthew J.; Schindler, John F.; Day, Jacqueline E.; Williams, Jennifer M.; Kiefer, James R.; Lightle, Sandra A.; Harris, Melissa S.; Guru, Siradanahalli; Fischer, H. David; Tomasselli, Alfredo G. (Pfizer)

    2012-05-29

    Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69 mg per 20 L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3 mg per 10 L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K{sub m} and k{sub cat}) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4 {angstrom}. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

  1. Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation.

    Science.gov (United States)

    Choi, Eunsil; Kang, Nalae; Jeon, Young; Pai, Hyun-Sook; Kim, Sung-Gun; Hwang, Jihwan

    2016-09-01

    The unique Escherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg(2+) concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium; two pathogenic bacteria, Staphylococcus aureus and Neisseria gonorrhoeae; and the extremophile Deinococcus radiodurans and then evaluated whether they could functionally complement the E. coli der-null phenotype. Only K. pneumoniae and S Typhimurium Der proteins enabled the E. coli der-null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression of K. pneumoniae and S Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused by E. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of the der-null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit. In Escherichia coli, Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study

  2. Expression of recombinant human colony stimulating factor (rhG-CSF) in Escherichia coli.

    OpenAIRE

    Fernanda Resende Gomes

    2010-01-01

    O Fator estimulador de colônias de granulócitos humano recombinante (rhG-CSF) produzido em Escherichia coli é uma proteína não glicosilada com 175 aminoácidos, de grande importância clínica para o tratamento de neutropenias. O presente trabalho propõe a construção de dois sistemas de expressão em E. coli, um sistema para obtenção do rhG-CSF no citoplasma e outro para secreção da proteína recombinante no meio de cultura utilizando a sequência sinal da L-asparaginase II. Os dois sistemas de exp...

  3. Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli.

    Science.gov (United States)

    Wang, Longgang; Wang, Wu

    2007-05-01

    The gene (choB(b)), encoding cholesterol oxidase from Brevibacterium sp. CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs and encodes a protein of 551 amino acids. ChoB(b) exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia coli BL21-CodonPlus (DE3)-RP grown at 23 degrees C in Luria-Bertani medium containing 50 microM riboflavin, the precursor of the FAD coenzyme of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a-choB(b).

  4. Diversification of gene expression during formation of static submerged biofilms by Escherichia coli

    Directory of Open Access Journals (Sweden)

    Olga Besharova

    2016-10-01

    Full Text Available Many bacteria primarily exist in nature as structured multicellular communities, so called biofilms. Biofilm formation is a highly regulated process that includes the transition from the motile planktonic to sessile biofilm lifestyle. Cellular differentiation within a biofilm is a commonly accepted concept but it remains largely unclear when, where and how exactly such differentiation arises. Here we used fluorescent transcriptional reporters to quantitatively analyze spatio-temporal expression patterns of several groups of genes during the formation of submerged Escherichia coli biofilms in an open static system. We first confirm that formation of such submerged biofilms as well as pellicles at the liquid-air interface requires the major matrix component, curli, and flagella-mediated motility. We further demonstrate that in this system, diversification of gene expression leads to emergence of at least three distinct subpopulations of E. coli, which differ in their levels of curli and flagella expression, and in the activity of the stationary phase sigma factor σS. Our study reveals mutually exclusive expression of curli fibers and flagella at the single cell level, with high curli levels being confined to dense cell aggregates/microcolonies and flagella expression showing an opposite expression pattern. Interestingly, despite the known σS-dependence of curli induction, there was only a partial correlation between the σS activity and curli expression, with subpopulations of cells having high σS activity but low curli expression and vice versa. Finally, consistent with different physiology of the observed subpopulations, we show striking differences between the growth rates of cells within and outside of aggregates.

  5. Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Chan, J W; Winhold, H; Corzett, M H; Ulloa, J M; Cosman, M; Balhorn, R; Huser, T

    2007-01-09

    Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in

  6. Cloning, Expression and Purification of Subunit H of Vacuolar H+-ATPase from Mythimna separata Walker (Lepidoptera: Noctuidae

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    Lina Lu

    2014-09-01

    Full Text Available The vacuolar (H+-ATPase (V-ATPase of insect, which is composed of membrane-bound V0 complex and peripheral V1 complex, participates in lots of important physiological process. Subunit H, as a subunit of V1 complex, plays a vital role in bridging the communication between V1 and V0 complexes and interaction with other proteins. Yeast subunit H has been successfully crystallized through expression in E. coli, but little is known about the structure of insect subunit H. In this study, we cloned, expressed and purified the subunit H from midgut of Mythimna separata Walker. Through RACE (rapidly amplification of cDNA ends technique, we got 1807 bp full length of subunit H, and to keep the nature structure of subunit H, we constructed Baculovirus expression vector with His-tag in the C-terminal and expressed the recombinant protein in insect sf9 cells, thereafter, purified the recombinant protein by Ni-NTA columns. Results of SDS-PAGE, western blotting and mass spectrometry showed that the recombinant protein was successfully expressed. The method of expressing and purifying M. separata subunit H will provide a foundation for obtaining the crystal of subunit H and further study of the design of novel insecticides based on its structure and function.

  7. Expression, purification, crystallization and preliminary crystallographic analysis of the proliferation-associated protein Ebp1

    Energy Technology Data Exchange (ETDEWEB)

    Kowalinski, Eva; Bange, Gert; Wild, Klemens; Sinning, Irmgard, E-mail: irmi.sinning@bzh.uni-heidelberg.de [Heidelberg University Biochemistry Center, INF 328, D-69120 Heidelberg (Germany)

    2007-09-01

    Preliminary X-ray analysis of the proliferation-associated protein Ebp1 from Homo sapiens is provided. ErbB-3-binding protein 1 (Ebp1) is a member of the family of proliferation-associated 2G4 proteins (PA2G4s) and plays a role in cellular growth and differentiation. Ligand-induced activation of the transmembrane receptor ErbB3 leads to dissociation of Ebp1 from the receptor in a phosphorylation-dependent manner. The non-associated protein is involved in transcriptional and translational regulation in the cell. Here, the overexpression, purification, crystallization and preliminary crystallographic studies of Ebp1 from Homo sapiens are reported. Initially observed crystals were improved by serial seeding to single crystals suitable for data collection. The optimized crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2 and diffracted to a resolution of 1.6 Å.

  8. Purification and Characterization of Recombinant Deinococcus radiodurans RNA Polymerase.

    Science.gov (United States)

    Esyunina, D M; Kulbachinskiy, A V

    2015-10-01

    The radioresistant bacterium Deinococcus radiodurans is one of the most interesting models for studies of cell stress resistance. Analysis of the mechanisms of gene expression in D. radiodurans revealed some specific features of the transcription apparatus that might play a role in cell resistance to DNA-damaging conditions. In particular, RNA polymerase from D. radiodurans forms unstable promoter complexes and during transcription elongation has a much higher rate of RNA cleavage than RNA polymerase from Escherichia coli. Analysis of the structure and functions of D. radiodurans RNA polymerase is complicated due to the absence of convenient genetic systems for making mutations in the RNA polymerase genes and difficulties with enzyme purification. In this work, we developed a system for expression of D. radiodurans RNA polymerase in E. coli cells. We obtained an expression vector encoding all core RNA polymerase subunits and defined optimal conditions for the expression and purification of the RNA polymerase. It was found that D. radiodurans RNA polymerase has much higher rates of RNA cleavage than E. coli RNA polymerase under a wide range of conditions, including variations in the concentration of catalytic magnesium ions and pH values of the reaction buffer. The expression system can be used for further studies of the RNA cleavage reaction and the mechanisms of transcription regulation in D. radiodurans, including analysis of mutant RNA polymerase variants.

  9. Discovering functional gene expression patterns in the metabolic network of Escherichia coli with wavelets transforms

    Directory of Open Access Journals (Sweden)

    Zapatka Marc

    2006-03-01

    Full Text Available Abstract Background Microarray technology produces gene expression data on a genomic scale for an endless variety of organisms and conditions. However, this vast amount of information needs to be extracted in a reasonable way and funneled into manageable and functionally meaningful patterns. Genes may be reasonably combined using knowledge about their interaction behaviour. On a proteomic level, biochemical research has elucidated an increasingly complete image of the metabolic architecture, especially for less complex organisms like the well studied bacterium Escherichia coli. Results We sought to discover central components of the metabolic network, regulated by the expression of associated genes under changing conditions. We mapped gene expression data from E. coli under aerobic and anaerobic conditions onto the enzymatic reaction nodes of its metabolic network. An adjacency matrix of the metabolites was created from this graph. A consecutive ones clustering method was used to obtain network clusters in the matrix. The wavelet method was applied on the adjacency matrices of these clusters to collect features for the classifier. With a feature extraction method the most discriminating features were selected. We yielded network sub-graphs from these top ranking features representing formate fermentation, in good agreement with the anaerobic response of hetero-fermentative bacteria. Furthermore, we found a switch in the starting point for NAD biosynthesis, and an adaptation of the l-aspartate metabolism, in accordance with its higher abundance under anaerobic conditions. Conclusion We developed and tested a novel method, based on a combination of rationally chosen machine learning methods, to analyse gene expression data on the basis of interaction data, using a metabolic network of enzymes. As a case study, we applied our method to E. coli under oxygen deprived conditions and extracted physiologically relevant patterns that represent an

  10. Expression, purification and preliminary X-ray characterization of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1

    Energy Technology Data Exchange (ETDEWEB)

    Omi, Rie [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Department of Chemistry, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Jitsumori, Keiji; Yamauchi, Takahiro; Ichiyama, Susumu; Kurihara, Tatsuo; Esaki, Nobuyoshi [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kamiya, Nobuo; Hirotsu, Ken, E-mail: hirotsu@sci.osaka-cu.ac.jp; Miyahara, Ikuko [Department of Chemistry, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2007-07-01

    A recombinant form of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1 has been expressed in E. coli, purified and crystallized. The crystal belongs to space group P6{sub 3}. Diffraction data have been collected to 1.75 Å resolution. dl-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (dl-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of dl-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P6{sub 3}, with unit-cell parameters a = b = 186.2, c = 114.4 Å. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V{sub M} value of 4.20–2.10 Å{sup 3} Da{sup −1}. A self-rotation function revealed peaks on the χ = 180° section. X-ray data have been collected to 1.75 Å resolution.

  11. Expression and purification of a new recombinant camel hepcidin able to promote the degradation of the iron exporter ferroportin1.

    Science.gov (United States)

    Boumaiza, Mohamed; Jaouen, Maryse; Deschemin, Jean-Christophe; Ezzine, Aymen; Ben Khalaf, Noureddine; Vaulont, Sophie; Marzouki, Mohamed Nèjib; Sari, Marie Agnès

    2015-11-01

    Hepcidin, a 25-amino-acid and highly disulfide bonded antimicrobial peptide, is the central regulator of iron homeostasis. This hormone is expressed in response to iron and inflammation and interacts with ferroportin1 (FPN1), the only known iron exporter in vertebrates, inducing its internalization and degradation. Thus, the export of iron from cells to plasma will be significantly diminished. Thereby, hepcidin has become the target of intense research studies due to its profound biomedical significance. This study describes the functional expression of recombinant camel hepcidin in Escherichia coli. Biologically active recombinant camel hepcidin was obtained thanks to the production of a hepcidin-thioredoxin fusion protein (TRX-HepcD) and a purified camel hepcidin, with an extra methionine at the N-terminus, was obtained after enterokinase cleavage of the fusion protein. Presence of the four disulfide bridges was verified using MALDI-ToF spectrometry. The recombinant camel hepcidin was compared to related synthetic bioactive peptides, including human hepcidin, and was found equally able to promote ferroportin degradation of mouse macrophages. Furthermore, camel hepcidins exhibits a high capacity to inhibit the growth of Leishmania major promastigotes. These results proved that production of functional camel hepcidin can be achieved in E. coli, this is a major interest for the production of cysteine rich peptides or proteins that can be purified under their functional form without the need of a refolding process. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase.

    Science.gov (United States)

    Yaoi, Katsuro; Mitsuishi, Yasushi

    2002-12-13

    A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp. M128. Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG-RCBH had exoglucanase activity. It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain. The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein. The deduced protein showed approximately 35% identity to members of glycoside hydrolase family 74. The cDNA encoding OXG-RCBH was then expressed in Escherichia coli. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained.

  13. Expression of ornithine decarboxylase of Coccidioides immitis in three Escherichia coli strains carrying the lambda DE3 lysogen and an E. coli EWH319 strain odc- null mutant.

    Science.gov (United States)

    Pantoja-Hernández, Miguel Angel; Muñoz-Sánchez, Claudia Ivonne; Guevara-González, Ramón Gerardo; Botello-Alvarez, Enrique; González-Chavira, Mario Martín; Torres-Pacheco, Irineo; Guevara-Olvera, Lorenzo

    2004-01-01

    Ornithine decarboxylase from respiratory fungal pathogen, Coccidioides immitis, cloned in the pETCiODC plasmid under control of T7lac promoter, was produced in E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3) and EWH319 transformant strains. E. coli BL21(DE3)pLysS-pETCiODC expressed the highest specific activity of ODC, suggesting that this strain could be successfully used for protein structure and drug testing studies.

  14. Expression, purification and characterization of the chitinolytic beta-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis.

    Science.gov (United States)

    Liu, Tian; Liu, Fengyi; Yang, Qing; Yang, Jun

    2009-11-01

    Insect beta-N-acetyl-D-hexosaminidases are of particular interest due to their multiple physiological roles in many life processes. Chitinolytic beta-N-acetyl-D-hexosaminidases, which function only in chitin degradation in insects, have long been regarded as species-specific target potentials in developing environmental friendly pesticides. Here the chitinolytic beta-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis was cloned and expressed in the yeast strain, Pichia pastoris, to meet the demands of biochemical studies and drug development. Enzymatic assay as well as Western blot confirmed that the high-level expression could be achieved after the induction of methanol for 120 h. Through the sequential combination of ammonium sulfate precipitation, metal chelating chromatography as well as anion exchange chromatography, 7.7 mg of the recombinant OfHex1 with high purity was obtained from 1 liter of culture supernatant. The recombinant OfHex1, characterized as a homodimer with molecular weight of 130 kDa, exhibited the same enzymatic activities as its native form, which could efficiently degrade the chitooligosaccharide substrate (GlcNAc)2 and release 4-methylumbelliferone (4MU) from substrates, 4MU-beta-GlcNAc and 4MU-beta-GalNAc. This work provides a low-costing and high-efficient purification procedure for the preparation of insect beta-N-acetyl-D-hexosaminidases.

  15. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the aspartate transcarbamoylase domain of human CAD.

    Science.gov (United States)

    Ruiz-Ramos, Alba; Lallous, Nada; Grande-García, Araceli; Ramón-Maiques, Santiago

    2013-12-01

    Aspartate transcarbamoylase (ATCase) catalyzes the synthesis of N-carbamoyl-L-aspartate from carbamoyl phosphate and aspartate in the second step of the de novo biosynthesis of pyrimidines. In prokaryotes, the first three activities of the pathway, namely carbamoyl phosphate synthetase (CPSase), ATCase and dihydroorotase (DHOase), are encoded as distinct proteins that function independently or in noncovalent association. In animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD. Up-regulation of CAD is essential for normal and tumour cell proliferation. Although the structures of numerous prokaryotic ATCases have been determined, there is no structural information about any eukaryotic ATCase. In fact, the only detailed structural information about CAD is that it self-assembles into hexamers and trimers through interactions of the ATCase domains. Here, the expression, purification and crystallization of the ATCase domain of human CAD is reported. The recombinant protein, which was expressed in bacteria and purified with good yield, formed homotrimers in solution. Crystallization experiments both in the absence and in the presence of the inhibitor PALA yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. The crystals appeared to belong to the hexagonal space group P6(3)22, and Matthews coefficient calculation indicated the presence of one ATCase subunit per asymmetric unit, with a solvent content of 48%. However, analysis of the intensity statistics suggests a special case of the P21 lattice with pseudo-symmetry and possibly twinning.

  16. Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

    Directory of Open Access Journals (Sweden)

    Pierre-Olivier Buclez

    2016-01-01

    Full Text Available Recombinant adeno-associated viruses (rAAV are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology.

  17. Expression, Purification, and Functional Characterization of Atypical Xenocin, Its Immunity Protein, and Their Domains from Xenorhabdus nematophila

    Directory of Open Access Journals (Sweden)

    Jitendra Singh Rathore

    2013-01-01

    Full Text Available Xenorhabdus nematophila, a gram-negative bacterium belonging to the family Enterobacteriaceae is a natural symbiont of a soil nematode from the family Steinernematidae. In this study cloning, expression, and purification of broad range iron regulated multidomain bacteriocin called xenocin from X. nematophila (66 kDa, encoded by xcinA gene and its multidomain immunity protein (42 kDa, encoded by ximB gene have been done. xcinA-ximB (N′ terminal 270 bp, translocation, and translocation-receptor domain of xcinA, ximB, and its hemolysin domain were cloned, expressed, and purified by single step Ni-NTA chromatography under native conditions. In the functional characterization, neutralization of xcinA toxicity by immunity domain of ximB gene was determined by endogenous assay. Exogenous toxic assays results showed that only the purified recombinant xenocin-immunity domain (10 kDa protein complex had toxic activity. Atypical cognate immunity protein (42 kDa of xenocin was fusion of immunity domain (10 kDa and hemolysin domain (32 kDa. In silico analysis of immunity protein revealed its similarity with hemolysin and purine NTPase like proteins. Hemolytic activity was not observed in immunity protein or in its various domains; however, full-length immunity protein lacking Walker motif showed ATPase activity. Finally, using circular dichroism performed secondary structural analyses of all the recombinant proteins/protein complexes.

  18. Expression, purification, crystallization and preliminary X-ray diffraction analysis of α-11 giardin from Giardia lamblia

    Energy Technology Data Exchange (ETDEWEB)

    Pathuri, Puja; Nguyen, Emily Tam [Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697 (United States); Luecke, Hartmut, E-mail: hudel@uci.edu [Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697 (United States); Department of Physiology and Biophysics, University of California, Irvine, CA 92697 (United States); Department of Information and Computer Sciences, University of California, Irvine, CA 92697 (United States)

    2006-11-01

    α-11 giardin from the intestinal protozoan parasite, G. lamblia has been cloned, expressed, purified and crystallized under two different conditions and in two different space groups. Crystals from the first condition diffracted to 1.1 Å and belong to a primitive orthorhombic space group and crystals obtained in the second condition diffracted to 2.93 Å and belong to a primitive monoclinic space group. α-11 Giardin, a protein from the annexin superfamily, is a 35.0 kDa protein from the intestinal protozoan parasite Giardia lamblia which triggers a form of diarrhea called giardiasis. Here, the cloning, expression, purification and the crystallization of α-11 giardin under two different conditions and in two different space groups is reported. Crystals from the first condition diffracted to 1.1 Å and belong to a primitive orthorhombic space group, while crystals from the second condition, which included calcium in the crystallization solution, diffracted to 2.93 Å and belong to a primitive monoclinic space group. Determination of the detailed atomic structure of α-11 giardin will provide a better insight into its biological function and might establish whether this class of proteins is a potential drug target against giardiasis.

  19. Expression and Purification of Functional Human Vascular Endothelial Growth Factor-A121; the Most Important Angiogenesis Factor

    Directory of Open Access Journals (Sweden)

    Fatemeh Kazemi-Lomedasht

    2014-12-01

    Full Text Available Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated. Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro. Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.

  20. Intestinal Microbiota-Derived GABA Mediates Interleukin-17 Expression during EnterotoxigenicEscherichia coliInfection.

    Science.gov (United States)

    Ren, Wenkai; Yin, Jie; Xiao, Hao; Chen, Shuai; Liu, Gang; Tan, Bie; Li, Nengzhang; Peng, Yuanyi; Li, Tiejun; Zeng, Benhua; Li, Wenxia; Wei, Hong; Yin, Zhinan; Wu, Guoyao; Hardwidge, Philip R; Yin, Yulong

    2016-01-01

    Intestinal microbiota has critical importance in pathogenesis of intestinal infection; however, the role of intestinal microbiota in intestinal immunity during enterotoxigenic Escherichia coli (ETEC) infection is poorly understood. The present study tested the hypothesis that the intestinal microbiota is associated with intestinal interleukin-17 (IL-17) expression in response to ETEC infection. Here, we found ETEC infection induced expression of intestinal IL-17 and dysbiosis of intestinal microbiota, increasing abundance of γ-aminobutyric acid (GABA)-producing Lactococcus lactis subsp. lactis . Antibiotics treatment in mice lowered the expression of intestinal IL-17 during ETEC infection, while GABA or L. lactis subsp. lactis administration restored the expression of intestinal IL-17. L. lactis subsp. lactis administration also promoted expression of intestinal IL-17 in germ-free mice during ETEC infection. GABA enhanced intestinal IL-17 expression in the context of ETEC infection through activating mechanistic target of rapamycin complex 1 (mTORC1)-ribosomal protein S6 kinase 1 (S6K1) signaling. GABA-mTORC1 signaling also affected intestinal IL-17 expression in response to Citrobacter rodentium infection and in drug-induced model of intestinal inflammation. These findings highlight the importance of intestinal GABA signaling in intestinal IL-17 expression during intestinal infection and indicate the potential of intestinal microbiota-GABA signaling in IL-17-associated intestinal diseases.

  1. Host Inflammatory Response Inhibits Escherichia coli O157:H7 Adhesion to Gut Epithelium through Augmentation of Mucin Expression

    Science.gov (United States)

    Xue, Yansong; Zhang, Hanying; Wang, Hui; Hu, Jia; Du, Min

    2014-01-01

    Escherichia coli O157:H7, a major Shiga toxin-producing pathogen, has a low infectious dose and causes serious illness in humans. The gastrointestinal tract of cattle is the primary reservoir of E. coli O157:H7, and thus, it is critical to eliminate or reduce E. coli O157:H7 gut colonization. Given that E. coli O157:H7 produces effectors that attenuate inflammatory signaling, we hypothesized that the host inflammatory response acts to perturb E. coli O157:H7 intestinal colonization. Tumor necrosis factor alpha (TNF-α) treatment of HT-29 cells resulted in increased expression of inflammatory cytokine interleukin 1β (IL-1β), IL-8, and TNF-α genes and increased IL-8 protein and resulted in decreased adhesion of E. coli O157:H7. Similarly, E. coli O157:H7 adhesion to cattle colonic explants was reduced by TNF-α treatment. Irrespective of the presence of E. coli O157:H7, TNF-α enhanced activation of p65, the key mediator of NF-κB inflammatory signaling, whereas E. coli O157:H7 infection suppressed this pathway by inhibiting p65 activation in HT-29 cells. To further explore the mechanisms linking the inflammatory response to attenuated E. coli O157:H7 adhesion, mucin 2 (MUC2) expression was analyzed, considering that the intestinal mucus layer is the first defense against enteric pathogens and MUC2 is the major secretory mucin in the intestine. MUC2 expression in HT-29 cells was increased by TNF-α treatment and by E. coli O157:H7 infection. However, reducing mucin expression by blocking mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinases 1/2 (ERK1/2) and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling increased E. coli O157:H7 adherence to HT-29 cells. These data suggest that the inflammatory cytokine response acts to protect host epithelial cells against E. coli O157:H7 colonization, at least in part, by promoting mucin production. PMID:24566630

  2. A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

    Directory of Open Access Journals (Sweden)

    Ramos C.R.R.

    2004-01-01

    Full Text Available We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET, expression of a short 6XHis tag at N-terminus (pET3-His and a high copy number of plasmid (pRSET. The small size of the vector (2.8 kb and the high copy number/cell (200-250 copies facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture. In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site. Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

  3. Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein {alpha}-subunit

    Energy Technology Data Exchange (ETDEWEB)

    Abdulaev, Najmoutin G. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Zhang Cheng; Dinh, Andy [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States); Ngo, Tony; Bryan, Philip N. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Brabazon, Danielle M. [Loyola College in Maryland, Department of Chemistry (United States); Marino, John P. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States)], E-mail: marino@carb.nist.gov; Ridge, Kevin D. [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States)

    2005-05-15

    Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein {alpha}-subunit (G{sub {alpha}}) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G{sub {alpha}} chimera ({approx}40 kDa polypeptide) has been tested. The results show that a prodomain fused G{sub {alpha}} chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G{sub {alpha}} isolated from natural sources. To assay for the functional integrity of the purified G{sub {alpha}} chimera at NMR concentrations and probe for changes in the structure and dynamics of G{sub {alpha}} that result from activation, {sup 15}N-HSQC spectra of the GDP/Mg{sup 2+} bound form of G{sub {alpha}} obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the {sup 15}N-HSQC spectra reveals a number of changes in chemical shifts of the {sup 1}HN, {sup 15}N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G{sub {alpha}} activation.

  4. Zinc oxide nanoparticle reduced biofilm formation and antigen 43 expressions in uropathogenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ali Shakerimoghaddam

    2017-04-01

    Full Text Available Objective(s: This study aimed to investigate the effect of zinc oxide nanoparticles (ZnO-np on biofilm formation and expression of the flu gene in uropathogenic Escherichia coli (UPEC strains. Materials and Methods: Minimum inhibitory concentration (MIC of ZnO-np was determined by agar dilution method. The effect of MIC and sub-MIC concentrations of ZnO-np on biofilm formation were determined by microtiter plate assay. The expression level of the flu gene was assessed by Real-Time PCR assay. Results: MIC and sub-MIC ZnO-np concentrations reduced biofilm formation by 50% and 33.4%, respectively. Sub-MIC ZnO-np concentration significantly reduced the flu gene expression in the UPEC isolates (P

  5. Intracellular polyamine pools, oligopeptide-binding protein A expression, and resistance to aminoglycosides in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Maria BR Acosta

    2005-11-01

    Full Text Available The role of intracellular free polyamine (putrescine and spermidine pools in multiple resistance to aminoglycoside antibiotics was investigated among in vitro selected kanamycin-resistant Escherichia coli J53 mutants expressing diminished oligopeptide-binding protein (OppA levels and/or defective ornithine decarboxylase (ODC activity. The results suggest that diminished OppA content, but not defective ODC activity expression, increased the relative concentration of free spermidine as compared to the wild type strain. Moreover, by adding exogenous polyamines or polyamine synthesis inhibitors to cultures with different mutant strains, a direct relationship between the intracellular OppA levels and resistance to kanamycin was revealed. Collectively these results further suggest a complex relation among OppA expression, aminoglycoside resistance and polyamine metabolism.

  6. Rare codons effect on expression of recombinant gene cassette in Escherichia coli BL21(DE3

    Directory of Open Access Journals (Sweden)

    Aghil Esmaeili-Bandboni

    2017-11-01

    Full Text Available Objective: To demonstrate the sensitivity of expression of fusion genes to existence of a large number of rare codons in recombinant gene sequenced. Methods: Primers for amplification of cholera toxin B, Shiga toxin B and gfp genes were designed by Primer3 software and synthesized. All of these 3 genes were cloned. Then the genes were fused together by restriction sites and enzymatic method. Two linkers were used as a flexible bridge in connection of these genes. Results: Cloning and fusion of cholera toxin B, Shiga toxin B and gfp genes were done correctly. After that, expression of the recombinant gene construction was surveyed. Conclusions: According to what was seen, because of the accumulation of 12 rare codons of Shiga toxin B and 19 rare codons of cholera toxin B in this gene cassette, the expression of the recombinant gene cassette, in Escherichia coli BL21, failed.

  7. Impact of intramammary treatment on gene expression profiles in bovine Escherichia coli mastitis.

    Science.gov (United States)

    Sipka, Anja; Klaessig, Suzanne; Duhamel, Gerald E; Swinkels, Jantijn; Rainard, Pascal; Schukken, Ynte

    2014-01-01

    Clinical mastitis caused by E. coli accounts for significant production losses and animal welfare concerns on dairy farms worldwide. The benefits of therapeutic intervention in mild to moderate cases are incompletely understood. We investigated the effect of intramammary treatment with cefapirin alone or in combination with prednisolone on gene expression profiles in experimentally-induced E. coli mastitis in six mid-lactating Holstein Friesian cows. Cows were challenged with E. coli in 3 quarters and received 4 doses of 300 mg cefapirin in one quarter and 4 doses of 300 mg cefapirin together with 20 mg prednisolone in another quarter. At 24 h (n = 3) or 48 h (n = 3) post-challenge, tissue samples from control and treated quarters were collected for microarray analysis. Gene expression analysis of challenged, un-treated quarters revealed an up-regulation of transcripts associated with immune response functions compared to un-challenged quarters. Both treatments resulted in down-regulation of these transcripts compared to challenged, un-treated quarters most prominently for genes representing Chemokine and TLR-signaling pathways. Gene expression of Lipopolysaccharide Binding Protein (LBP), CCL2 and CXCL2 were only significantly down-regulated in cefapirin-prednisolone-treated quarters compared to un-treated controls. Down-regulation of chemokines was further confirmed on the basis of protein levels in milk whey for CXCL1, CXCL2 and CXCL8 in both treatments with a greater decrease in cefapirin-prednisolone-treated quarters. The data reveal a significant effect of treatment on cell recruitment with a more pronounced effect in cefapirin-prednisolone treated quarters. Provided a rapid bacteriological clearance, combination therapy may prevent neutrophil-induced tissue damage and promote recovery of the gland.

  8. Impact of intramammary treatment on gene expression profiles in bovine Escherichia coli mastitis.

    Directory of Open Access Journals (Sweden)

    Anja Sipka

    Full Text Available Clinical mastitis caused by E. coli accounts for significant production losses and animal welfare concerns on dairy farms worldwide. The benefits of therapeutic intervention in mild to moderate cases are incompletely understood. We investigated the effect of intramammary treatment with cefapirin alone or in combination with prednisolone on gene expression profiles in experimentally-induced E. coli mastitis in six mid-lactating Holstein Friesian cows. Cows were challenged with E. coli in 3 quarters and received 4 doses of 300 mg cefapirin in one quarter and 4 doses of 300 mg cefapirin together with 20 mg prednisolone in another quarter. At 24 h (n = 3 or 48 h (n = 3 post-challenge, tissue samples from control and treated quarters were collected for microarray analysis. Gene expression analysis of challenged, un-treated quarters revealed an up-regulation of transcripts associated with immune response functions compared to un-challenged quarters. Both treatments resulted in down-regulation of these transcripts compared to challenged, un-treated quarters most prominently for genes representing Chemokine and TLR-signaling pathways. Gene expression of Lipopolysaccharide Binding Protein (LBP, CCL2 and CXCL2 were only significantly down-regulated in cefapirin-prednisolone-treated quarters compared to un-treated controls. Down-regulation of chemokines was further confirmed on the basis of protein levels in milk whey for CXCL1, CXCL2 and CXCL8 in both treatments with a greater decrease in cefapirin-prednisolone-treated quarters. The data reveal a significant effect of treatment on cell recruitment with a more pronounced effect in cefapirin-prednisolone treated quarters. Provided a rapid bacteriological clearance, combination therapy may prevent neutrophil-induced tissue damage and promote recovery of the gland.

  9. Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Beatriz Lázaro

    Full Text Available Triglycerides (TAGs, the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT. With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3-fold in the monounsaturated fatty acids (MUFA occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids.

  10. Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded genetic code.

    Science.gov (United States)

    Liu, Chang C; Cellitti, Susan E; Geierstanger, Bernhard H; Schultz, Peter G

    2009-01-01

    Tyrosine sulfation is an important post-translational modification that occurs in higher eukaryotes and is involved in cell-cell communication, viral entry and adhesion. We describe a protocol for the heterologous expression of selectively tyrosine-sulfated proteins in Escherichia coli through the use of an expanded genetic code that co-translationally inserts sulfotyrosine in response to the amber nonsense codon, TAG. The components required for this process, an orthogonal aminoacyl-tRNA synthetase specific for sulfotyrosine and its cognate orthogonal tRNA that recognizes the amber codon, are encoded on the plasmid pSUPAR6-L3-3SY, and their use, along with a simple chemical synthesis of sulfotyrosine, are outlined in this protocol. Specifically, the gene for a protein of interest is mutated such that the codon corresponding to the desired location of tyrosine sulfate is TAG. Co-transformation of an expression vector containing this gene and pSUPAR6-L3-3SY into an appropriate E. coli strain allows the overexpression of the site-specifically sulfated protein with high efficiency and fidelity. The resulting protein contains tyrosine sulfate at any location specified by a TAG codon, making this method significantly simpler and more versatile than competing methods such as in vitro enzymatic sulfation, chemical sulfation and peptide synthesis. Once the proper expression vectors are cloned, our protocol should allow the production of the desired sulfated proteins in <1 week.

  11. High-yield Escherichia coli-based cell-free expression of human proteins

    Energy Technology Data Exchange (ETDEWEB)

    Michel, Erich; Wuethrich, Kurt, E-mail: wuthrich@mol.biol.ethz.ch [ETH Zurich, Institute of Molecular Biology and Biophysics (Switzerland)

    2012-05-15

    Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [{sup 15}N,{sup 1}H]-HSQC NMR.

  12. Expression of Caenorhabditis elegans antimicrobial peptide NLP-31 in Escherichia coli

    Science.gov (United States)

    Lim, Mei-Perng; Nathan, Sheila

    2014-09-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a fulminant disease endemic in Southeast Asia and Northern Australia. The standardized form of therapy is antibiotics treatment; however, the bacterium has become increasingly resistant to these antibiotics. This has spurred the need to search for alternative therapeutic agents. Antimicrobial peptides (AMPs) are small proteins that possess broad-spectrum antimicrobial activity. In a previous study, the nematode Caenorhabditis elegans was infected by B. pseudomallei and a whole animal transcriptome analysis identified a number of AMP-encoded genes which were induced significantly in the infected worms. One of the AMPs identified is NLP-31 and to date, there are no reports of anti-B. pseudomallei activity demonstrated by NLP-31. To produce NLP-31 protein for future studies, the gene encoding for NLP-31 was cloned into the pET32b expression vector and transformed into Escherichia coli BL21(DE3). Protein expression was induced with 1 mM IPTG for 20 hours at 20°C and recombinant NLP-31 was detected in the soluble fraction. Taken together, a simple optimized heterologous production of AMPs in an E. coli expression system has been successfully developed.

  13. Vitreoscilla hemoglobin expression in engineered Escherichia coli: improved performance in high cell-density batch cultivations.

    Science.gov (United States)

    Pablos, Tania E; Mora, Eugenio Meza; Le Borgne, Sylvie; Ramírez, Octavio T; Gosset, Guillermo; Lara, Alvaro R

    2011-08-01

    High cell-density cultivations are the preferred system for biomolecules production by Escherichia coli. It has been previously demonstrated that a strain of E. coli with a modified substrate transport system is able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. The use of elevated amounts of glucose from the beginning of the cultivation, eliminates the existence of substrate gradients due to deficient mixing at large-scale. However, the large amounts of oxygen demanded resulted in microaerobic conditions after some hours of cultivation, even at small-scale. In this work, the effect of expressing the Vitreoscilla hemoglobin (VHb) in the engineered strain during batch cultures using high-glucose concentrations was tested. Together, the expression of VHb and the modified substrate transport system resulted in a 33% increase of biomass production compared to the parental strain (W3110) lacking the VHb in batch cultivations using 25 g/L of glucose. When 50 g/L of glucose were used, expression of VHb in the modified strain led to 11% higher biomass production compared to W3110. The VHb also increased the growth rates of the strains by about 30% in the aerobic phase and more than 200% in the microaerobic phase of batch cultivation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Optimization of the Expression of Reteplase in Escherichia coli TOP10 Using Arabinose Promoter

    OpenAIRE

    Shafiee, Fatemeh; Moazen, Fatemeh; Rabbani, Mahammad; Mir Mohammad Sadeghi, Hamid

    2015-01-01

    Background: Reteplase is a mutant version of t-PA (tissue plasminogen activator) with prolonged half-life. In the present study, E. coli Top 10 bacteria were utilized in the production of reteplase, which is the nonglycosylated active domain of t-PA. Reteplase gene was ligated into pBAD/gIII plasmid which, allows secretion of this protein in periplasmic space. It would allow the correct formation of disulfide bonds in protein structure. Objectives: This study aimed at expression of reteplase ...

  15. Uropathogenic Escherichia coli Express Type 1 Fimbriae Only in Surface Adherent Populations Under Physiological Growth Conditions.

    Science.gov (United States)

    Stærk, Kristian; Khandige, Surabhi; Kolmos, Hans Jørn; Møller-Jensen, Jakob; Andersen, Thomas Emil

    2016-02-01

    Most uropathogenic Escherichia coli (UPEC) strains harbor genes encoding adhesive type 1 fimbria (T1F). T1F is a key factor for successful establishment of urinary tract infection. However, UPEC strains typically do not express T1F in the bladder urine, and little is understood about its induction in vivo. A flow chamber infection model was used to grow UPEC under conditions simulating distinct infection niches in the bladder. Type 1 fimbriation on isolated UPEC was subsequently determined by yeast cell agglutination and immunofluorescence microscopy, and the results were correlated with the ability to adhere to and invade cultured human bladder cells. Although inactive during planktonic growth in urine, T1F expression occurs when UPEC settles on and infects bladder epithelial cells or colonizes catheters. As a result, UPEC in these sessile populations enhances bladder cell adhesion and invasion potential. Only T1F-negative UPEC are subsequently released to the urine, thus limiting T1F expression to surface-associated UPEC alone. Our results demonstrate that T1F expression is strictly regulated under physiological growth conditions with increased expression during surface growth adaptation and infection of uroepithelial cells. This leads to separation of UPEC into low-expression planktonic populations and high-expression sessile populations. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  16. Targeted amino-terminal acetylation of recombinant proteins in E. coli.

    Directory of Open Access Journals (Sweden)

    Matthew Johnson

    Full Text Available One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co-expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.

  17. [Prokaryotic expression, purification and antigenicity identification of human renal cell carcinoma-associated antigen G250].

    Science.gov (United States)

    Xiao, Yi; Gao, Jiangping; Gao, Kun; Yan, Jinqi; Zhang, Liang; Wang, Yu; Xu, Yuanji; Wang, Wei; Wang, Xiaoxiong; Yu, Jiyun

    2013-03-01

    To amplify human renal cell carcinoma (RCC)-associated antigen G250 gene and construct a recombinant plasmid pET-42a-hG250, express and purify human G250 protein and identify its antigenicity. The gene of human G250 was amplified from pGEM-T-G250 by PCR. After sequencing, the PCR product (112-1242 bp) was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-hG250. The plasmid was transformed into BL21 (DE3) and human G250 protein was expressed under the induction of IPTG. The fusion protein was purified and identified by SDS-PAGE, Western blotting and ELISA sequentially. The human G250 prokaryotic expression vector pET-42a-hG250 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. After transformation into BL21 (DE3), the target protein was successfully induced to express and purified as expected. Western blotting and ELISA demonstrated that the purified human G250 protein had a desirable immunogenicity. The recombinant prokaryotic expression vector pET-42a-hG250 has been constructed successfully. The purified human G250 protein has a good antigenicity.

  18. Expression, purification and characterization of an atypical 2-Cys peroxiredoxin from the silkworm, Bombyx mori.

    Science.gov (United States)

    Zhang, L; Lu, Z

    2015-04-01

    Peroxiredoxins (Prxs) play important roles in protecting organisms against damage caused by reactive oxygen species (ROS). In this study, we cloned a cDNA of Bombyx mori peroxiredoxin 5 (BmPrx5), which contained a 565-bp open reading frame for a 188-residue protein. Sequence analysis indicated that BmPrx5 belongs to the atypical 2-Cys peroxiredoxin family. Recombinant BmPrx5 purified from Escherichia coli showed antioxidant activity that removes H2 O2 and protects DNA from oxidative damage. Quantitative real-time PCR showed that the level of BmPrx5 mRNA in haemocytes increased early and decreased by 24 h after injection of H2 O2 whereas, in the fat body, the transcript level decreased at 6 h and increased at 12 h. Pseudomonas aeruginosa and Staphylococcus aureus infection resulted in higher levels of H2 O2 in the haemolymph and of BmPrx5 mRNA in haemocytes at 8 h postinfection. These data suggest that BmPrx5 acts as an antioxidant enzyme to protect the silkworm from oxidative damage induced by bacterial infection. Further study is needed to elucidate the exact role of BmPrx5 in the silkworm immune system. © 2014 The Royal Entomological Society.

  19. The use of hipO, encoding benzoylglycine amidohydrolase (hippuricase), as a reporter of gene expression in Campylobacter coli.

    Science.gov (United States)

    Park, S F

    1999-04-01

    A novel integrative promoter probe vector which utilizes the Campylobacter jejuni hipO gene as a reporter of gene expression was developed as a genetic tool in Campylobacter coli. The utility of the system was demonstrated by coupling expression of the hipO reporter to the promoters of flaA, flaB and katA. Subsequently, expression of these genes could be monitored accurately from chromosomally-borne transcriptional fusions using a simple non-destructive colorimetric assay. The system should serve as a useful tool for studying the response of Camp. coli to environmental stresses.

  20. [Prokaryotic expression, purification of human LINGO-1(aa76-319) and preparation of its polyclonal antibody].

    Science.gov (United States)

    Lv, Jun; Lu, Xin; Jiang, Xiao-Dan; Hu, Chang-Chen; Cai, Ying-Qian; DU, Mou-Xuan; Zou, Yu-Xi; Qin, Ling-Sha

    2009-11-01

    To express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb). The 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting. The prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity. The fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.

  1. Role of curli and cellulose expression in adherence of Escherichia coli O157:H7 to spinach leaves.

    Science.gov (United States)

    Macarisin, Dumitru; Patel, Jitendra; Bauchan, Gary; Giron, Jorge A; Sharma, Vijay K

    2012-02-01

    Shiga-toxigenic Escherichia coli O157:H7 outbreaks have been linked to consumption of fresh produce. It is generally recognized that bacterial attachment to vegetal matrices constitutes the first step in contamination of fresh produce. Cellular appendages, such as curli fibers, and cellulose, a constituent of extracellular matrix, have been suggested to be involved in E. coli attachment and persistence in fresh produce. A comparative evaluation was conducted on the ability of Shiga toxin-producing E. coli O157:H7 strains EDL933 and 86-24, linked to two independent foodborne disease outbreaks in humans, and their mutants deficient in curli and/or cellulose expression to colonize and to firmly attach to spinach leaf. Inoculated spinach leaves were incubated at 22°C, and at 0, 24, and 48 h after incubation loosely and strongly attached E. coli O157:H7 populations were determined. Curli-expressing E. coli O157:H7 strains developed stronger association with leaf surface, whereas curli-deficient mutants attached to spinach at significantly (pspinach leaves was not significantly different from that of curliated strains. The relative attachment strength of E. coli O157:H7 to spinach increased with incubation time for the curli-expressing strains. Laser scanning confocal microscopy (LSCM) analysis of inoculated leaves revealed that curli-expressing E. coli O157:H7 were surrounded by extracellular structures strongly immunostained with anti-curli antibodies. Production of cellulose was not required to develop strong attachment to spinach leaf. These results indicate that curli fibers are essential for strong attachment of E. coli O157:H7 to spinach whereas cellulose is dispensable.

  2. Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

    CERN Document Server

    Foulon, V; Croes, K; Waelkens, E

    1999-01-01

    Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

  3. An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains.

    Science.gov (United States)

    Fleetwood, Filippa; Andersson, Ken G; Ståhl, Stefan; Löfblom, John

    2014-12-30

    Cell display technologies (e.g. bacterial display) are attractive in directed evolution as they provide the option to use flow-cytometric cell sorting for selection from combinatorial libraries. The aim of this study was to engineer and investigate an expression vector system with dual functionalities: i) recombinant display of Affibody libraries on Escherichia coli for directed evolution and ii) small scale secreted production of candidate affinity proteins, allowing initial downstream characterizations prior to subcloning. Autotransporters form a class of surface proteins in Gram-negative bacteria that have potential for efficient translocation and tethering of recombinant passenger proteins to the outer membrane. We engineered a bacterial display vector based on the E. coli AIDA-I autotransporter for anchoring to the bacterial surface. Potential advantages of employing autotransporters combined with E. coli as host include: high surface expression level, high transformation frequency, alternative promoter systems available, efficient translocation to the outer membrane and tolerance for large multi-domain passenger proteins. The new vector was designed to comprise an expression cassette encoding for an Affibody molecule, three albumin binding domains for monitoring of surface expression levels, an Outer membrane Protease T (OmpT) recognition site for potential protease-mediated secretion of displayed affinity proteins and a histidine-tag for purification. A panel of vectors with different promoters were generated and evaluated, and suitable cultivation conditions were investigated. The results demonstrated a high surface expression level of the different evaluated Affibody molecules, high correlation between target binding and surface expression level, high signal-to-background ratio, efficient secretion and purification of binders in OmpT-positive hosts as well as tight regulation of surface expression for the titratable promoters. Importantly, a mock selection

  4. The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide

    Directory of Open Access Journals (Sweden)

    Liu Song

    2011-12-01

    Full Text Available Abstract Background Streptomyces transglutaminase (TGase is naturally synthesized as zymogen (pro-TGase, which is then processed to produce active enzyme by the removal of its N-terminal pro-peptide. This pro-peptide is found to be essential for overexpression of soluble TGase in E. coli. However, expression of pro-TGase by E. coli requires protease-mediated activation in vitro. In this study, we developed a novel co- expression method for the direct production of active TGase in E. coli. Results A TGase from S. hygroscopicus was expressed in E. coli only after fusing with the pelB signal peptide, but fusion with the signal peptide induced insoluble enzyme. Therefore, alternative protocol was designed by co-expressing the TGase and its pro-peptide as independent polypeptides under a single T7 promoter using vector pET-22b(+. Although the pro-peptide was co-expressed, the TGase fused without the signal peptide was undetectable in both soluble and insoluble fractions of the recombinant cells. Similarly, when both genes were expressed in the order of the TGase and the pro-peptide, the solubility of TGase fused with the signal peptide was not improved by the co-expression with its pro-peptide. Interestingly, active TGase was only produced by the cells in which the pro-peptide and the TGase were fused with the signal peptide and sequentially expressed. The purified recombinant and native TGase shared the similar catalytic properties. Conclusions Our results indicated that the pro-peptide can assist correct folding of the TGase inter-molecularly in E. coli, and expression of pro-peptide prior to that of TGase was essential for the production of active TGase. The co-expression strategy based on optimizing the order of gene expression could be useful for the expression of other functional proteins that are synthesized as a precursor.

  5. Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae

    OpenAIRE

    Kumar, Manoj; Saravanan, P.; S.K.Jalali

    2015-01-01

    Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although ...

  6. Expression and purification of human full-length N Oct-3, a transcription factor involved in melanoma growth.

    Science.gov (United States)

    Cabos-Siguier, Béatrice; Steunou, Anne-Lise; Joseph, Gérard; Alazard, Robert; Ducoux-Petit, Manuelle; Nieto, Laurence; Monsarrat, Bernard; Erard, Monique; Clottes, Eric

    2009-03-01

    This report describes the first purification procedure of the human full-length N Oct-3 protein in amounts suitable for structural studies and proteomic investigations. N Oct-3 is a transcription factor member of the POU protein family. It possesses a large N-terminal transactivation domain and a DNA-binding domain (DBD) which is composed of two subdomains, POUs and POUh, which are joined by a linker peptide. N Oct-3 is a master gene for central nervous system development but also for melanoma progression. Previous structural studies have all been performed using N Oct-3 DBD only. In this study, the full-length N Oct-3 protein was bacterially expressed and purified to homogeneity. The purified protein gave a single band at approximately 53 kDa on SDS-PAGE, while cDNA sequence analysis revealed a calculated molecular mass of 47 kDa confirmed by mass spectroscopy. Size-exclusion chromatography experiments indicated that in solution, full-length N Oct-3 was a monomer. Circular dichroïsm and intrinsic tryptophan fluorescence showed that full-length N Oct-3 was folded, with a significant alpha-helix content probably located in its DBD. Comparison with the purified N Oct-3 DBD demonstrated that, at least in vitro, the affinity of the protein for its DNA targets was similar. This suggests that the transactivation domain of N Oct-3 was not involved in N Oct-3 DNA interaction.

  7. S-peptide epitope tagging for protein purification, expression monitoring, and localization in mammalian cells.

    Science.gov (United States)

    Hackbarth, Jennifer S; Lee, Sun-Hee; Meng, Xue Wei; Vroman, Benjamin T; Kaufmann, Scott H; Karnitz, Larry M

    2004-11-01

    Epitope tags are widely used in cell biology and biochemistry research. The S-peptide/S-protein interaction has previously been utilized to purify polypeptides expressed in bacteria. We have now re-engineered the S-peptide/S-protein system to allow isolation of S-peptide-tagged polypeptides and their binding partners from eukaryotic cells with S-protein-agarose. In addition, two anti-S-peptide monoclonal antibodies have been generated for analysis of expression and subcellular localization of S-peptide-tagged polypeptides. These reagents make the S-peptide/S-protein system an attractive alternative to currently available epitope tagging methods.

  8. Expression, purification and insights into structure and folding of the ADAM22 pro domain

    DEFF Research Database (Denmark)

    Sørensen, Hans Peter; Jacobsen, Jonas; Nielbo, Steen

    2008-01-01

    and NMR spectroscopy. An ADAM22-P fragment encoding residues 26-199 could be expressed in high amounts, remained soluble above 1 mM, and was suitable for structural studies by NMR spectroscopy. CD spectroscopy and predictions suggest that the secondary structure in ADAM22-P consists of beta......-strands. Furthermore, our data indicate that the pro domains of ADAMs are expressed as two subdomains. The most N-terminal subdomain (ADAM22-P(N)) was found to be susceptible to proteolysis and was required for folding stability of the second subdomain (ADAM22-P(C))....

  9. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jarmander Johan

    2012-09-01

    Full Text Available Abstract Background The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium. Conclusions Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent

  10. Acquisition of Carbapenem Resistance by Plasmid-Encoded-AmpC-Expressing Escherichia coli.

    Science.gov (United States)

    van Boxtel, Ria; Wattel, Agnes A; Arenas, Jesús; Goessens, Wil H F; Tommassen, Jan

    2017-01-01

    Although AmpC β-lactamases can barely degrade carbapenems, if at all, they can sequester them and prevent them from reaching their targets. Thus, carbapenem resistance in Escherichia coli and other Enterobacteriaceae can result from AmpC production and simultaneous reduction of antibiotic influx into the periplasm by mutations in the porin genes. Here we investigated the route and genetic mechanisms of acquisition of carbapenem resistance in a clinical E. coli isolate carrying bla CMY-2 on a plasmid by selecting for mutants that are resistant to increasing concentrations of meropenem. In the first step, the expression of OmpC, the only porin produced in the strain under laboratory conditions, was lost, leading to reduced susceptibility to meropenem. In the second step, the expression of the CMY-2 β-lactamase was upregulated, leading to resistance to meropenem. The loss of OmpC was due to the insertion of an IS1 element into the ompC gene or to frameshift mutations and premature stop codons in this gene. The bla CMY-2 gene was found to be located on an IncIγ plasmid, and overproduction of the CMY-2 enzyme resulted from an increased plasmid copy number due to a nucleotide substitution in the inc gene. The clinical relevance of these genetic mechanisms became evident from the analysis of previously isolated carbapenem-resistant clinical isolates, which appeared to carry similar mutations. Copyright © 2016 American Society for Microbiology.

  11. Gene envY of Escherichia coli K-12 affects thermoregulation of major porin expression.

    Science.gov (United States)

    Lundrigan, M D; Earhart, C F

    1984-01-01

    The temperature-dependent expression of OmpF and OmpC, the major channel-forming proteins of the Escherichia coli K-12 outer membrane, was studied. In wild-type cells, decreasing growth temperatures resulted in increased amounts of OmpF protein and correspondingly decreased quantities of OmpC protein. Bacteria deleted for the 13-min chromosomal region did not exhibit this temperature-dependent fluctuation in porin proteins. Plasmid pML22, which consists of pBR322 containing a 0.5-megadalton E. coli chromosomal DNA insert, complemented the thermoregulatory defect. The regulatory gene was named envY. In minicells, pML22 directed the synthesis of an envelope polypeptide (EnvY) having an apparent molecular weight of 25,000. The EnvY protein was synthesized in minicells in greater amounts at 27 degrees C than at 37 degrees C, and a reducing agent was necessary in the solubilization buffer for its subsequent detection on polyacrylamide gels. The results describe the initial characterization of a regulatory system which, along with proteins of the ompB operon, the cyclic AMP system, and the tolC gene product, is involved in a complex network affecting major porin expression. Images PMID:6317653

  12. Improvement of penicillin G acylase expression in Escherichia coli through UV induced mutations

    Directory of Open Access Journals (Sweden)

    Rubina Arshad

    2010-12-01

    Full Text Available We used ultraviolet (UV radiation to induce mutation in three locally isolated strains of Escherichia coli. Different dilutions of bacterial cultures were exposed to UV lamp of 254 nm wavelength for different time intervals at varied distances ranging from 5 to 210 sec and 5 to 100 cm. Viable colonies were screened for mutants with an increased production of penicillin G acylase (PGA and a reduced production of β-lactamase, which are the desired properties of PGA producing industrial strains. A survival curve was made to get optimum exposure time and distance. The survival percentage for each exposure period was calculated and 1-5% survival was found useful for obtaining mutants with desired change. Screening for PGA and β-lactamase constitutive and/or deficient mutants was made by Serratia marcescens overlay test. A total of 100 survivors were selected of which 49% expressed PGA activity higher than the parent strain. Frequency of β-lactamase constitutive and deficient mutants was 48 and 52%, respectively. The best hyper-producing mutant (BDCS-N-M74, with almost negligible expression of β-lactamase, exhibited three-fold (22.5 mg 6-APA h-1 mg-1 wet cells increase in PGA activity compared with that in the parent strain (6.7 mg 6-APA h-1 mg-1 wet cells. The results indicated the successful induction of UV mediated mutation in E. coli for PGA hyper-producing mutants lacking β-lactamase activity.

  13. The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

    Directory of Open Access Journals (Sweden)

    Caroline A Magalhães

    2011-03-01

    Full Text Available Typical and atypical enteropathogenic Escherichia coli (EPEC are considered important bacterial causes of diarrhoea. Considering the repertoire of virulence genes, atypical EPEC (aEPEC is a heterogeneous group, harbouring genes that are found in other diarrheagenic E. coli pathotypes, such as those encoding haemolysins. Haemolysins are cytolytic toxins that lyse host cells disrupting the function of the plasma membrane. In addition, these cytolysins mediate a connection to vascular tissue and/or blood components, such as plasma and cellular fibronectin. Therefore, we investigated the haemolytic activity of 72 aEPEC isolates and determined the correlation of this phenotype with the presence of genes encoding enterohaemolysins (Ehly and cytolysin A (ClyA. In addition, the correlation between the expression of haemolysins and the ability of these secreted proteins to adhere to extracellular matrix (ECM components was also assessed in this study. Our findings demonstrate that a subset of aEPEC presents haemolytic activity due to the expression of Ehlys and/or ClyA and that this activity is closely related to the ability of these isolates to bind to ECM components.

  14. Characterization of a novel Acinetobacter baumannii xanthine dehydrogenase expressed in Escherichia coli.

    Science.gov (United States)

    Wang, Cheng-Hua; Zhao, Tong-Xin; Li, Mei; Zhang, Chong; Xing, Xin-Hui

    2016-02-01

    To characterize a novel xanthine dehydrogenase (XDH) from Acinetobacter baumannii by recombinant expression in Escherichia coli and to assess its potential for industrial applications. The XDH gene cluster was cloned from A. baumannii CICC 10254, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant XDH consisted of two subunits with the respective molecular weights of 87 kDa and 56 kDa according to SDS-PAGE. XDH catalysis was optimum at pH 8.5 and 40-45 °C, was stable under alkaline conditions (pH 7-11) and the half-inactivation temperature was 60 °C. The K m, turnover number and catalytic efficiency for xanthine were 25 μM, 69 s(-1) and 2.7 μM(-1) s(-1), respectively, which is an improvement over XDHs characterized previously. A. baumannii XDH is less than 50 % identical to previously identified XDH orthologs from other species, and is the first from the Acinetobacter genus to be characterized. The novel A. baumannii enzyme was found to be among the most active, thermostable and alkaline-tolerant XDH enzymes reported to date and has potential for use in industrial applications.

  15. Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128.

    Science.gov (United States)

    Yaoi, Katsuro; Mitsuishi, Yasushi

    2004-02-27

    A novel xyloglucan-specific endo-beta-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-beta-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (-2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained.

  16. Expression, purification and preliminary X-ray characterization of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1

    Science.gov (United States)

    Omi, Rie; Jitsumori, Keiji; Yamauchi, Takahiro; Ichiyama, Susumu; Kurihara, Tatsuo; Esaki, Nobuyoshi; Kamiya, Nobuo; Hirotsu, Ken; Miyahara, Ikuko

    2007-01-01

    dl-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (dl-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of dl-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P63, with unit-cell parameters a = b = 186.2, c = 114.4 Å. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V M value of 4.20–2.10 Å3 Da−1. A self-rotation function revealed peaks on the χ = 180° section. X-ray data have been collected to 1.75 Å resolution. PMID:17620717

  17. Identification of novel isoprene synthases through genome mining and expression in Escherichia coli.

    Science.gov (United States)

    Ilmén, Marja; Oja, Merja; Huuskonen, Anne; Lee, Sangmin; Ruohonen, Laura; Jung, Simon

    2015-09-01

    Isoprene is a naturally produced hydrocarbon emitted into the atmosphere by green plants. It is also a constituent of synthetic rubber and a potential biofuel. Microbial production of isoprene can become a sustainable alternative to the prevailing chemical production of isoprene from petroleum. In this work, sequence homology searches were conducted to find novel isoprene synthases. Candidate sequences were functionally expressed in Escherichia coli and the desired enzymes were identified based on an isoprene production assay. The activity of three enzymes was shown for the first time: expression of the candidate genes from Ipomoea batatas, Mangifera indica, and Elaeocarpus photiniifolius resulted in isoprene formation. The Ipomoea batatas isoprene synthase produced the highest amounts of isoprene in all experiments, exceeding the isoprene levels obtained by the previously known Populus alba and Pueraria montana isoprene synthases that were studied in parallel as controls. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  18. Division genes in Escherichia coli are expressed coordinately to cell septum requirements by gearbox promoters.

    Science.gov (United States)

    Aldea, M; Garrido, T; Pla, J; Vicente, M

    1990-11-01

    The cell division ftsQAZ cluster and the ftsZ-dependent bolA morphogene of Escherichia coli are found to be driven by gearboxes, a distinct class of promoters characterized by showing an activity that is inversely dependent on growth rate. These promoters contain specific sequences upstream from the mRNA start point, and their -10 region is essential for the inverse growth rate dependence. Gearbox promoters are essential for driving ftsQAZ and bolA gene expression so that the encoded products are synthesized at constant amounts per cell independently of cell size. This mode of regulation would be expected for the expression of proteins that either play a regulatory role in cell division or form a stoichiometric component of the septum, a structure that, independently of cell size and growth rate, is produced once per cell cycle.

  19. Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

    Directory of Open Access Journals (Sweden)

    Adam Bajinting

    2017-06-01

    Full Text Available Fibroblast growth factor receptors (FGFRs are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

  20. Cloning, expression, purification and characterization of Leishmania tropica PDI-2 protein

    Directory of Open Access Journals (Sweden)

    Ali Dina

    2016-01-01

    Full Text Available In Leishmania species, protein disulfide isomerase (PDI is an essential enzyme that catalyzes thiol-disulfide interchange. The present work describes the isolation, cloning, sequencing and expression of the pdI-2 gene. Initially, the gene was amplified from L. tropica genomic DNA by PCR using specific primers before cloning into the expression vector pET-15b. The construct pET/pdI-2 was transformed into BL21(DE3 cells and induced for the protein expression. SDS-PAGE and western blot analysis showed that the expressed protein is about 51 kDa. Cloned gene sequence analysis revealed that the deduced amino acid sequence showed significant homology with those of several parasites PDIs. Finally, recombinant protein was purified with a metal-chelating affinity column. The putative protein was confirmed as a thiol - disulfide oxidoreductase by detecting its activity in an oxidoreductase assay. Assay result of assay suggested that the PDI-2 protein is required for both oxidation and reduction of disulfide bonds in vitro. Antibodies reactive with this 51 kDa protein were detected by Western blot analysis in sera from human infected with L. tropica. This work describes for the first time the enzymatic activity of recombinant L. tropica PDI-2 protein and suggests a role for this protein as an antigen for the detection of leishmaniasis infection.

  1. Expression and purification of Nod factor receptors - Initial characterization of ligand binding

    DEFF Research Database (Denmark)

    Broghammer, Angelique

    chemical composition, Man3XylFucGlcNAc4. In an early attempt to investigate the Nod factor perception mechanism, direct binding of Nod factor was visualized in a bead assay with fluorescently labeled Nod factor and GFP-tagged fusion protein purified from the membrane fraction of leaf material, expressing...

  2. Expression screening, protein purification and NMR analysis of human protein domains for structural genomics

    NARCIS (Netherlands)

    Folkers, G.E.|info:eu-repo/dai/nl/162277202; van Buuren, B.N.M.; Kaptein, R.|info:eu-repo/dai/nl/074334603

    2004-01-01

    Structural genomics, the determination of protein structures on a genome-wide scale, is still in its infancy for eukaryotes due to the number and size of their genes. Low protein expression and solubility of eukaryotic geneproducts are the major bottlenecks in high-throughput (HTP) recombinant

  3. Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris.

    Science.gov (United States)

    Lee, Pey Yee; Yong, Voon Chen; Rosli, Rozita; Gam, Lay Harn; Chong, Pei Pei

    2014-02-01

    Squalene synthase (SS) is the key precursor and first committed enzyme of the sterol biosynthesis pathway. In a previous work, SS has been identified as one of the immunogenic proteins that could be a potential diagnostic candidate for the pathogenic fungus Candida tropicalis. In this study, SS from C. tropicalis was cloned and expressed as recombinant protein in Pichia pastoris to investigate its reactivity with serum antibodies. ERG9 gene that encodes for SS was amplified by PCR and cloned in-frame into pPICZB expression vector. The recombinant construct was then transformed into P. pastoris GS115 host strain. Expression of the recombinant protein was confirmed by SDS-PAGE and Western blot analysis using anti-His tag probe. Optimal protein production was achieved by cultivating the culture with 1.0% methanol for 72h. The recombinant protein was purified to approximately 97% pure in a single step immobilized metal affinity chromatography with a yield of 70.3%. Besides, the purified protein exhibited specific reactivity with immune sera on Western blot. This is the first report on heterologous expression of antigenic SS from C. tropicalis in P. pastoris which can be exploited for large-scale production and further research. The results also suggested that the protein might be of great value as antigen candidate for serodiagnosis of Candida infection. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Successful Validation of RNA Purification and Quantitative Real-Time PCR Analysis of Gene Expression on the International Space Station

    Science.gov (United States)

    Tran, L.; Parra, Macarena P.; Jung, J.; Boone, T.; Schonfeld, Julie; Almeida, Eduardo

    2017-01-01

    The NASA Ames WetLab-2 system was developed to offer new on-orbit gene expression analysis capabilities to ISS researchers and can be used to conduct on-orbit RNA isolation and quantitative real time PCR (RT-qPCR) analysis of gene expression from a wide range of biological samples ranging from microbes to mammalian tissues. On orbit validation included three quantitative PCR (qPCR) runs using an E. coli genomic DNA template pre-loaded at three different concentrations. The flight Ct values for the DNA standards showed no statistically significant differences relative to ground controls although there was increased noise in Ct curves, likely due to microgravity-related bubble retention in the optical windows. RNA was successfully purified from both E. coli and mouse liver samples and successfully generated singleplex, duplex and triplex data although with higher standard deviations than ground controls, also likely due to bubbles. Using volunteer science activities, a potential bubble reduction strategy was tested and resulted in smooth amplification curves and tighter Cts between replicates. The WetLab-2 validation experiment demonstrates a novel molecular biology workbench on ISS which allows scientists to purify and stabilize RNA, and to conduct RT-qPCR analyses on-orbit with rapid results. This novel ability is an important step towards utilizing ISS as a National Laboratory facility with the capability to conduct and adjust science experiments in real time without sample return, and opens new possibilities for rapid medical diagnostics and biological environmental monitoring on ISS.

  5. Expression of nattokinase in Escherichia coli and renaturation of its inclusion body.

    Science.gov (United States)

    Ni, He; Guo, Peng-Cheng; Jiang, Wei-Ling; Fan, Xiao-Min; Luo, Xiang-Yu; Li, Hai-Hang

    2016-08-10

    Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies. The recombinant cells were lysed with 0.1mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100 to purify the inclusion bodies. The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10. The refolded proteins showed 42.8IU/mg and 79.3IU/mg fibrinolytic activity by the traditional dilution method (20-fold dilution into RS-10) and the directly mixing the protein solution with equal volume RS-10, respectively, compared to the 52.0IU/mg of total water-soluble proteins from B. subtilis var. natto. This work demonstrated that the inclusion body of recombinant nattokinase expressed in E. coli could be simply refolded to the natural enzyme activity level by directly mixing the protein solution with equal volume refolding solution. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Soluble expression of disulfide bond containing proteins FGF15 and FGF19 in the cytoplasm of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Bo Kong

    Full Text Available Fibroblast growth factor 19 (FGF19 is the human ortholog of mouse FGF15, and both proteins function as an endocrine signal to regulate various liver functions. FGF15/FGF19 protein contains two disulfide bonds. It is unfavorable to form disulfide bonds in Escherichia coli (E. coli cytoplasm because of the bacterial cytoplasmic reducing environment. Modification of the cytoplasmic reducing environment and/or co-expression of protein chaperones are common strategies to express disulfide bond containing proteins in E. coli. In the current study, we report a method to produce soluble FGF15/FGF19 protein in cytoplasm of E. coli. Several commercial available strains with the disruption of thiol-redox pathways, and/or co-expression of redoxase or refolding chaperones were used to develop this novel method for expression of FGF15/FGF19 in E. coli. Mutation of the thiol-disulfide bond reducing pathway in E. coli or N-terminal fusion of thioredox (TRX alone is not enough to support disulfide bond formation in FGF15/19 proteins. However, TRX fusion protein improved FGF19 solubility in strains of thiol-redox system mutants. In addition, DsbC co-expressed in thiol-redox system mutants alone improved and further enhanced FGF19 solubility with combination of TRX fusion tag. The soluble FGF19 proteins were easily purified through Ni-NTA affinity chromatography and anion exchange chromatography, and the purified protein maintained its biological activities, confirmed by suppressing hepatic Cyp7a1 gene transcription in mice and by activating ERK1/2 signaling pathway in HepG2 cells. In contrast, soluble FGF15 protein in cytoplasm remained very low using these strategies. In summary, we have successfully developed a method to express functional FGF19 protein in prokaryotic cells, and this strategy may be adapted for the expression of other disulfide-containing proteins.

  7. Expression and purification of a recombinant Fip-fve protein from Flammulina velutipes in baculovirus-infected insect cells.

    Science.gov (United States)

    Wu, C-M; Wu, T-Y; Kao, S-S; Ko, J-L; Jinn, T-R

    2008-05-01

    To develop an efficient and facile expression system supply of high purity and stable activity of rFip-fve for oral administration, medicinal study and applications. A recombinant virus that contained the chimera gene, encoding a bombyxin signal peptide sequence fused to a Fip-fve-6His sequence, was constructed. The rFip-fve was purified from the supernatant of the infected Sf21 cells using a nickel-chelated affinity column, and was verified by Western blot and MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analyses. Results showed that a glycosylated mature rFip-fve was produced and secreted into the infected cell supernatant. The immunomodulatory activity of rFip-fve was evaluated by measuring the amount of interleukin-2 released from murine splenocytes. A reliable scheme to express and purify active rFip-fve in a baculovirus/insect cell system for medicinal applications and genetic study is a feasible means of solving potential problems related to the production and activity of rFip-fve protein. The rFip-fve expressed in insect cells was processed and modified in a manner more similar to that of its native counterpart than that in bacterial cells. Therefore, the potential applications of rFip-fve that is generated in Sf21 cells can be more effectively evaluated that produced in Escherichia coli.

  8. Construction, expression, purification, and characterization of a dual-targeting PD-1/VEGF-A fusion protein (P-V).

    Science.gov (United States)

    Qian, Niliang; Gao, Liucun; Dong, Lihou; Liu, Hongchuan; Fu, Jie; Meng, Dan; Gao, Xin; Zhang, Jing; Gao, Yucai; Song, Haifeng

    2015-05-01

    Targeting programmed death-1 (PD-1) is regarded as a novel and promising means for the treatment of many types of solid tumor. In the tumor microenvironment (TME), VEGF expression is dramatically up-regulated, and compounds that neutralize VEGF or block the interaction of VEGF with its receptors exhibit potent antitumor activity, and blocking PD-1 might promote T cell infiltration into TME and significantly enhance local immune activation. Thus, we fused domain II and domain III of kinase-insert domain receptor (KDR), the receptor of VEGF-A, to the Fc side of an anti-PD-1 monoclonal antibody with a (Gly4Ser)3 linker to generate a dual targeting fusion protein. The recombinant plasmid was successfully constructed and the fusion protein was expressed in 293E cells. Protein purification was performed in a single step by using protein A affinity chromatography. The molecular weight of the fusion protein was approximately 220kDa, and the yield was approximately 2.97g/L. Specific binding of recombinant protein to PD-1 and VEGF was detected by enzyme-linked immunosorbent assay (ELISA) analysis. Half maximal effective concentration (EC50) values were 0.561nM for PD-1 and 0.682nM for VEGF-A; accordingly, half maximal inhibitory concentration (IC50) values were 0.914nM and 0.583nM, respectively. Proliferation inhibition assays indicated that the fusion protein could inhibit the growth of human umbilical vein endothelial cells effectively. Taken together, the results indicate that this novel fusion protein can simultaneously target PD-1 and VEGF and may be beneficial for combining anti-angiogenesis with immunotherapeutic approaches for the treatment of patients with cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Cloning and expression in Escherichia coli of a gene coding for a chondroitin lyase from Bacteroides thetaiotaomicron

    Energy Technology Data Exchange (ETDEWEB)

    Guthrie, E.P.; Shoemaker, N.B.; Salyers, A.A.

    1985-11-01

    The authors cloned the gene for one of the two chondroitin lyases of Bacteroides thetaiotaomicron into the cosmid vector pHC79 and subcloned it into pBR328. No proteins the size of B. thetaiotamicron chondroitin lyase I or II (104 to 108 kilodaltons) were detectable in maxicell or in vitro transcription-translation preparations. However, partial purification of the chondroitin lyase activity from the Escherichia coli subclone showed that its properties were similar to those of the B. thetaiotaomicron chondroitin lyases. Antibodies to the chondroitin lyase that was produced in E. coli cross-reacted with the B. thetaiotaomicron chondroitin lyase II but not with chondroitin lyase I. The molecular weight of the enzyme produced in E. coli was slightly lower than those of the two chondroitin lyases from B. thetaiotaomicron. The chondroitin lyase gene was located on the subcloned 7.8-kilobase EcoRI fragment. The size of the gene was approximately 3.3 kilobases, as expected for a protein with a molecular weight of 104,000.

  10. Expression of Immune-Related Genes of Ducks Infected with Avian Pathogenic Escherichia coli (APEC).

    Science.gov (United States)

    Li, Rong; Li, Ning; Zhang, Jinzhou; Wang, Yao; Liu, Jiyuan; Cai, Yumei; Chai, Tongjie; Wei, Liangmeng

    2016-01-01

    Avian pathogenic Escherichia coli (APEC) can cause severe disease in ducks, characterized by perihepatitis, pericarditis, and airsacculitis. Although the studies of bacteria isolation and methods of detection have been reported, host immune responses to APEC infection remain unclear. In response, we systemically examined the expression of immune-related genes and bacteria distribution in APEC-infected ducks. Results demonstrated that APEC can quickly replicate in the liver, spleen, and brain, with the highest bacteria content at 2 days post infection. The expression of toll-like receptors (TLRs), avian β-defensins (AvBDs) and major histocompatibility complex (MHC) were tested in the liver, spleen, and brain of infected ducks. TLR2, TLR4, TLR5, and TLR15 showed different expression patterns, which indicated that they all responded to APEC infection. The expression of AvBD2 was upregulated in all tested tissues during the 3 days of testing, whereas the expression of AvBD4, AvBD5, AvBD7, and AvBD9 were downregulated, and though MHC-I was upregulated on all test days, MHC-II was dramatically downregulated. Overall, our results suggest that APEC can replicate in various tissues in a short time, and the activation of host immune responses begins at onset of infection. These findings thus clarify duck immune responses to APEC infection and offer insights into its pathogenesis.

  11. Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Davoud Koolivand

    2016-10-01

    Full Text Available The genomic region of Grapevine fanleaf virus (GFLV encoding the movement protein (MP was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3 to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG concentrations (1, 1.5, and 2 mM each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

  12. Expression of Immune-Related Genes of Ducks Infected with Avian Pathogenic Escherichia coli (APEC

    Directory of Open Access Journals (Sweden)

    Rong eLi

    2016-05-01

    Full Text Available Avian pathogenic Escherichia coli (APEC can cause severe disease in ducks, characterized by perihepatitis, pericarditis and airsacculitis. Although the studies of bacteria isolation and methods of detection have been reported, host immune responses to APEC infection remain unclear. In response, we systemically examined the expression of immune-related genes and bacteria distribution in APEC-infected ducks. Results demonstrated that APEC can quickly replicate in the liver, spleen and brain, with the highest bacteria content at 2 day post infection. The expression of Toll-like receptors (TLRs, avian β-defensins (AvBDs and major histocompatibility complex (MHC were tested in the liver, spleen and brain of infected ducks. TLR2, TLR4, TLR5 and TLR15 showed different expression patterns, which indicated that they all responded to APEC infection. The expression of AvBD2 was upregulated in all tested tissues during the 3 days of testing, whereas the expression of AvBD4, AvBD5, AvBD7 and AvBD9 were downregulated, and though MHC-I was upregulated on all test days, MHC-II was dramatically downregulated. Overall, our results suggest that APEC can replicate in various tissues in a short time, and the activation of host immune responses begins at onset of infection. These findings thus clarify duck immune responses to APEC infection and offer insights into its pathogenesis.

  13. Expression and purification of human papillomavirus 18 L1 virus-like particle from saccharomyces cerevisiae.

    Science.gov (United States)

    Woo, Mi-Kyung; An, Jung-Mo; Kim, Jun-Dong; Park, Sue-Nie; Kim, Hong-Jin

    2008-02-01

    Cervical cancer caused by human papillomavirus (HPV) might be successfully prevented by HPV vaccination and screening. HPV vaccination and HPV serology assays have been investigated using HPV virus-like particles (VLPs). In this study we produced HPV18 L1 VLPs in Saccharomyces cerevisiae and purified them. The HPV18 L1 gene was cloned into the yeast expression vector YEGalpha-HIR525, and transformed into Saccharomyces cerevisiae. Expression of HPV18 L1 protein was demonstrated by Western blotting. The HPV18 L1 protein was purified by ultracentrifugation, size-exclusion chromatography and cation-exchange chromatography, and was up to 95% pure. We showed by transmission electron microscopy that the purified protein self-assembled into VLPs. These findings should be useful for establishing vaccine efficacy as well as characterizing vaccine candidates, and may provide an international reference standard for HPV serology assays.

  14. Expression and Purification of Z Protein from Junín Virus

    Directory of Open Access Journals (Sweden)

    S. E. Goñi

    2010-01-01

    Full Text Available Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z. The arenavirus Junín (JUNV is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one. One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.

  15. Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli

    Science.gov (United States)

    2010-01-01

    Background Hydrogenases catalyze reversible reaction between hydrogen (H2) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H2 production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hyaA and hyaB genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H2 producing ability. Results Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H2 (12.5 mL H2/(h·L) in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H2 even when formate was added as substrate for formate hydrogenlyase (FHL) pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (~65% of total cell fraction), based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified E. coli [NiFe]-hydrogenase 1 using his6-tag displayed oxygen-tolerant activity of ~12 nmol H2/(min·mg protein) under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition. Conclusions This is the first report on physiological function of E. coli [NiFe]-hydrogenase 1 for H2 production. We found that [NiFe]-hydrogenase 1 has H2 production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is not necessary to protect

  16. Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Kim Jaoon YH

    2010-07-01

    Full Text Available Abstract Background Hydrogenases catalyze reversible reaction between hydrogen (H2 and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H2 production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hyaA and hyaB genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H2 producing ability. Results Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H2 (12.5 mL H2/(h·L in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H2 even when formate was added as substrate for formate hydrogenlyase (FHL pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (~65% of total cell fraction, based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified E. coli [NiFe]-hydrogenase 1 using his6-tag displayed oxygen-tolerant activity of ~12 nmol H2/(min·mg protein under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition. Conclusions This is the first report on physiological function of E. coli [NiFe]-hydrogenase 1 for H2 production. We found that [NiFe]-hydrogenase 1 has H2 production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is

  17. Purification and characterization of the first recombinant bird pancreatic lipase expressed in Pichia pastoris: The turkey

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    Fendri Ahmed

    2011-01-01

    Full Text Available Abstract Background The turkey pancreatic lipase (TPL was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC4 in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast Pichia pastoris in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme. Results The recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on Pichia genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined. Conclusions Our results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships.

  18. EXPRESSION, PURIFICATION ET ÉTUDE STRUCTURALE PAR RMN DE PEBP DE DROSOPHILE

    OpenAIRE

    Gilles, Rautureau

    2006-01-01

    Phosphatidylethanolamine Binding Proteins (PEBPs) are proteins of about 20 kDa highly conserved proteins in all three kingdoms from bacteria to mammals. They are characterized by their capacity to bind anionic ligands. In addition PEBPs seem to be involved in numerous cellular processes (mostly in signalling pathways), and physiological processes (such as spermatogenesis) of the healthy organism. However modifications of the level of PEBPs expression are correlated with pathological situation...

  19. A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri

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    Giordanni C. Dantas

    2016-06-01

    Full Text Available Abstract Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac, is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.

  20. A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri.

    Science.gov (United States)

    Dantas, Giordanni C; Martins, Paula M M; Martins, Daniela A B; Gomes, Eleni; Ferreira, Henrique

    2016-01-01

    Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  1. Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

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    Y. Cui

    2008-05-01

    Full Text Available Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1 allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+, expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG, 267-277 (NYHAVNIVGYG and 284-303 (YWIVRNSWDTTWGDSGYGYF. Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%, an extended strand (17.13%, a ß turn (5.61%, and a random coil (43.30%. A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

  2. Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by Recombinant E. coli.

    Science.gov (United States)

    Fakruddin, Md; Mohammad Mazumdar, Reaz; Bin Mannan, Khanjada Shahnewaj; Chowdhury, Abhijit; Hossain, Md Nur

    2013-01-01

    E. coli is the most frequently used host for production of enzymes and other proteins by recombinant DNA technology. E. coli is preferable for its relative simplicity, inexpensive and fast high-density cultivation, well-known genetics, and large number of compatible molecular tools available. Despite all these advantages, expression and production of recombinant enzymes are not always successful and often result in insoluble and nonfunctional proteins. There are many factors that affect the success of cloning, expression, and mass production of enzymes by recombinant E. coli. In this paper, these critical factors and approaches to overcome these obstacles are summarized focusing controlled expression of target protein/enzyme in an unmodified form at industrial level.

  3. Genes regulated by the Escherichia coli SOS repressor LexA exhibit heterogenous expression

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    Gillor Osnat

    2010-11-01

    Full Text Available Abstract Background Phenotypic heterogeneity may ensure that a small fraction of a population survives environmental perturbations or may result in lysis in a subpopulation, to increase the survival of siblings. Genes involved in DNA repair and population dynamics play key roles in rapid responses to environmental conditions. In Escherichia coli the transcriptional repressor LexA controls a coordinated cellular response to DNA damage designated the SOS response. Expression of LexA regulated genes, e.g. colicin encoding genes, recA, lexA and umuDC, was examined utilizing transcription fusions with the promoterless gfp at the single cell level. Results The investigated LexA regulated genes exhibited heterogeneity, as only in a small fraction of the population more intense fluorescence was observed. Unlike recA and lexA, the pore forming and nuclease colicin activity genes as well as umuDC, exhibited no basal level activity. However, in a lexA defective strain high level expression of the gene fusions was observed in the large majority of the cells. All of the investigated genes were expressed in a recA defective strain, albeit at lower levels, revealing expression in the absence of a spontaneous SOS response. In addition, the simultaneous expression of cka, encoding the pore forming colicin K, and lexA, investigated at the single cell level revealed high level expression of only cka in rare individual cells. Conclusion LexA regulated genes exhibit phenotypic heterogeneity as high level expression is observed in only a small subpopulation of cells. Heterogenous expression is established primarily by stochastic factors and the binding affinity of LexA to SOS boxes.

  4. DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli.

    Science.gov (United States)

    Liu, Yamei; Stepanov, Victor G; Strych, Ulrich; Willson, Richard C; Jackson, George W; Fox, George E

    2010-12-06

    Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.

  5. Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197.

    Science.gov (United States)

    Roth, R; van Zyl, P; Tsekoa, T; Stoychev, S; Mamputha, S; Buthelezi, S; Crampton, M

    2017-05-01

    To investigate the production of soluble cross-reacting material 197 (CRM197 ) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines. The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulphide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM197 , at a production rate ~10% of the production of insoluble CRM197 , in equivalent small-scale cultures. Structural analysis of the purified CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favourably on all structural levels. SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197 in E. coli, which was previously unachievable. Previous literature reports have shown that CRM197 can be expressed in E. coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM197 . © 2017 The Society for Applied Microbiology.

  6. Expression, purification, crystallization and preliminary crystallographic analysis of the PAB0955 gene product.

    Science.gov (United States)

    Gras, Stéphanie; Fernandez, Bernard; Chaumont, Valérie; Carpentier, Philippe; Armengaud, Jean; Housset, Dominique

    2005-02-01

    PAB0955 from Pyrococcus abyssi is a prototype of a new Walker-type ATPase/GTPase conserved in archaea and eukaryota but not found in bacteria. PAB0955 has been expressed, purified and crystallized, and it has been shown that this thermostable protein is dimeric in reductive conditions. Crystals have been obtained either without nucleotide or in the presence of GDP or GTPgammaS. Preliminary X-ray crystallographic data up to 2.08 A resolution have been collected from these crystals.

  7. Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens

    OpenAIRE

    Shima Mahmoudi; Setareh Mamishi; Mona Ghazi; Reihaneh Hosseinpour Sadeghi; Babak Pourakbari

    2013-01-01

    Background and Objectives ESAT-6 (6-kDaearly secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) have been described as dominant antigens recognized by T-cells and considered as virulence factors in Mycobacterium tuberculosis. The aim of this study was to clone, express and purify recombinant ESAT-6 andCFP-10 proteins of M. tuberculosis in soluble form. Materials and Methods ESAT-6 andCFP-10 genes were amplified by PCR, cloned into pET32a (+) vector, and overexpress-ed us...

  8. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach

    2009-01-01

    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed......, purified and the kinetic properties have been studied and are compared. Substrate inhibition by tryptophan is observed for isoform 1 but not for isoform 2. Large differences are observed in the K m,tetrahydrobiopterin values for the two isoforms, being >10 times larger for isoform 1 compared to isoform 2....

  9. PURIFICATION OF PENICILLIN-BINDING PROTEIN-4 OF ESCHERICHIA-COLI AS A SOLUBLE-PROTEIN BY DYE-AFFINITY CHROMATOGRAPHY

    NARCIS (Netherlands)

    MOTTL, H; KECK, W

    1991-01-01

    The dacB gene of Escherichia coli, coding for penicillin-binding protein 4 (PBP4) was cloned under the control of the phage lambda-p(R) promoter and cro gene translation signals. Derepression of the phage lambda promoter for 2 h at 42-degrees-C in E. coli led to the maximum over-production of PBP4

  10. Cloning and expression of Che a 1, the major allergen of Chenopodium album in Escherichia coli.

    Science.gov (United States)

    Vahedi, Fatemeh; Sankian, Mojtaba; Moghadam, Malihe; Mohaddesfar, Maryam; Ghobadi, Sirous; Varasteh, Abdol Reza

    2011-04-01

    Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b+ vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant protein by means of Ni-NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1.

  11. Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

    Directory of Open Access Journals (Sweden)

    Tianxiang Gong

    2011-09-01

    Full Text Available Mature mouse beta defensin 2 (mBD2 is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA, pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli Rosseta-gami (2 for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%. After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans and Cryptococcus neoformans (C. neoformans was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV, the protective rate for Madin-Darby canine kidney cells (MDCK was 93.86% at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.

  12. Growth and recombinant protein expression with Escherichia coli in different batch cultivation media.

    Science.gov (United States)

    Hortsch, Ralf; Weuster-Botz, Dirk

    2011-04-01

    Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L(-1). EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L(-1). The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.