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Sample records for coli cells expressing

  1. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    Science.gov (United States)

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  2. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  3. Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida.

    Science.gov (United States)

    Yamada, Mamoru; Okada, Yukiyoshi; Yoshida, Toyokazu; Nagasawa, Toru

    2008-04-01

    The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20 degrees C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.

  4. Expression in E. coli systems

    DEFF Research Database (Denmark)

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

    2003-01-01

    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......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...

  5. Factors limiting SOS expression in log-phase cells of Escherichia coli.

    Science.gov (United States)

    Massoni, Shawn C; Leeson, Michael C; Long, Jarukit Edward; Gemme, Kristin; Mui, Alice; Sandler, Steven J

    2012-10-01

    In Escherichia coli, RecA-single-stranded DNA (RecA-ssDNA) filaments catalyze DNA repair, recombination, and induction of the SOS response. It has been shown that, while many (15 to 25%) log-phase cells have RecA filaments, few (about 1%) are induced for SOS. It is hypothesized that RecA's ability to induce SOS expression in log-phase cells is repressed because of the potentially detrimental effects of SOS mutagenesis. To test this, mutations were sought to produce a population where the number of cells with SOS expression more closely equaled the number of RecA filaments. Here, it is shown that deleting radA (important for resolution of recombination structures) and increasing recA transcription 2- to 3-fold with a recAo1403 operator mutation act independently to minimally satisfy this condition. This allows 24% of mutant cells to have elevated levels of SOS expression, a percentage similar to that of cells with RecA-green fluorescent protein (RecA-GFP) foci. In an xthA (exonuclease III gene) mutant where there are 3-fold more RecA loading events, recX (a destabilizer of RecA filaments) must be additionally deleted to achieve a population of cells where the percentage having elevated SOS expression (91%) nearly equals the percentage with at least one RecA-GFP focus (83%). It is proposed that, in the xthA mutant, there are three independent mechanisms that repress SOS expression in log-phase cells. These are the rapid processing of RecA filaments by RadA, maintaining the concentration of RecA below a critical level, and the destabilizing of RecA filaments by RecX. Only the first two mechanisms operate independently in a wild-type cell.

  6. Synergy between type 1 fimbriae expression and C3 opsonisation increases internalisation of E. coli by human tubular epithelial cells.

    Science.gov (United States)

    Li, Ke; Zhou, Wuding; Hong, Yuzhi; Sacks, Steven H; Sheerin, Neil S

    2009-03-31

    Bacterial infection of the urinary tract is a common clinical problem with E. coli being the most common urinary pathogen. Bacterial uptake into epithelial cells is increasingly recognised as an important feature of infection. Bacterial virulence factors, especially fimbrial adhesins, have been conclusively shown to promote host cell invasion. Our recent study reported that C3 opsonisation markedly increases the ability of E. coli strain J96 to internalise into human proximal tubular epithelial cells via CD46, a complement regulatory protein expressed on host cell membrane. In this study, we further assessed whether C3-dependent internalisation by human tubular epithelial cells is a general feature of uropathogenic E. coli and investigated features of the bacterial phenotype that may account for any heterogeneity. In 31 clinical isolates of E. coli tested, C3-dependent internalisation was evident in 10 isolates. Type 1 fimbriae mediated-binding is essential for C3-dependent internalisation as shown by phenotypic association, type 1 fimbrial blockade with soluble ligand (mannose) and by assessment of a type 1 fimbrial mutant. we propose that efficient internalisation of uropathogenic E. coli by the human urinary tract depends on co-operation between type 1 fimbriae-mediated adhesion and C3 receptor -ligand interaction.

  7. Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

    International Nuclear Information System (INIS)

    Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.; Yankovskii, N.K.; Debabov, V.G.

    1989-01-01

    Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector λpSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB + clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA + transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmids consistently transduced the markers thrB + and lysA + . The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes

  8. A Spatial Control for Correct Timing of Gene Expression during the Escherichia coli Cell Cycle

    Directory of Open Access Journals (Sweden)

    Yuan Yao

    2016-12-01

    Full Text Available Temporal transcriptions of genes are achieved by different mechanisms such as dynamic interaction of activator and repressor proteins with promoters, and accumulation and/or degradation of key regulators as a function of cell cycle. We find that the TorR protein localizes to the old poles of the Escherichia coli cells, forming a functional focus. The TorR focus co-localizes with the nucleoid in a cell-cycle-dependent manner, and consequently regulates transcription of a number of genes. Formation of one TorR focus at the old poles of cells requires interaction with the MreB and DnaK proteins, and ATP, suggesting that TorR delivery requires cytoskeleton organization and ATP. Further, absence of the protein–protein interactions and ATP leads to loss in function of TorR as a transcription factor. We propose a mechanism for timing of cell-cycle-dependent gene transcription, where a transcription factor interacts with its target genes during a specific period of the cell cycle by limiting its own spatial distribution.

  9. Expression of maize prolamins in Escherichia Coli

    International Nuclear Information System (INIS)

    Wang, Szu-zhen; Esen, Asim

    1985-01-01

    We have constructed a cDNA expression library of developing corn (Zea manys L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250-900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with 32 P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed. (author)

  10. High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli.

    Science.gov (United States)

    Su, Lin; Chen, Song-Sen; Yang, Ke-Gong; Liu, Chang-Zheng; Zhang, Yan-Li; Liang, Zhi-Quan

    2006-06-01

    Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.

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

    African Journals Online (AJOL)

    Administrator

    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 ...

  12. [Effect of eicosapentaenoic acid on mRNA expression of tight junction protein ZO-1 in intestinal epithelial cells after Escherichia coli LF82 infection].

    Science.gov (United States)

    Hao, Li-Jun; Lin, Yan; Zhang, Wei; Tian, Jiao; Wang, Ya; Chen, Peng-De; Hu, Chong-Kang; Zeng, Ling-Chao; Yang, Jie; Wang, Bao-Xi; Jiang, Xun

    2017-06-01

    To investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection. The Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 μmol/L EPA) and EPA (0, 25, 50, 100, and 200 μmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant. After EPA treatment (25 and 50 μmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (PE.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (PE.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (PE.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated

  13. [Expression of the genes for lysine biosynthesis of Bacillus subtilis in Escherichia coli cells].

    Science.gov (United States)

    Shevchenko, T N; Okunev, O V; Aleksieva, Z M; Maliuta, S S

    1984-01-01

    Hybrid plasmids pLRS33 and pLRB4 containing Bac. subtilis genes coding lysin biosynthesis were subjected to genetical analysis. It is shown that after pLRS33- and pLRB4- transformation of E. coli strains, auxotrophic relative to lysin and diaminopimelic acid, there occurs complementation of dapA, dapB, dapC, dapD, dapE, lysA mutations by plasmid pLRS33 and of dapC, dapB, lysA mutations by plasmid pLRB4. The plasmids are studied for their influence on the level of lysin and its precurror synthesis in E. coli strains.

  14. Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7.

    Science.gov (United States)

    Roe, Andrew J; Naylor, Stuart W; Spears, Kevin J; Yull, Helen M; Dransfield, Tracy A; Oxford, Matthew; McKendrick, Iain J; Porter, Megan; Woodward, Martin J; Smith, David G E; Gally, David L

    2004-10-01

    Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

  15. Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell.

    Science.gov (United States)

    Chang, Fei; Zhang, Xianbing; Pan, Yu; Lu, Youxue; Fang, Wei; Fang, Zemin; Xiao, Yazhong

    2017-11-07

    β-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted the industrial applications of β-glucosidase. Thus development of new strategies to express β-glucosidases with cost-effective and simple procedure to meet the increasing demands on enzymes for biocatalysis is of paramount importance. Light activated cassette YF1/FixJ and the SRRz lysis system were successfully constructed to produce Bgl1A(A24S/F297Y), a mutant β-glucosidase tolerant to both glucose and ethanol. By optimizing the parameters for light induction, Bgl1A(A24S/F297Y) activity reached 33.22 ± 2.0 U/mL and 249.92 ± 12.25 U/mL in 250-mL flask and 3-L fermentation tank, respectively, comparable to the controls of 34.02 ± 1.96 U/mL and 322.21 ± 10.16 U/mL under similar culture conditions with IPTG induction. To further simplify the production of our target protein, the SRRz lysis gene cassette from bacteriophage Lambda was introduced to trigger cell autolysis. As high as 84.53 ± 6.79% and 77.21 ± 4.79% of the total β-glucosidase were released into the lysate after cell autolysis in 250 mL flasks and 3-L scale fermentation with lactose as inducer of SRRz. In order to reduce the cost of protein purification, a cellulose-binding module (CBM) from Clostridium thermocellum was fused into the C-terminal of Bgl1A(A24S/F297Y) and cellulose was used as an economic material to adsorb the fusion enzyme from the lysate. The yield of the fusion protein could reach 92.20 ± 2.27% after one-hour adsorption at 25 °C. We have developed an efficient and inexpensive way to produce β-glucosidase for potential industrial applications by using the combination of light induction, cell autolysis, and CBM purification strategy.

  16. 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...

  17. Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana.

    Science.gov (United States)

    Hong, Young-Ho; Lee, Dong-Woo; Lee, Sang-Jae; Choe, Eun-Ah; Kim, Seong-Bo; Lee, Yoon-Hee; Cheigh, Chan-Ick; Pyun, Yu-Ryang

    2007-04-01

    Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t (1/2) = 43 days at 70 degrees C) in a continuous recycling mode at 70 degrees C produced 49 and 38 g D-tagatose/l from 180 and 90 g D-galactose/l, respectively, within 12 h.

  18. Hyper-regulation of pyr-gene expression in Escherichia coli cells with slow ribosomes. Evidence for RNA polymerase pausing in vivo

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1988-01-01

    UTP-modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate...... transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper-regulated by the UTP pool in this type of cells...

  19. Escherichia coli LF82 differentially regulates ROS production and mucin expression in intestinal epithelial T84 cells: implication of NOX1.

    Science.gov (United States)

    Elatrech, Imen; Marzaioli, Viviana; Boukemara, Hanane; Bournier, Odile; Neut, Christel; Darfeuille-Michaud, Arlette; Luis, José; Dubuquoy, Laurent; El-Benna, Jamel; My-Chan Dang, Pham; Marie, Jean-Claude

    2015-05-01

    Increased reactive oxygen species (ROS) production is associated with inflamed ileal lesions in Crohn's disease colonized by pathogenic adherent-invasive Escherichia coli LF82. We investigated whether such ileal bacteria can modulate ROS production by epithelial cells, thus impacting on inflammation and mucin expression. Ileal bacteria from patients with Crohn's disease were incubated with cultured epithelial T84 cells, and ROS production was assayed using the luminol-amplified chemiluminescence method. The gentamicin protection assay was used for bacterial invasion of T84 cell. The expression of NADPH oxidase (NOX) subunits, mucin, and IL-8 was analyzed by quantitative real-time PCR and Western blots. Involvement of NOX and ROS was analyzed using diphenyleneiodonium (DPI) and N-acetylcysteine (NAC). Among different bacteria tested, only LF82 induced an increase of ROS production by T84 cells in a dose-dependent manner. This response was inhibited by DPI and NAC. Heat- or ethanol-attenuated LF82 bacteria and the mutant LF82ΔFimA, which does not express pili type 1 and poorly adheres to epithelial cells, did not induce the oxidative response. The LF82-induced oxidative response coincides with its invasion in T84 cells, and both processes were inhibited by DPI. Also, we observed an increased expression of NOX1 and NOXO1 in response to LF82 bacteria versus the mutant LF82ΔFimA. Furthermore, LF82 inhibited mucin gene expression (MUC2 and MUC5AC) in T84 cells while increasing the chemotactic IL-8 expression, both in a DPI-sensitive manner. Adherent-invasive E. coli LF82 induced ROS production by intestinal NADPH oxidase and altered mucin and IL-8 expression, leading to perpetuation of inflammatory lesions in Crohn's disease.

  20. Whole-cell bioreduction of aromatic α-keto esters using Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase co-expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Egger Sigrid

    2008-12-01

    Full Text Available Abstract Background Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP+ or NAD+ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH. The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR. Results Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular CtXR and CbFDH activity per gram of dry E. coli cell mass (gCDW. The maximum conversion rate (rS for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/gCDWh, suggesting that the activity of CbFDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of CtXR and CbFDH in the presence of the α-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions (100 mM substrate; 40 gCDW/L, we obtained ethyl R-4-cyanomandelate with an enantiomeric excess (e.e. of 97.2% in a yield of 82

  1. Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

    Science.gov (United States)

    Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

    2013-03-01

    Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Expression in E. coli systems

    DEFF Research Database (Denmark)

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

    2003-01-01

    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...

  3. Contribution of the Pmra Promoter to Expression of Genes in the Escherichia coli mra Cluster of Cell Envelope Biosynthesis and Cell Division Genes

    Science.gov (United States)

    Mengin-Lecreulx, Dominique; Ayala, Juan; Bouhss, Ahmed; van Heijenoort, Jean; Parquet, Claudine; Hara, Hiroshi

    1998-01-01

    Recently, a promoter for the essential gene ftsI, which encodes penicillin-binding protein 3 of Escherichia coli, was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802–5811, 1997). Disruption of this promoter (Pmra) on the chromosome and its replacement by the lac promoter (Pmra::Plac) led to isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from Pmra to ftsW, the fifth gene downstream from ftsI, was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which Pmra::Plac promoter expression was repressed or fully induced. It was confirmed that the Pmra promoter is required for expression of the first nine genes of the mra cluster: mraZ (orfC), mraW (orfB), ftsL (mraR), ftsI, murE, murF, mraY, murD, and ftsW. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced Pmra::Plac cells. This was correlated with an accumulation of the nucleotide precursors UDP–N-acetylglucosamine and UDP–N-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP–N-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ, the penultimate gene from this cluster, was significantly reduced when Pmra expression was repressed. It was concluded that the transcription of the genes located downstream from ftsW in the mra cluster, from murG to ftsZ, is also mainly (but not exclusively) dependent on the Pmra promoter. PMID:9721276

  4. Co-Expression of ORFCma with PHB Depolymerase (PhaZCma ) in Escherichia coli Induces Efficient Whole-Cell Biodegradation of Polyesters.

    Science.gov (United States)

    Lee, Ming-Chieh; Liu, En-Jung; Yang, Cheng-Han; Hsiao, Li-Jung; Wu, Tzong-Ming; Li, Si-Yu

    2018-04-01

    Whole-cell degradation of polyesters not only avoids the tedious process of enzyme separation, but also allows the degraded product to be reused as a carbon source. In this study, Escherichia coli BL21(DE3) harboring phaZ Cma , a gene encoding poly(3-hydroxybutyrate) (PHB) depolymerase from Caldimonas manganoxidans, is constructed. The extra-cellular fraction of E. coli/pPHAZ exhibits a fast PHB degradation rate where it only took 35 h to completely degrade PHB films, while C. manganoxidans takes 81 h to do the same. The co-expression of ORF Cma (a putative periplasmic substrate binding protein that is within the same operon of phaZ Cma ) further improves the PHB degradation. While 28 h is needed for E. coli/pPHAZ to cause an 80% weight loss in PHB films, E. coli/pORFPHAZ needs only 21 h. Furthermore, it is able to degrade at-least four different polyesters, PHB, poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(butylene succinate-co-adipate) (PBSA). Testing of the time course of 3-hydroxybutyrate concentration and the turbidity of the degradation solutions over time shows that PhaZ Cma has both exo- and endo-enzymatic activity. The whole-cell E. coli/pORFPHAZ can be used for recycling various polyesters while ORF Cma can potentially be a universal element for enhancing the secretion of recombinant protein. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

    Directory of Open Access Journals (Sweden)

    Mädje Katharina

    2012-01-01

    Full Text Available Abstract Background Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(PH. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH, presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S-1-(2-chlorophenylethanol from o-chloroacetophenone. Results As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3 that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by

  6. Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas.

    Science.gov (United States)

    Harris, L. C.; Margison, G. P.

    1993-01-01

    V79 Chinese hamster cells expressing either the O6-alkylguanine-DNA-alkyltransferase (ATase) encoded by the E. coli ogt gene or a truncated version of the E. coli ada gene have been exposed to various alkylnitrosoureas to investigate the contribution of ATase repairable lesions to the toxicity of these compounds. Both ATases are able to repair O6-alkylguanine (O6-AlkG) and O4-alkylthymine (O4-AlkT) but the ogt ATase is more efficient in the repair of O4-methylthymine (O4-MeT) and higher alkyl derivatives of O6-AlkG than is the ada ATase. Expression of the ogt ATase provided greater protection against the toxic effects of the alkylating agents then the ada ATase particularly with N-ethyl-N-nitrosourea (ENU) and N-butyl-N-nitrosourea (BNU) to which the ada ATase expressing cells were as sensitive as parent vector transfected cells. Although ogt was expressed at slightly higher levels than the truncated ada in the transfected cells, this could not account for the differential protection observed. For-N-methyl-N-nitrosourea (MNU) the increased protection in ogt-transfected cells is consistent with O4-MeT acting as a toxic lesion. For the longer chain alkylating agents and chloroethylating agents, the protection afforded by the ogt protein may be a consequence of the more efficient repair of O6-AlkG, O4-AlkT or both of these lesions in comparison with the ada-encoded ATase. Images Figure 2 Figure 3 PMID:8512805

  7. The oxidation of alkylaryl sulfides and benzo[b]thiophenes by Escherichia coli cells expressing wild-type and engineered styrene monooxygenase from Pseudomonas putida CA-3.

    Science.gov (United States)

    Nikodinovic-Runic, Jasmina; Coulombel, Lydie; Francuski, Djordje; Sharma, Narain D; Boyd, Derek R; Ferrall, Rory Moore O; O'Connor, Kevin E

    2013-06-01

    Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 μmoles min(-1) g cell dry weight(-1) resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite (S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10-23 %). The rate of benzo[b]thiophene and 2-methylbenzo[b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[b]thiophene and 2-methylbenzo[b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group.

  8. Multiple linear B-cell epitopes of classical swine fever virus glycoprotein E2 expressed in E.coli as multiple epitope vaccine induces a protective immune response

    Directory of Open Access Journals (Sweden)

    Wei Jian-Chao

    2011-07-01

    Full Text Available Abstract Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. Epitope-based vaccines is one of the current focuses in the development of new vaccines against classical swine fever virus (CSFV. Two B-cell linear epitopes rE2-ba from the E2 glycoprotein of CSFV, rE2-a (CFRREKPFPHRMDCVTTTVENED, aa844-865 and rE2-b (CKEDYRYAISSTNEIGLLGAGGLT, aa693-716, were constructed and heterologously expressed in Escherichia coli as multiple epitope vaccine. Fifteen 6-week-old specified-pathogen-free (SPF piglets were intramuscularly immunized with epitopes twice at 2-week intervals. All epitope-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:16 to 1:256. At this time, the pigs were subjected to challenge infection with a dose of 1 × 106 TCID50 virulent CSFV strain. After challenge infection, all of the rE2-ba-immunized pigs were alive and without symptoms or signs of CSF. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 5 days post challenge infection. The data from in vivo experiments shown that the multiple epitope rE2-ba shown a greater protection (similar to that of HCLV vaccine than that of mono-epitope peptide(rE2-a or rE2-b. Therefore, The results demonstrated that this multiple epitope peptide expressed in a prokaryotic system can be used as a potential DIVA (differentiating infected from vaccinated animals vaccine. The E.coli-expressed E2 multiple B-cell linear epitopes retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.

  9. Expression of maize prolamins in Escherichia Coli. [Zea mays L

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Szu-zhen; Esen, Asim

    1985-12-02

    A cDNA expression library of developing corn (Zea mays L.) endosperm has been constructed using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250-900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with /sup 32/P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, it was conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed. 21 references.

  10. Antibacterial activity of defensin PaDef from avocado fruit (Persea americana var. drymifolia) expressed in endothelial cells against Escherichia coli and Staphylococcus aureus.

    Science.gov (United States)

    Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Suárez-Rodríguez, Luis M; Salgado-Garciglia, Rafael; Rodríguez-Zapata, Luis C; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

    2013-01-01

    Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP) represent attractive control agents. Mexican avocado (Persea americana var. drymifolia) is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp) encoding a protein (78 aa) homologous with plant defensins (>80%). We expressed the defensin PaDef cDNA (pBME3) in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 μg/mL of total protein from clones (>55%). Also, S. aureus viability was inhibited from 50 μg/mL total protein (27-38%) but was more evident at 100 μg/mL (52-65%). This inhibition was higher than the effect showed by polyclonal population (~23%). Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

  11. Antibacterial Activity of Defensin PaDef from Avocado Fruit (Persea americana var. drymifolia Expressed in Endothelial Cells against Escherichia coli and Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Jaquelina Julia Guzmán-Rodríguez

    2013-01-01

    Full Text Available Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP represent attractive control agents. Mexican avocado (Persea americana var. drymifolia is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp encoding a protein (78 aa homologous with plant defensins (>80%. We expressed the defensin PaDef cDNA (pBME3 in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 μg/mL of total protein from clones (>55%. Also, S. aureus viability was inhibited from 50 μg/mL total protein (27–38% but was more evident at 100 μg/mL (52–65%. This inhibition was higher than the effect showed by polyclonal population (~23%. Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

  12. The oxygen effect in E. coli cells

    International Nuclear Information System (INIS)

    Myasnik, M.N.; Skvortsov, V.G.; Sokolov, V.A.

    1982-01-01

    In experiments on E. coli strains deficient in some stages of DNA repair from radiation damages, it was demonstrated that the value of the oxygen effect, under optimal conditions for manifestation thereof, decreases in the following order: E. coli WP2 (the wild type) → E. coli WP2 exr - and E. coli B → E. coli WP2 uvr A6 → E. coli WP2 rec Al and E. coli WP2 hcr - exr - . It was detected that 0.14 M NaCl solution sensitizes the anoxic cells of some E. coli strains to the effect of γ-radiation. It was established that mutation of the uvr A-gene increases sharply the sensitivity of cells to iradiation under the anoxic conditions in the presence of NaCl, the reverse'' oxygen effect being observed

  13. Expression and purification of soluble recombinant Hexastatin in E. coli

    International Nuclear Information System (INIS)

    He Xin; Wen Lei; Song Naling; Wang Dezhi; Zhao Qiren

    2012-01-01

    Purpose: To construct the expression vector of Hexastatin gene, to express and to purify the recombinant protein for further activity research. Methods: The human Hexastatin gene was isolated by RTPCR from EC9706 cells total RNA and cloned into pMD18-T for sequencing. Then the Hexastatin gene was subcloned into pMAL-c4x expression vector and induced to express by IPTG. The recombinant fusion protein was purified with Amylose Resin Heads. Results: RT-PCR product was about 687 bp and its sequence was the same as that of Hexastatin reported. The recombinant protein was expressed in E. coli BL21 with high level and the soluble protein accounted for 24.8% of the total bacterial protein. The purification of recombinant protein purified with Amylose Resin Heads reached more than 90%. Conclusion: The cloning, expression and purification of human Hexastatin have laid a foundation for its anti-angiogenesis therapy for tumor. (authors)

  14. Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene

    Directory of Open Access Journals (Sweden)

    Makino Takuya

    2012-07-01

    Full Text Available Abstract Background Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds. Results Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2, were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl flavanones were respectively converted to apigenin and (hydroxyl flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms, and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds. Conclusion We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity for various small molecules, allowing the biosynthesis of not only flavones (from flavanones but also a variety of

  15. 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.

  16. Genome engineering for improved recombinant protein expression in Escherichia coli.

    Science.gov (United States)

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-12-19

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

  17. Transcriptome of E. coli K1 bound to human brain microvascular endothelial cells

    OpenAIRE

    Xie, Yi; Parthasarathy, Geetha; Di Cello, Francescopaolo; Teng, Ching-Hao; Paul-Satyaseela, Maneesh; Kim, Kwang Sik

    2007-01-01

    Escherichia coli K1 is the most common Gram-negative organism causing neonatal meningitis. Binding to human brain microvascdular endothelial cells (HBMEC) is an essential step for E. coli K1 traversal of the blood-brain barrier. In this study, we examined expression profiles of E. coli K1 strain RS218 during its binding to HBMEC. Comparison of HBMEC-bound E. coli K1 with collagen-bound E. coli revealed more than one hundred genes whose expression patterns were significantly changed in HBMEC-b...

  18. Expression of fully functional tetrameric human hemoglobin in Escherichia coli

    International Nuclear Information System (INIS)

    Hoffman, S.J.; Looker, D.L.; Roehrich, J.M.; Cozart, P.E.; Durfee, S.L.; Tedesco, J.L.; Stetler, G.L.

    1990-01-01

    Synthesis genes encoding the human α- and β-globin polypeptides have been expressed from a single operon in Escherichia coli. The α- and β-globin polypeptides associate into soluble tetramers, incorporate heme, and accumulate to >5% of the total cellular protein. Purified recombinant hemoglobin has the correct stoichiometry of α- and β-globin chains and contains a full complement of heme. Each globin chain also contains an additional methionine as an extension to the amino terminus. The recombinant hemoglobin has a C 4 reversed-phase HPLC profile essentially identical to that of human hemoglobin A 0 and comigrates with hemoglobin A 0 on SDS/PAGE. The visible spectrum and oxygen affinity are similar to that of native human hemoglobin A 0 . The authors have also expressed the α- and β-globin genes separately and found that the expression of the α-globin gene alone results in a marked decrease in the accumulation of α-globin in the cell. Separate expression of the β-globin gene results in high levels of insoluble β-globin. These observations suggest that the presence of α- and β-globin in the same cell stabilizes α-globin and aids the correct folding of β-globin. This system provides a simple method for expressing large quantities of recombinant hemoglobin and allows facile manipulation of the genes encoding hemoglobin to produce functionally altered forms of this protein

  19. Cytolysin a expressing E. coli a promising candidate for imageable therapeutic probe

    International Nuclear Information System (INIS)

    Nguyen, Vu Hong; Phan, Thuy Xuan; Hong, Yeoung Jin; Min, Jung Joon

    2007-01-01

    Using bacteria for cancer treatment has a long history. Discovery of optical reporter genes consisting of fluorescent and luminescent protein facilitates the monitor of bacteria in vivo, non-invasively and repeatedly. E. coli, the natural enteric bacteria possessing capacity of tumor-targeting ability, seems to be suitable candidate for cancer treatment. In this study, we established the strain light-emitting E. coli for diagnostic purpose and Cytolysin A (Cly A) expressing E. coli for therapeutic purpose. E. coli (MG1655, wild type strain) was transformed plasmid pUC19 carrying lux gene to create the light expressing bacteria and test the tumor targeting-capacity by injecting the bacteria into CT26-tumor bearing mice via tail vein. On the other hand, for therapeutic purpose, plasmid containing Cly A gene, which is encoded for a pore-forming protein toxin, was introduced into E. coli. The toxicity of Cly A was evaluated in vitro by inoculating the bacteria with various cultured cancer cell lines. On the other hand, to test the therapeutic effect, the bacteria were injected intratumorally and intravenously into s.c.CT26-bearing as well as CT26-lung metastasized Balb/c mice. In vivo imaging data showed that the E. coli strains selectively located in the tumor. The in vitro result showed that the number of death cells were significantly higher in the samples containing E. coli expressing Cly A (E. coli Cly A) compared with the samples containing wild type strain. The growth of tumors was repressed in mice injected with either E. coli Cly A (significantly) or wild type E. coli (mildly), while tumors in no treatment group still grew fast. Furthermore, the tumors inoculated with E. coli cly A were necrotized but not with wild type E. coli. In the CT26-lung metastasized mouse model, the life span of mice was elongated when inject E. coli and longer in the group injected with E. coli cly A. Cly A expressing E. coli can become an effective candidate for imageable

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

    KAUST Repository

    Aljassim, Nada I.; Mantilla-Calderon, David; Wang, Tiannyu; Hong, Pei-Ying

    2017-01-01

    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. 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.

  2. Production of 10S-hydroxy-8(E)-octadecenoic acid from oleic acid by whole recombinant Escherichia coli cells expressing 10S-dioxygenase from Nostoc punctiforme PCC 73102 with the aid of a chaperone.

    Science.gov (United States)

    Kim, Min-Ji; Seo, Min-Ju; Shin, Kyung-Chul; Oh, Deok-Kun

    2017-01-01

    To increase the production of 10S-hydroxy-8(E)-octadecenoic acid from oleic acid by whole recombinant Escherichia coli cells expressing Nostoc punctiforme 10S-dioxygenase with the aid of a chaperone. The optimal conditions for 10S-hydroxy-8(E)-octadecenoic acid production by recombinant cells co-expressing chaperone plasmid were pH 9, 35 °C, 15 % (v/v) dimethyl sulfoxide, 40 g cells l -1 , and 10 g oleic acid l -1 . Under these conditions, recombinant cells co-expressing chaperone plasmid produced 7.2 g 10S-hydroxy-8(E)-octadecenoic acid l -1 within 30 min, with a conversion yield of 72 % (w/w) and a volumetric productivity of 14.4 g l -1 h -1 . The activity of recombinant cells expressing 10S-dioxygenase was increased by 200 % with the aid of a chaperone, demonstrating the first biotechnological production of 10S-hydroxy-8(E)-octadecenoic acid using recombinant cells expressing 10S-dioxygenase.

  3. ENTRAPMENT OF FLUORESCENT E. COLI CELLS IN ALGINATE GEL

    Directory of Open Access Journals (Sweden)

    T. VINTILA

    2009-05-01

    Full Text Available By this experiment we will demonstrate the possibility to obtain genetically modified microbial strains that can be used as markers in different studies. The trait transferred in this study is the fluorescence in UV light expressed by a gene isolated from jellyfish. This gene was insered into a plasmid carrying ampiciline resistance and in the operon for arabinose fermentation. The plasmid was called pGLO. E coli HB101 K-12, ampicillin resistant colonies has been obtained. The colonies on the LB/amp/ara plate fluoresce green under UV light and the transformed colonies can grow on ampicillin. Transformation efficiency = 362 transformed colonies/ μg DNA. The cells where immobilized by entrapment in alginate gel to study the phenomenon involved in cells immobilization. After immobilization in alginate gel, 5x104 cells of E. coli pGLO / capsule and 1,4 x 105 cells of E. coli HB101/capsule has been found. Fluorescent microscopy revealed the presence of pGLO carrying cells into the capsules. After cultivation of alginate capsules containing E. coli in LB broth, and fluorescent microscopy of the capsule sections, several observations of the phenomenon involved in continuous fermentation using biocatalysts in has been made. These cells grow and migrate to the cortical part of the matrix where they are immobilized.

  4. Importance of the High-Expression of Proline Transporter PutP to the Adaptation of Escherichia coli to High Salinity.

    Science.gov (United States)

    Sasaki, Hideaki; Sato, Daichi; Oshima, Akinobu

    2017-01-01

     The effect of the amount of the proline transporter PutP expression on the mechanism of adaptation of E. coli cells to high salinity was analyzed. The PutP gene derived from the E. coli expression plasmid was introduced into the E. coli cell, and a high PutP expression strain was developed. At 1.2 M NaCl culture condition, the growth of normal E. coli cells was inhibited, whereas high ProP expression cells showed growth under 2.5 M NaCl conditions. The uptake of proline by E. coli as a compatible solute and substrate for metabolization was in good accordance with those seen in cell growth. These data suggested that the amount of the proline transporter PutP expression played an important role in the adaptation of E. coli cells to high saline conditions.

  5. Biodegradation of dioxins by recombinant Escherichia coli expressing rat CYP1A1 or its mutant

    Energy Technology Data Exchange (ETDEWEB)

    Shinkyo, Raku; Inouye, Kuniyo [Kyoto Univ. (Japan). Div. of Food Science and Biotechnology; Kamakura, Masaki; Ikushiro, Shin-ichi; Sakaki, Toshiyuki [Toyama Prefectural Univ. (Japan). Biotechnology Research Center

    2006-09-15

    Among polychlorinated dibenzo-p-dioxins (PCDDs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is the most toxic one. Recently, we reported that rat CYP1A1 mutant, F240A, expressed in yeast showed metabolic activity toward 2,3,7,8-TetraCDD. In this study, we successfully expressed N-terminal truncated P450s ({delta}1A1 and {delta}F240A) in Escherichia coli cells. Kinetic analysis using membrane fractions prepared from the recombinant E. coli cells revealed that {delta}F240A has enzymatic properties similar to F240A expressed in yeast. The metabolism of PCDDs by recombinant E. coli cells expressing both {delta}F240A and human NADPH-P450 reductase was also examined. When 2,3,7-TriCDD was added to the E. coli cell culture at a final concentration of 10 {mu}M, approximately 90% of the 2,3,7-TriCDD was converted into multiple metabolites within 8 h. These results indicate the possible application of prokaryotic cells expressing {delta}F240A to the bioremediation of PCDD-contaminated soil. (orig.)

  6. Expression, purification, and characterization of a dentin phosphoprotein produced by Escherichia coli, and its odontoblastic differentiation effects on human dental pulp cells.

    Science.gov (United States)

    Yun, Ye-Rang; Jeon, Eunyi; Lee, Sujin; Kang, Wonmo; Kim, Sang-Gi; Kim, Hae-Won; Suh, Chang Kook; Jang, Jun-Hyeog

    2012-08-01

    To investigate the functions of recombinant human dentin phosphoprotein (rhDPP), we examined cell adhesion, viability and the odontoblastic differentiation activity of human dental pulp cells (hDPCs). Firstly, rhDPP was constructed using pBAD-HisA plasmid in Escherichia coli. Cell adhesion and viability of hDPCs by rhDPP was examined using a crystal violet assay and a MTT assay, ALP, mineralization activity and odontoblastic differentiation-related mRNA levels of hDPCs were measured to elucidate the odontoblastic differentiation effect of rhDPP on hDPCs. Initially, rhDPP significantly and dose-dependently increased hDPCs adhesion versus the untreated control (p < 0.05). Cell viability was also significantly increased by rhDPP at 5 days (p < 0.001). Furthermore, the odontoblastic differentiation effect of rhDPP was verified by measuring ALP activity, mineralization activity and the mRNA levels of odontoblastic differentiation markers. Taken together, rhDPP is expected to play an important role on hDPCs, thereby suggesting its potential use for tooth repair and regeneration.

  7. 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

  8. Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli.

    Science.gov (United States)

    Fujimitsu, Kazuyuki; Su'etsugu, Masayuki; Yamaguchi, Yoko; Mazda, Kensaku; Fu, Nisi; Kawakami, Hironori; Katayama, Tsutomu

    2008-08-01

    The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

  9. Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells.

    Science.gov (United States)

    Selmin, Ornella I; Fang, Changming; Lyon, Adam M; Doetschman, Tom C; Thompson, Patricia A; Martinez, Jesse D; Smith, Jeffrey W; Lance, Peter M; Romagnolo, Donato F

    2016-02-01

    The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. We investigated how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation. Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J and Apc-deficient (Apc(Min) (/+)) male mice, respectively. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC. In Apc(Min) (/+) mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp and increased Cox-2 expression in apparently normal adjacent mucosa and colon tumors. Apc knockdown in HCT-116 cells increased cellular myelocytomatosis (c-MYC) and lowered (∼50%) FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells

  10. Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli

    DEFF Research Database (Denmark)

    Grant, Chris; Woodley, John; Baganz, Frank

    2011-01-01

    , successful n-dodecane oxidation for the production of 1-dodecanol or dodecanoic acid has proven elusive in the past when using alkB-expressing recombinants. This article demonstrates, for the first time in vivo, by using the Escherichia coli GEC137 pGEc47ΔJ strain, that n-dodecane oxidation using this enzyme......The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C5–C12 alkanes to primary alcohols both in the wild-type organism by growth on C5–C12 alkanes as sole carbon source and in vitro. Despite this...... aqueous phase and 200mL of n-dodecane as a second phase. The maximum volumetric rate of combined alcohol and acid production achieved was 1.9g/Lorganic/h (0.35g/Ltotal/h). The maximum specific activity of combined alcohol and acid production was 7-fold lower on n-dodecane (3.5μmol/min/gdcw) than on n...

  11. 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.

  12. in Escherichia coli with native cholesterol oxidase expressed

    African Journals Online (AJOL)

    The structure and bio-activity of an endogenous cholesterol oxidase from Brevibacterium sp. was compared to the same enzyme exogenously expressed in Escherichia coli BL21 (DE3) with and without N- or C-terminal his-tags. The different proteins were purified with affinity and subtractive protocols. The specific activity of ...

  13. 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...

  14. Short-chain flavor ester synthesis in organic media by an E. coli whole-cell biocatalyst expressing a newly characterized heterologous lipase.

    Directory of Open Access Journals (Sweden)

    Guillaume Brault

    Full Text Available Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to 'naturally' produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into E. coli BL21 (DE3 for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2 showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w. The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media.

  15. Engineering cell wall synthesis mechanism for enhanced PHB accumulation in E. coli.

    Science.gov (United States)

    Zhang, Xing-Chen; Guo, Yingying; Liu, Xu; Chen, Xin-Guang; Wu, Qiong; Chen, Guo-Qiang

    2018-01-01

    The rigidity of bacterial cell walls synthesized by a complicated pathway limit the cell shapes as coccus, bar or ellipse or even fibers. A less rigid bacterium could be beneficial for intracellular accumulation of poly-3-hydroxybutyrate (PHB) as granular inclusion bodies. To understand how cell rigidity affects PHB accumulation, E. coli cell wall synthesis pathway was reinforced and weakened, respectively. Cell rigidity was achieved by thickening the cell walls via insertion of a constitutive gltA (encoding citrate synthase) promoter in front of a series of cell wall synthesis genes on the chromosome of several E. coli derivatives, resulting in 1.32-1.60 folds increase of Young's modulus in mechanical strength for longer E. coli cells over-expressing fission ring FtsZ protein inhibiting gene sulA. Cell rigidity was weakened by down regulating expressions of ten genes in the cell wall synthesis pathway using CRISPRi, leading to elastic cells with more spaces for PHB accumulation. The regulation on cell wall synthesis changes the cell rigidity: E. coli with thickened cell walls accumulated only 25% PHB while cell wall weakened E. coli produced 93% PHB. Manipulation on cell wall synthesis mechanism adds another possibility to morphology engineering of microorganisms. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  16. 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 antimicr......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...... 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...... in expression have no current defined function. These genes, as well as those induced by stresses relevant to biofilm growth such as oxygen and nutrient limitation, may be important factors that trigger enhanced resistance mechanisms of sessile communities to antibiotics and hydrodynamic shear forces....

  17. Expression, purification and crystallization of the Cmi immunity protein from Escherichia coli

    International Nuclear Information System (INIS)

    Römer, Christin; Patzer, Silke I.; Albrecht, Reinhard; Zeth, Kornelius; Braun, Volkmar

    2011-01-01

    The colicin M immunity protein Cmi protects E. coli cells against killing by colicin M. The Cmi protein was produced for structure determination and crystals were obtained which diffracted to high resolution. Many bacteria kill related bacteria by secretion of bacteriocins. In Escherichia coli, the colicin M protein kills E. coli after uptake into the periplasm. Self-protection from destruction is provided by the co-expressed immunity protein. The colicin M immunity protein (Cmi) was cloned, overexpressed and purified to homogeneity. The correct fold of purified Cmi was analyzed by activity tests and circular-dichroism spectroscopy. Crystallization trials yielded crystals, one of which diffracted to a resolution of 1.9 Å in the orthorhombic space group C222 1 . The crystal packing, with unit-cell parameters a = 66.02, b = 83.47, c = 38.30 Å, indicated the presence of one monomer in the asymmetric unit with a solvent content of 53%

  18. 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.

  19. 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...... a protocol for expressing Hbs with low intrinsic solubilities. Since the alpha- and beta-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression......-translational modifications. CONCLUSION/SIGNIFICANCE: Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need...

  20. Regulation of gene expression in Escherichia coli and its bacteriophage

    International Nuclear Information System (INIS)

    Higgins, C.F.

    1986-01-01

    This chapter reviews the study of prokaryotic gene expression beginning with a look at the regulation of the lactose operon and the mechanism of attenuation in the tryptophan operon to the more recent development of recombinant DNA technology. The chapter deals almost entirely with escherichia coli and its bacteriophage. The only experimental technique which the authors explore in some detail is the construction and use of gene and operon fusions which have revolutionized the study of gene expression. Various mechanisms by which E. Coli regulate the cellular levels of individual messenger-RNA species are described. Translational regulation of the cellular levels of messenger-RNA include signals encoded within the messenger-RNA molecule itself and regulatory molecules which interact with the messenger-RNA and alter it translational efficiency

  1. 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.

  2. 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.

  3. Expression and purification of recombinant hemoglobin in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Chandrasekhar Natarajan

    Full Text Available 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 a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus, a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

  4. Expression and purification of recombinant hemoglobin in Escherichia coli.

    Science.gov (United States)

    Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela; Weber, Roy E; Moriyama, Hideaki; Storz, Jay F

    2011-01-01

    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 a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species. As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications. Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

  5. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

    DEFF Research Database (Denmark)

    Sørensen, Hans; Mortensen, Kim

    2005-01-01

    Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed...... molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target...... for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible...

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

    OpenAIRE

    Connell, I; Agace, W; Klemm, P; Schembri, M; Mărild, S; Svanborg, C

    1996-01-01

    Type 1 fimbriae are adhesion organelles expressed by many Gram-negative bacteria. They facilitate adherence to mucosal surfaces and inflammatory cells in vitro, but their contribution to virulence has not been defined. This study presents evidence that type 1 fimbriae increase the virulence of Escherichia coli for the urinary tract by promoting bacterial persistence and enhancing the inflammatory response to infection. In a clinical study, we observed that disease severity was greater in chil...

  7. 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.

  8. The SOS response is permitted in Escherichia coli strains deficient in the expression of the mazEF pathway.

    Science.gov (United States)

    Kalderon, Ziva; Kumar, Sathish; Engelberg-Kulka, Hanna

    2014-01-01

    The Escherichia coli (E. coli) SOS response is the largest, most complex, and best characterized bacterial network induced by DNA damage. It is controlled by a complex network involving the RecA and LexA proteins. We have previously shown that the SOS response to DNA damage is inhibited by various elements involved in the expression of the E. coli toxin-antitoxin mazEF pathway. Since the mazEF module is present on the chromosomes of most E. coli strains, here we asked: Why is the SOS response found in so many E. coli strains? Is the mazEF module present but inactive in those strains? We examined three E. coli strains used for studies of the SOS response, strains AB1932, BW25113, and MG1655. We found that each of these strains is either missing or inhibiting one of several elements involved in the expression of the mazEF-mediated death pathway. Thus, the SOS response only takes place in E. coli cells in which one or more elements of the E. coli toxin-antitoxin module mazEF or its downstream pathway is not functioning.

  9. Expression, purification, and activity assay of peptide deformylase from Escherichia coli and Staphylococcus aureus.

    Science.gov (United States)

    Che, Xuchun; Hu, Jinwei; Wang, Lijuan; Zhu, Zhifeng; Xu, Qiong; Lv, Junqiang; Fu, Zheng; Sun, Yajun; Sun, Jia; Lin, Gang; Lu, Rong; Yao, Zhi

    2011-11-01

    Peptide deformylase (PDF) is considered an attractive target for screening novel antibiotics. The PDF from Escherichia coli and Staphylococcus aureus are representative of the gram-negative species type of PDF (type I PDF) and the gram-positive species type of PDF (type II PDF), respectively. They could be used for screening broad-spectrum antibiotics. Herein, we cloned the def gene by PCR, inserted it into plasmid pET-22b-def, and transformed the plasmid into E. coli BL21 (DE3) cells, then the cells were induced by IPTG to express PDF. E. coli Ni(2+)-PDF was extracted and purified by ion-exchange chromatography and gel filtration chromatography. S. aureus PDFs were extracted and purified using the MagExtractor kit. The nickel form of S. aureus PDF was obtained by adding NiCl(2) to all reagents used for purification. Iron-enriched S. aureus PDF was obtained by adding FeCl(3) to the growth medium for E. coli BL21 (DE3) cells and adding FeCl(3) and catalase to all reagents used for purification. The activities of PDFs were analyzed, compared, and grouped according to the experimental conditions that produced optimal activity, and we used actinonin as an inhibitor of PDF and calculated the IC(50) value. We obtained high expression of E. coli and S. aureus PDF with high activity and stability. The function of PDFs was inhibited by actinonin in a dose-dependent manner. Results may be helpful for future mechanistic investigations of PDF as well as high-throughput screening for other PDF inhibitors.

  10. The new pLAI (lux regulon based auto-inducible expression system for recombinant protein production in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Nocadello Salvatore

    2012-01-01

    Full Text Available Abstract Background After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. Result The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS system. The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. Conclusion Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene

  11. Amaranthus caudatus extract inhibits the invasion of E. coli into uroepithelial cells.

    Science.gov (United States)

    Mohanty, Soumitra; Zambrana, Silvia; Dieulouard, Soizic; Kamolvit, Witchuda; Nilsén, Vera; Gonzales, Eduardo; Östenson, Claes-Göran; Brauner, Annelie

    2018-06-28

    Amaranthus caudatus is traditionally used to treat infections. Based on its traditional usage, we investigated the effect of A. caudatus on the bladder epithelial cells in the protection of E. coli infection. The direct antimicrobial effects of A. caudatus on uropathogenic bacteria were investigated using minimum inhibitory concentration (MIC) assay. Bladder epithelial cell lines T24 and 5637 and uropathogenic E. coli strain #12 were used to investigate the effect of A. caudatus. Bacterial adhesion and invasion into bladder cells treated with A. caudatus was analyzed. Expression of uroplakin-1a (UPK1A), β1 integrin (ITGB1), caveolin-1 (CAV1) and the antimicrobial peptides human β defensin-2 (DEFB4A) and LL-37 (CAMP) was evaluated using RT-PCR. No direct antibacterial effect on E. coli or any of the tested uropathogenic strains was observed by A. caudatus. However, we demonstrated reduced mRNA expression of uroplakin-1a and caveolin-1, but not β1 integrin after treatment of uroepithelial cells, mirrored by the decreased adhesion and invasion of E. coli. A. caudatus treatment did not induce increased gene expression of the antimicrobial peptides, LL-37 and human β-defensin-2. Our results showed that A. caudatus has a protective role on bladder epithelial cells against uropathogenic E. coli infection by decreasing the bacterial adhesion and invasion, thereby preventing infection. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. 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.

  13. EFFECTS OF THE ANTIMUTAGENS VANILLIN AND CINNAMALDEHYDE ON SPONTANEOUS MUTATION IN E. COLI LACL STRAINS AND ON GLOBAL GENE EXPRESSION IN SALMONELLA TA104 AND HUMAN HEPG2 CELLS

    Science.gov (United States)

    Effects of the Antimutagens Vanillin and Cinnamaldehyde on Spontaneous Mutation in E. coli lacI Strains and on Global Gene Epression in Salmonella TAlO4 and Human HepG2 Cells In previous work we have shown that vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutag...

  14. Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Shimada, Ichio

    2010-01-01

    The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.

  15. 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.

  16. Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

    Science.gov (United States)

    Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

    2015-01-01

    In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively. © 2014 Wiley Periodicals, Inc.

  17. A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically-Modified Neural Stem Cells Expressing E.Coli Cytosine Deaminase for Treatment of Recurrent High Grade Gliomas

    Science.gov (United States)

    2017-11-07

    Adult Anaplastic Astrocytoma; Recurrent Grade III Glioma; Recurrent Grade IV Glioma; Adult Anaplastic Oligodendroglioma; Adult Brain Tumor; Adult Giant Cell Glioblastoma; Adult Glioblastoma; Adult Gliosarcoma; Adult Mixed Glioma; Recurrent Adult Brain Tumor; Adult Anaplastic Oligoastrocytoma; Recurrent High Grade Glioma

  18. Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli

    International Nuclear Information System (INIS)

    Coghlan, V.M.; Vickery, L.E.

    1989-01-01

    A cDNA fragment encoding human ferredoxin, a mitochondrial [2Fe-2S] protein, was introduced into Escherichia coli by using an expression vector based on the approach of Nagai and Thogersen. Expression was under control of the λP L promoter and resulted in production of ferredoxin as a cleavable fusion protein with an amino-terminal fragment derived from bacteriophage λcII protein. The fusion protein was isolated from the soluble fraction of induced cells and was specifically cleaved to yield mature recombinant ferredoxin. The recombinant protein was shown to be identical in size to ferredoxin isolated from human placenta (13,546 Da) by NaDodSO 4 /PAGE and partial amino acid sequencing. E. coli cells expressing human ferredoxin were brown in color, and absorbance and electron paramagnetic resonance spectra of the purified recombinant protein established that the [2Fe-2S]center was assembled and incorporated into ferredoxin in vivo. Recombinant ferredoxin was active in steroid hydroxylations when reconstituted with cytochromes P-450 sec and P-450 11β and exhibited rates comparable to those observed for ferredoxin isolated from human placenta. This expression system should be useful in production of native and structurally altered forms of human ferredoxin for studies of ferredoxin structure and function

  19. 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

  20. Induction of genetic recombination in the lambda bacteriophage by ultraviolet radiation of the Escherichia Coli cells

    International Nuclear Information System (INIS)

    Alcantara D, D.

    1986-12-01

    In this work there are reported the results that show that although the stimulation of the recombination of the Lambda bacteriophage, by UV irradiation of the cells of Escherichia Coli, it looks to be the result of the high expression of the functions of the SOS system, doesn't keep some relationship with the high concentration of protein reached RecA. (Author)

  1. Alterations induced in Escherichia Coli cells by gamma radiation

    International Nuclear Information System (INIS)

    Kappke, J.; Schelin, H.R.; Paschuk, S.A.; Denyak, V.; Silva, E.R. da; Jesus, E.F.O. de; Lopes, R.T.; Carlin, N.; Toledo, E.S.

    2007-01-01

    Modifications occurred in Escherichia coli cells exposed to gamma radiation ( 60 Co source) were investigated. The irradiations were done at the LIN-COPPE laboratory of the UFRJ and the analysis at the Biology Department of the UTFPR. The E. coli cells were irradiated with 30, 60, 90, 120, 150, 180, 210, 240, 300, 480, 600 e 750 Gy doses. The samples were analyzed with Gram-stain, biochemical tests in EPM, MIO and Lysine Broth, Simmons Cytrate Medium and Rhamnose Broth, antibiogram and isolation of auxotrophic mutants. It was observed that for the received doses the E. coli did not show morphological alterations in the tests. Some E. Coli cells showed to be able to deaminade the L-tryptophan or they changed their sensibility for amoxillin and cephaloonine after the irradiation. The existence of aauxotrophic mutants after irradiation was also verified. (author)

  2. High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli

    Czech Academy of Sciences Publication Activity Database

    Rozbeský, Daniel; Kavan, Daniel; Chmelík, Josef; Novák, Petr; Vaněk, Ondřej; Bezouška, Karel

    2011-01-01

    Roč. 77, č. 2 (2011), s. 178-184 ISSN 1046-5928 R&D Projects: GA ČR GA303/09/0477; GA ČR GD305/09/H008; GA ČR GAP207/10/1040; GA AV ČR IAA500200620; GA MŠk LC07017; GA MŠk LC545; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50200510 Keywords : Natural killer cell * NKR-P1C receptor * NK1.1 antigen Subject RIV: EC - Immunology Impact factor: 1.587, year: 2011

  3. Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli

    DEFF Research Database (Denmark)

    Lundemo, M. T.; Notonier, S.; Striedner, G.

    2016-01-01

    fatty acids at the terminal position. ω-Hydroxylated fatty acids can be used in the field of high-end polymers and in the cosmetic and fragrance industry. Here, we have identified the limitations for implementation of a whole-cell P450-catalyzed reaction by characterizing the chosen biocatalyst as well......Cytochrome P450s are interesting biocatalysts due to their ability to hydroxylate non-activated hydrocarbons in a selective manner. However, to date only a few P450-catalyzed processes have been implemented in industry due to the difficulty of developing economically feasible processes...

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

    Directory of Open Access Journals (Sweden)

    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

  5. An Engineered Survival-Selection Assay for Extracellular Protein Expression Uncovers Hypersecretory Phenotypes in Escherichia coli.

    Science.gov (United States)

    Natarajan, Aravind; Haitjema, Charles H; Lee, Robert; Boock, Jason T; DeLisa, Matthew P

    2017-05-19

    The extracellular expression of recombinant proteins using laboratory strains of Escherichia coli is now routinely achieved using naturally secreted substrates, such as YebF or the osmotically inducible protein Y (OsmY), as carrier molecules. However, secretion efficiency through these pathways needs to be improved for most synthetic biology and metabolic engineering applications. To address this challenge, we developed a generalizable survival-based selection strategy that effectively couples extracellular protein secretion to antibiotic resistance and enables facile isolation of rare mutants from very large populations (i.e., 10 10-12 clones) based simply on cell growth. Using this strategy in the context of the YebF pathway, a comprehensive library of E. coli single-gene knockout mutants was screened and several gain-of-function mutations were isolated that increased the efficiency of extracellular expression without compromising the integrity of the outer membrane. We anticipate that this user-friendly strategy could be leveraged to better understand the YebF pathway and other secretory mechanisms-enabling the exploration of protein secretion in pathogenesis as well as the creation of designer E. coli strains with greatly expanded secretomes-all without the need for expensive exogenous reagents, assay instruments, or robotic automation.

  6. 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.

  7. GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

    Science.gov (United States)

    Goyal, Megha; Chaudhuri, Tapan K

    2015-07-01

    Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Integration of AI-2 Based Cell-Cell Signaling with Metabolic Cues in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Arindam Mitra

    Full Text Available The quorum sensing molecule Autoinducer-2 (AI-2 is generated as a byproduct of activated methyl cycle by the action of LuxS in Escherichia coli. AI-2 is synthesized, released and later internalized in a cell-density dependent manner. Here, by mutational analysis of the genes, uvrY and csrA, we describe a regulatory circuit of accumulation and uptake of AI-2. We constructed a single-copy chromosomal luxS-lacZ fusion in a luxS + merodiploid strain and evaluated its relative expression in uvrY and csrA mutants. At the entry of stationary phase, the expression of the fusion and AI-2 accumulation was positively regulated by uvrY and negatively regulated by csrA respectively. A deletion of csrA altered message stability of the luxS transcript and CsrA protein exhibited weak binding to 5' luxS regulatory region. DNA protein interaction and chromatin immunoprecipitation analysis confirmed direct interaction of UvrY with the luxS promoter. Additionally, reduced expression of the fusion in hfq deletion mutant suggested involvement of small RNA interactions in luxS regulation. In contrast, the expression of lsrA operon involved in AI-2 uptake, is negatively regulated by uvrY and positively by csrA in a cell-density dependent manner. The dual role of csrA in AI-2 synthesis and uptake suggested a regulatory crosstalk of cell signaling with carbon regulation in Escherichia coli. We found that the cAMP-CRP mediated catabolite repression of luxS expression was uvrY dependent. This study suggests that luxS expression is complex and regulated at the level of transcription and translation. The multifactorial regulation supports the notion that cell-cell communication requires interaction and integration of multiple metabolic signals.

  9. Toxicity of cadmium sulfide (CdS) nanoparticles against Escherichia coli and HeLa cells

    International Nuclear Information System (INIS)

    Hossain, Sk Tofajjen; Mukherjee, Samir Kumar

    2013-01-01

    Highlights: • Toxic effect of CdS NPs on the growth and cell division in E. coli was studied. • CdS NPs affected cell surface topology and cell division. • Downregulation of both FtsZ and FtsQ was observed due to NPs exposure. • CdS NPs affected HeLa cell morphology with fragmented nuclei. • All such effects might be due to elevated oxidative stress. -- Abstract: The present study endeavours to assess the toxic effect of synthesized CdS nanoparticles (NPs) on Escherichia coli and HeLa cells. The CdS NPs were characterized by DLS, XRD, TEM and AFM studies and the average size of NPs was revealed as ∼3 nm. On CdS NPs exposure bacterial cells changed morphological features to filamentous form and damage of the cell surface was found by AFM study. The expression of two conserved cell division components namely ftsZ and ftsQ in E. coli was decreased both at transcriptional and translational levels upon CdS NPs exposure. CdS NPs inhibited proper cell septum formation without affecting the nucleoid segregation. Viability of HeLa cells declined with increasing concentration of CdS NPs and the IC 50 value was found to be 4 μg/mL. NPs treated HeLa cells showed changed morphology with condensed and fragmented nuclei. Increased level of reactive oxygen species (ROS) was found both in E. coli and HeLa cells on CdS NPs exposure. The inverse correlation between declined cell viabilities and elevated ROS level suggested that oxidative stress seems to be the key event by which NPs induce toxicity both in E. coli and HeLa cells

  10. Biodesulfurization of dibenzothiophene in Escherichia coli is enhanced by expression of a Vibrio harveyi oxidoreductase gene.

    Science.gov (United States)

    Reichmuth, D S; Hittle, J L; Blanch, H W; Keasling, J D

    2000-01-05

    One possible alternative to current fuel hydrodesulfurization methods is the use of microorganisms to remove sulfur compounds. Biodesulfurization requires much milder processing conditions, gives higher specificity, and does not require molecular hydrogen. In the present work we have produced two compatible plasmids: pDSR3, which allows Escherichia coli to convert dibenzothiophene (DBT) to hydroxybiphenyl (HBP), and pDSR2, which produces a Vibrio harveyi flavin oxidoreductase. We show that the flavin oxidoreductase enhances the rate of DBT removal when co-expressed in vivo with the desulfurization enzymes. The plasmids pDSR2 and pDSR3 were co-expressed in growing cultures. The expression of oxidoreductase caused an increase in the rate of DBT removal but a decrease in the rate of HBP production. The maximum rate of DBT removal was 8 mg/h. g dry cell weight. Experiments were also conducted using resting cells with the addition of various carbon sources. It was found that the addition of glucose or glycerol to cultures with oxidoreductase expression produced the highest DBT removal rate (51 mg/h. g dry cell weight). The culture with acetate and no oxidoreductase expression had the highest level of HBP production. For all carbon sources, the DBT removal rate was faster and the HBP generation rate slower with the expression of the oxidoreductase. Analysis of desulfurization intermediates indicates that the last enzyme in the pathway may be limiting. Copyright 2000 John Wiley & Sons, Inc.

  11. High Level Expression and Purification of the Clinically Active Antimicrobial Peptide P-113 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Kuang-Ting Cheng

    2018-03-01

    Full Text Available P-113, which was originally derived from the human saliva protein histatin 5, is a histidine-rich antimicrobial peptide with the sequence AKRHHGYKRKFH. P-113 is currently undergoing phase II clinical trial as a pharmaceutical agent to fight against fungal infections in HIV patients with oral candidiasis. Previously, we developed a new procedure for the high-yield expression and purification of hG31P, an analogue and antagonist of human CXCL8. Moreover, we have successfully removed lipopolysaccharide (LPS, endotoxin associated with hG31P in the expression with Escherichia coli. In this paper, we have used hG31P as a novel fusion protein for the expression and purification of P-113. The purity of the expressed P-113 is more than 95% and the yield is 4 mg P-113 per liter of E. coli cell culture in Luria-Bertani (LB medium. The antimicrobial activity of the purified P-113 was tested. Furthermore, we used circular dichroism (CD and nuclear magnetic resonance (NMR spectroscopy to study the structural properties of P-113. Our results indicate that using hG31P as a fusion protein to obtain large quantities of P-113 is feasible and is easy to scale up for commercial production. An effective way of producing enough P-113 for future clinical studies is evident in this study.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Ma, Yi; Yu, Jieying; Lin, Jinglian; Wu, Shaomin; Li, Shan; Wang, Jufang

    2016-01-01

    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-H 10 . 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.

  14. 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.

  15. Phenotypic and gene expression responses of E. coli to antibiotics during spaceflight

    Science.gov (United States)

    Zea, Luis

    Bacterial susceptibility to antibiotics has been shown in vitro to be reduced during spaceflight; however, the underlying mechanisms responsible for this outcome are not fully understood. In particular, it is not yet clear whether this observed response is due to increased drug resistance (a microbial defense response) or decreased drug efficacy (a microgravity biophysical mass transport effect). To gain insight into the differentiation between these two potential causes, an investigation was undertaken onboard the International Space Station (ISS) in 2014 termed Antibiotic Effectiveness in Space-1 (AES-1). For this purpose, E. coli was challenged with two antibiotics, Gentamicin Sulfate and Colistin Sulfate, at concentrations higher than those needed to inhibit growth on Earth. Phenotypic parameters (cell size, cell envelope thickness, population density and lag phase duration) and gene expression were compared between the spaceflight samples and ground controls cultured in varying levels of drug concentration. It was observed that flight samples proliferated in antibiotic concentrations that were inhibitory on Earth, growing on average to a 13-fold greater concentration than matched 1g controls. Furthermore, at the highest drug concentrations in space, E. coli cells were observed to aggregate into visible clusters. In spaceflight, cell size was significantly reduced, translating to a decrease in cell surface area to about one half of the ground controls. Smaller cell surface area can in turn proportionally reduce the rate of antibiotic molecules reaching the cell. Additionally, it was observed that genes --- in some cases more than 2000 --- were overexpressed in space with respect to ground controls. Up-regulated genes include poxB, which helps catabolize glucose into organic acids that alter acidity around and inside the cell, and the gadABC family genes, which confer resistance to extreme acid conditions. The next step is to characterize the mechanisms behind

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

    Directory of Open Access Journals (Sweden)

    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

  17. 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....

  18. Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology.

    Science.gov (United States)

    Shafiee, Fatemeh; Rabbani, Mohammad; Jahanian-Najafabadi, Ali

    2017-01-01

    The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium. Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli ( E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3), followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM). Finally, the best culture medium was selected. Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml). Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media. In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.

  19. Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Fatemeh Shafiee

    2017-01-01

    Full Text Available Background: The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium. Materials and Methods: Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli (E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3, followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM. Finally, the best culture medium was selected. Results: Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml. Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media. Conclusion: In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.

  20. Biodesulfurization of dibenzothiophene in Escherichia coli is enhanced by expression of a Vibrio harveyi oxidoreductase gene

    Energy Technology Data Exchange (ETDEWEB)

    Reichmuth, D.S.; Hittle, J.L.; Blanch, H.W.; Keasling, J.D.

    2000-01-05

    One possible alternative to current fuel hydrodesulfurization methods is the use of microorganisms to remove sulfur compounds. Biodesulfurization requires much milder processing conditions, gives higher specificity, and does not require molecular hydrogen. In the present work the authors have produced two compatible plasmids: pDSR3, which allows Escherichia coli to convert dibenzothiophene (DBT) to hydroxybiphenyl (HBP), and pDSR2, which produces a Vibrio harveyi flavin oxidoreductase. The authors show that the flavin oxidoreductase enhances the rate of DBT removal when co-expressed in vivo with the desulfurization enzymes. The plasmids pDSR2 and pDSR3 were co-expressed in growing cultures. The expression of oxidoreductase caused an increase in the rate of DBT removal but a decrease in the rate of HBP production. The maximum rate of DBT removal was 8 mg/h {center{underscore}dot} g dry cell weight. Experiments were also conducted using resting cells with the addition of various carbon sources. It was found that the addition of glucose or glycerol to cultures with oxidoreductase expression produced the highest DBT removal rate. The culture with acetate and no oxidoreductase expression had the highest level of HBP production. For all carbon sources, the DBT removal rate was faster and the HBP generation rate slower with the expression of the oxidoreductase. Analysis of desulfurization intermediates indicates that the last enzyme in the pathway may be limiting.

  1. 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....

  2. 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.

  3. Functional Expression of Spider Neurotoxic Peptide Huwentoxin-I in E. coli

    Science.gov (United States)

    Zhang, Hui; Liu, Yan-Bo; Peng, Kuan; Liang, Songping; Zhang, Dong-Yi

    2011-01-01

    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 Nav1.7 at an IC50 of 640 nmole/L, similar to that of the natural huwentoxin-I, which is 630 nmole/L. PMID:21731778

  4. [Expression, purification and immunogenicity of human papillomavirus type 11 virus-like particles from Escherichia coli].

    Science.gov (United States)

    Yan, Chunyan; Li, Shaowei; Wang, Jin; Wei, Minxi; Huang, Bo; Zhuang, Yudi; Li, Zhongyi; Pan, Huirong; Zhang, Jun; Xia, Ningshao

    2009-11-01

    To produce human papillomavirus type 11 virus-like particles (HPV11 VLPs) from Escherichia coli and to investigate its immunogenicity and type cross neutralization nature. We expressed the major capsid protein of HPV11 (HPV11-L1) in Escherichia coli ER2566 in non fusion fashion and purified by amino sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography, sequentially. Then we removed the reductant DTT to have the purified HPV11-L1 self-assemble into VLPs in vitro. We investigated the morphology of these VLPs with dynamic light scattering and transmission electron microscopy. We assayed the immunogenicity of the resultant HPV11 VLPs by vaccinations on mice and evaluated by HPV6/11/16/18 pseudovirion neutralization cell models. We expressed HPV11 L1 in Escherichia coli with two forms, soluble and inclusion body. The soluble HPV11 L1 with over 95% purity can self assemble to VLPs in high efficiency. Morphologically, these VLPs were globular, homogeneous and with a diameter of - 50 nm, which is quite similar with native HPV11 virions. The half effective dosage (ED50) of HPV11 VLPs is 0.031 microg, and the maximum titer of neutralizing antibody elicited is averaged to 10(6). The cross neutralization activity (against HPV6/16/18) of the anti-HPV11 serum was found to have exact correlation to the inter-type homology in amino acid alignment. We can provide HPV11 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV11.

  5. 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.

  6. 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.

  7. Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor.

    Science.gov (United States)

    Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho

    2017-09-15

    Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

  8. Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Reham Wasfi

    Full Text Available The purpose of this study was to: (i evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram against Escherichia coli; (ii investigate the effects of these honeys on bacterial ultrastructure; and (iii assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival in the test organism. The minimum inhibitory concentration (MIC of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM and quantitative real-time polymerase chain reaction (qPCR analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed "ghost" cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets.

  9. Induction of UMUC+ gene expression in Escherichia coli irradiated by near ultraviolet light

    International Nuclear Information System (INIS)

    Sato, N.; Ohnishi, T.; Tano, K.; Nozu, K.; Yamamoto, K.

    1985-01-01

    The induction of umu + gene expression caused by irradiation with near ultraviolet light (BLB; black light blue) was studied in Escherichia coli K-12 strains with special reference to the effects of SOS repair deficiencies. The umuC + gene expression was measured as the enzymic activity of β-galactosidase which is regulated by the promoter of the umuC + operon carried in a plasmid DNA carrying a promoter of umuC + operon, a umuD + gene and a umuC + -lacZ + gene fusion. A high induction of the umuC + gene expression was observed in the uvrA cells in the case of BLB or UV irradiation as compared with the parental wild-type cells. Caffeine inhibited the induction of the umuC + gene expression due to BLB or UV irradiation in both strains. There was very little induction in lexA and recA mutants. In contrast with UV irradiation, there was no killing of cells by BLB irradiation in any strain (wild, uvrA, lexA and recA). Possible implications of the experimental results were discussed. (author)

  10. 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 Ni 2+ . 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.

  11. How much territory can a single E. coli cell control?

    Directory of Open Access Journals (Sweden)

    Ziad W. El-Hajj

    2015-04-01

    Full Text Available Bacteria have been traditionally classified in terms of size and shape and are best known for their very small size. E. coli cells in particular are small rods, each 1-2 microns. However the size varies with the medium, and faster growing cells are larger because they must have more ribosomes to make more protoplasm per unit time, and ribosomes take up space. Indeed, Maaloe's experiments on how E. coli establishes its size began with shifts between rich and poor media.Recently much larger bacteria have been described, including Epulopiscium fishelsoni at 700 μm and Thiomargarita namibiensisis at 750 μm. These are not only much longer than E. coli cells but also much wider, necessitating considerable intracellular organization. Epulopiscium cells for instance, at 80 μm wide, enclose a large enough volume of cytoplasm to present it with major transport problems.This review surveys E. coli cells much longer than those which grow in nature and in usual lab cultures. These include cells mutated in a single gene (metK which are 2-4x longer than their nonmutated parent. This metK mutant stops dividing when slowly starved of S-adenosylmethionine but continues to elongate to 50 μm and more. FtsZ mutants have been routinely isolated as long cells which form during growth at 42°C. The SOS response is a well-characterized regulatory network that is activated in response to DNA damage and also results in cell elongation. Our champion elongated E. coli is a metK strain with a further, as yet unidentified mutation, which reaches 750 μm with no internal divisions and no increase in width.

  12. Optimalisation of expression conditions for production of round-leaf sundew chitinase (Drosera rotundifolia L. in three E. coli expression strains

    Directory of Open Access Journals (Sweden)

    Miroslav Rajninec

    2016-12-01

    Full Text Available Round-leaf sundew (Drosera rotundifolia L., family Droseraceae, genus Drosera, is one of a few plant species with a strong antifungal potential. Chitinases of carnivorous plants play an important role in decomposition of chitin-containing cell structures of insect prey. The cell wall of many phytopathogenic fungi also contains chitin, which can be utilized by chitinases, thus round-leaf sundew represents an interesting gene source for plant biotechnology. The purpose of this study was to compare the suitability of 3 different E. coli expression strains (E. coli BL21- CodonPlus® (DE3-RIPL, E. coli ArcticExpress (DE3RIL and E. coli SHuffle® T7 for production and isolation of heterologous round-leaf sundew chitinase (DrChit. Results showed that the recombinant protein was successfully expressed in all three strains, but occurred in insoluble protein fraction. To get the DrChit protein into soluble protein fraction some modifications concerning to induction temperatures and concentration of the IPTG inductor were tested. In addition, composition of lysis buffer has been modified with supplementation of strong non-ionic detergents, Triton® X100 and Tween® 20, respectively. As these modifications didn’t increase the amount of the DrChit protein in soluble fraction, therefore, its isolation under denaturing conditions and subsequent refolding for activity assays is recommended.

  13. [Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21].

    Science.gov (United States)

    Li, Guo-Hui; Fan, Yu-Zhen; Huang, Si-Yong; Liu, Qiang; Yin, Dan-Dan; Liu, Li; Chen, Ren-An; Hao, Miao-Wang; Liang, Ying-Min

    2014-06-01

    This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

  14. 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...... strains 83972 and VR50. Significant differences in expression levels were seen between the biofilm expression profiles of the two strains with the corresponding planktonic expression profiles in morpholinepropanesulfonic acid minimal laboratory medium and human urine; 417 and 355 genes were up- and down...

  15. Prophage induction and cell division in E. coli. Pt. 3

    International Nuclear Information System (INIS)

    George, J.; Castellazzi, M.; Buttin, G.

    1975-01-01

    In E. coli K12, cell filamentation promoted by tif is enhanced by the lon mutation; in contrast, prophage induction and repair of UV-irradiated phage lambda, also promoted by tif, are not affected by lon. From a tif lon double mutant, 'revertants' having recovered the ability to divide at 41 0 were isolated, among which most (95%) had also lost heir Lon filamentous phenotype after ultraviolet (UV) irradiation. From these 95% of revertants 94% are suppressed for the whole Tif phenotype, by additional mutations that render them deficient in DNA repair, as judged from their high UV sensitivity; some have been characterized as recA mutants. 1% have recovered a control on cell division at 41% or after UV irradiation by means of secondary mutations altering neither the other phenotypic properties of tif and lon, nor the repair and recombination ability of the cells: in particular, this class of 'revertants' remains thermoinducible upon lysogenisation; the mutations which specifically supress filamentation have been mapped at two loci, sfiA and sfiB, cotransducible respectively with pyrD and leu. In the remaining 5% of revertants that still exhibit an UV-induced filamentous growth, 3% can be tentatively classified as true tif + revertants; 2% behave as tif thermodependent revertants, showing suppression of Tif (and Lon) phenotype only at 41 0 : 2 recAts have been identified in this class. Non-lysogenic tif lon sfi and tif sfi strains remain viable during prolonged growth at 41 0 . Under these conditions, tif expresses mutator properties, which can be conveniently analyzed in this sfi background. The action of tif, lon and sfi mutations is tentatively interpreted on the basis of a negative control of cell division specifically associated with DNA repair. (orig.) [de

  16. Role of tetracycline speciation in the bioavailability to Escherichia coli for uptake and expression of antibiotic resistance.

    Science.gov (United States)

    Zhang, Yingjie; Boyd, Stephen A; Teppen, Brian J; Tiedje, James M; Li, Hui

    2014-05-06

    Tetracycline contains ionizable functional groups that manifest several species with charges at different locales and differing net charge; the fractional distribution of each species depends on pH-pKa relationship in the aqueous phase. In nature, these species interact with naturally abundant cations (e.g., Ca(2+) and Mg(2+)) to form metal-tetracycline complexes in water. In this study, we used Escherichia coli MC4100/pTGM whole-cell bioreporter to investigate tetracycline uptake from solution under varying conditions of pH, salt composition and concentration by quantifying the corresponding expression of antibiotic resistance gene. The expression of antibiotic resistance gene in the E. coli bioreporter responded linearly to intracellular tetracycline concentration. Less tetracycline entered E. coli cells at solution pH of 8.0 than at pH 6.0 or 7.0 indicating reduced bioavailability of the antibiotic at higher pH. Both Mg(2+) and Ca(2+) in solution formed metal-tetracycline complexes which reduced uptake of tetracycline by E. coli hence diminishing the bioresponse. Among the various tetracycline species present in solution, including both metal-complexed and free (noncomplexed) species, zwitterionic tetracycline was identified as the predominant species that most readily passed through the cell membrane eliciting activation of the antibiotic resistance gene in E. coli. The results indicate that the same total concentration of tetracycline in ambient solution can evoke very different expression of antibiotic resistance gene in the exposed bacteria due to differential antibiotic uptake. Accordingly, geochemical factors such as pH and metal cations can modulate the selective pressure exerted by tetracycline for development and enrichment of antibiotic resistant bacteria. We suggest that tetracycline speciation analysis should be incorporated into the risk assessment framework for evaluating environmental exposure and the corresponding development of antibiotic

  17. Modeling base excision repair in Escherichia coli bacterial cells

    International Nuclear Information System (INIS)

    Belov, O.V.

    2011-01-01

    A model describing the key processes in Escherichia coli bacterial cells during base excision repair is developed. The mechanism is modeled of damaged base elimination involving formamidopyrimidine DNA glycosylase (the Fpg protein), which possesses several types of activities. The modeling of the transitions between DNA states is based on a stochastic approach to the chemical reaction description

  18. Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid

    OpenAIRE

    Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

    2009-01-01

    Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane α-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lip...

  19. Expression and cytoprotective activity of the small GTPase RhoB induced by the Escherichia coli cytotoxic necrotizing factor 1

    DEFF Research Database (Denmark)

    Huelsenbeck, Stefanie C; Roggenkamp, Dennis; May, Martin

    2013-01-01

    B expression, based on the inactivation of Rho/Ras proteins. In this study, we report on a long lasting expression of RhoB in cultured cells upon activation of Rho proteins by the cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. The observations of this study highlight a new pathway involving Rac1...... without any signs of cell death. In conclusion, the cytoprotective RhoB response is not only evoked by bacterial protein toxins inactivating Rho/Ras proteins but also by the Rac1-activating toxin CNF1....

  20. [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

    Science.gov (United States)

    Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

    2007-01-01

    Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

  1. 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...

  2. Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression.

    Science.gov (United States)

    Yagnik, Darshna; Serafin, Vlad; J Shah, Ajit

    2018-01-29

    The global escalation in antibiotic resistance cases means alternative antimicrobials are essential. The aim of this study was to investigate the antimicrobial capacity of apple cider vinegar (ACV) against E. coli, S. aureus and C. albicans. The minimum dilution of ACV required for growth inhibition varied for each microbial species. For C. albicans, a 1/2 ACV had the strongest effect, S. aureus, a 1/25 dilution ACV was required, whereas for E-coli cultures, a 1/50 ACV dilution was required (p < 0.05). Monocyte co-culture with microbes alongside ACV resulted in dose dependent downregulation of inflammatory cytokines (TNFα, IL-6). Results are expressed as percentage decreases in cytokine secretion comparing ACV treated with non-ACV treated monocytes cultured with E-coli (TNFα, 99.2%; IL-6, 98%), S. aureus (TNFα, 90%; IL-6, 83%) and C. albicans (TNFα, 83.3%; IL-6, 90.1%) respectively. Proteomic analyses of microbes demonstrated that ACV impaired cell integrity, organelles and protein expression. ACV treatment resulted in an absence in expression of DNA starvation protein, citrate synthase, isocitrate and malate dehydrogenases in E-coli; chaperone protein DNak and ftsz in S. aureus and pyruvate kinase, 6-phosphogluconate dehydrogenase, fructose bisphosphate were among the enzymes absent in C.albican cultures. The results demonstrate ACV has multiple antimicrobial potential with clinical therapeutic implications.

  3. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    Science.gov (United States)

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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    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.

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

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    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

  6. 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.

  7. Translational errors in expression of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF-TOF and Orbitrap mass spectrometry

    Science.gov (United States)

    Introduction: Shiga toxin (Stx) is an AB5 toxin expressed by Shiga toxin-producing E. coli (STEC) and Shigella dysenteriae. The Stx holotoxin attaches to surface receptors of eukaryotic cells. After cellular envelopment, the toxin disrupts ribosomal protein synthesis causing cell death. Variations i...

  8. Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

    Science.gov (United States)

    Baek, A-Hyong; Jeon, Eun-Yeong; Lee, Sun-Mee; Park, Jin-Byung

    2015-05-01

    We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts. © 2014 Wiley Periodicals, Inc.

  9. 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

  10. Aspirin augments the expression of Adenomatous Polyposis Coli protein by suppression of IKKβ

    International Nuclear Information System (INIS)

    Ashida, Noboru; Kishihata, Masako; Tien, Dat Nguyen; Kamei, Kaeko; Kimura, Takeshi; Yokode, Masayuki

    2014-01-01

    Highlights: • Clinical studies revealed aspirin inhibits cancer, but the mechanism is not known. • Adenomatous Polyposis Coli (APC) is a well-known tumor-suppressing gene. • We found aspirin up-regulates the protein of APC. • Aspirin suppressed the expression of IKKβ, an essential kinase in NFκB activation. • The deletion of IKKβ significantly increases the expression of APC protein. - Abstract: Aspirin has been widely used as analgesic, antipyretic and anti-inflammatory medicine for long. In addition to these traditional effects, clinical studies suggest that aspirin can protect against cancer, but its mechanism has not been explored. To unveil it, we identified the proteins up- or down-regulated after incubation with aspirin by using proteomics analysis with Nano-flow LC/MALDI-TOF system. Interestingly, the analysis identified the protein of Adenomatous Polyposis Coli (APC) as one of the most up-regulated protein. APC regulates cell proliferation or angiogenesis, and is widely known as a tumor-suppressing gene which can cause colorectal cancer when it is mutated. Western blots confirmed this result, and real-time PCR indicated it is transcriptionally regulated. We further tried to elucidate the molecular mechanism with focusing on IKKβ. IKKβ is the essential kinase in activation of nuclear factor-kappa B (NF-κB), major transcriptional factors that regulate genes responsible for inflammation or immune response. Previous reports indicated that aspirin specifically inhibits IKKβ activity, and constitutively active form of IKKβ accelerates APC loss. We found that aspirin suppressed the expression of IKKβ, and the deletion of IKKβ by siRNA increases the expression of APC in HEK294 cells. Finally, we observed similar effects of aspirin in human umbilical vein endothelial cells. Taken together, these results reveal that aspirin up-regulates the expression of APC via the suppression of IKKβ. This can be a mechanism how aspirin prevents cancer at

  11. Aspirin augments the expression of Adenomatous Polyposis Coli protein by suppression of IKKβ

    Energy Technology Data Exchange (ETDEWEB)

    Ashida, Noboru, E-mail: nashida@kuhp.kyoto-u.ac.jp [Department of Clinical Innovative Medicine, Institute for Advancement of Clinical and Translational Science, Faculty of Medicine, Kyoto University, Kyoto (Japan); Kishihata, Masako [Department of Clinical Innovative Medicine, Institute for Advancement of Clinical and Translational Science, Faculty of Medicine, Kyoto University, Kyoto (Japan); Tien, Dat Nguyen [Department of Clinical Innovative Medicine, Institute for Advancement of Clinical and Translational Science, Faculty of Medicine, Kyoto University, Kyoto (Japan); Department of Biomolecular Engineering, Kyoto Institute of Technology, Kyoto (Japan); Kamei, Kaeko [Department of Biomolecular Engineering, Kyoto Institute of Technology, Kyoto (Japan); Kimura, Takeshi [Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto (Japan); Yokode, Masayuki [Department of Clinical Innovative Medicine, Institute for Advancement of Clinical and Translational Science, Faculty of Medicine, Kyoto University, Kyoto (Japan)

    2014-04-04

    Highlights: • Clinical studies revealed aspirin inhibits cancer, but the mechanism is not known. • Adenomatous Polyposis Coli (APC) is a well-known tumor-suppressing gene. • We found aspirin up-regulates the protein of APC. • Aspirin suppressed the expression of IKKβ, an essential kinase in NFκB activation. • The deletion of IKKβ significantly increases the expression of APC protein. - Abstract: Aspirin has been widely used as analgesic, antipyretic and anti-inflammatory medicine for long. In addition to these traditional effects, clinical studies suggest that aspirin can protect against cancer, but its mechanism has not been explored. To unveil it, we identified the proteins up- or down-regulated after incubation with aspirin by using proteomics analysis with Nano-flow LC/MALDI-TOF system. Interestingly, the analysis identified the protein of Adenomatous Polyposis Coli (APC) as one of the most up-regulated protein. APC regulates cell proliferation or angiogenesis, and is widely known as a tumor-suppressing gene which can cause colorectal cancer when it is mutated. Western blots confirmed this result, and real-time PCR indicated it is transcriptionally regulated. We further tried to elucidate the molecular mechanism with focusing on IKKβ. IKKβ is the essential kinase in activation of nuclear factor-kappa B (NF-κB), major transcriptional factors that regulate genes responsible for inflammation or immune response. Previous reports indicated that aspirin specifically inhibits IKKβ activity, and constitutively active form of IKKβ accelerates APC loss. We found that aspirin suppressed the expression of IKKβ, and the deletion of IKKβ by siRNA increases the expression of APC in HEK294 cells. Finally, we observed similar effects of aspirin in human umbilical vein endothelial cells. Taken together, these results reveal that aspirin up-regulates the expression of APC via the suppression of IKKβ. This can be a mechanism how aspirin prevents cancer at

  12. Functional Properties of Mouse Chitotriosidase Expressed in the Periplasmic Space of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Masahiro Kimura

    Full Text Available Chitotriosidase (Chit1 is an enzyme associated with various diseases, including Gaucher disease, chronic obstructive pulmonary disease, Alzheimer disease and cystic fibrosis. In this study, we first expressed mouse mature Chit1 fused with V5 and (His6 tags at the C-terminus (Chit1-V5-His in the cytoplasm of Escherichia coli and found that most of the expressed protein was insoluble. In contrast, Chit1 tagged with Protein A at the N-terminus and V5-His at the C-terminus, was expressed in the periplasmic space of E. coli as a soluble protein and successfully purified. We evaluated the chitinolytic properties of the recombinant enzyme using 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside [4NP-chitobioside, 4NP-(GlcNAc2] and found that its activity was comparable to CHO cells-expressed Chit1-V5-His. Optimal conditions for the E. coli-produced Chit1 were pH ~5.0 at 50°C. Chit1 was stable after 1 h incubation at pH 5.0~11.0 on ice and its chitinolytic activity was lost at pH 2.0, although the affinity to chitin remained unchanged. Chit1 efficiently cleaved crystalline and colloidal chitin substrates as well as oligomers of N-acetyl-D-glucosamine (GlcNAc releasing primarily (GlcNAc2 fragments at pH 5.0. On the other hand, (GlcNAc3 was relatively resistant to digestion by Chit1. The degradation of 4NP-(GlcNAc2 and (GlcNAc3 was less evident at pH 7.0~8.0, while (GlcNAc2 production from colloidal chitin and (GlcNAc6 at these pH conditions remained strong at the neutral conditions. Our results indicate that Chit1 degrades chitin substrates under physiological conditions and suggest its important pathophysiological roles in vivo.

  13. Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications.

    Directory of Open Access Journals (Sweden)

    Michael eGänzle

    2015-06-01

    Full Text Available High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

  14. Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications.

    Science.gov (United States)

    Gänzle, Michael; Liu, Yang

    2015-01-01

    High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

  15. Efficient biosynthesis of L-phenylglycine by an engineered Escherichia coli with a tunable multi-enzyme-coordinate expression system.

    Science.gov (United States)

    Liu, Qiaoli; Zhou, Junping; Yang, Taowei; Zhang, Xian; Xu, Meijuan; Rao, Zhiming

    2018-03-01

    Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of L-phenylglycine (L-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD + -dependent mutant formate dehydrogenase from Candida boidinii (CbFDH A10C ) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDH A10C and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was 28.4 mg L -1  min -1  g -1 dry cell weight for L-Phg production using whole-cell transformation, it's was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 °C), 60 g L -1 benzoylformic acid was completely converted to pure chiral L-Phg in 4.5 h with 10 g L -1 dry cells and 50.4 g L -1 ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved L-Phg productivity and demonstrated a novel low-cost method for enantiopure L-Phg production.

  16. Expression of a humanized SZ-63 McAb functional recombinant Fab fragment in E. Coli

    International Nuclear Information System (INIS)

    Xia Lijun; Gu Jianming; Zhang Xiaoming; Liu Yue; Wan Haiying; Li Peixia; Ruan Changgeng

    1995-06-01

    MRNA was selected on oligo(dT)-cellulose from total RNA isolated from SZ-63 hybridoma cells by CsCl ultracentrifugation. cDNA coding for heavy and light variable regions were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified fragments were then cloned and sequenced by the 32 P labelled sanger dideoxy-mediated chain-termination method. The nucleotides of VH and Vκ are 354 and 321 respectively, the amino acid sequence of heavy and light chain of SZ-63 were also deduced. Then, linking the variable genes of SZ-63 with human immunoglobulin γ 1 CH and κ VL genes, constructing pHEN1-63 Fab/Hu chimera for expression and transforming E. coli HB2151. The expressed chimeric SZ-63 Fab was soluble. Both ELISA and Western blot results showed the expression products could specifically bind with cross-linked fibrin and the content in expression culture was about 225 μg/L. (5 figs.)

  17. Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

    International Nuclear Information System (INIS)

    Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria

    2006-01-01

    FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl 2 , as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 ± 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K M value for FMN of 1.5 ± 0.3 μM. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast

  18. Regulation of gene expression: Cryptic β-glucoside (bgl operon of Escherichia coli as a paradigm

    Directory of Open Access Journals (Sweden)

    Dharmesh Harwani

    2014-12-01

    Full Text Available Bacteria have evolved various mechanisms to extract utilizable substrates from available resources and consequently acquire fitness advantage over competitors. One of the strategies is the exploitation of cryptic cellular functions encoded by genetic systems that are silent under laboratory conditions, such as the bgl (β-glucoside operon of E. coli. The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to regulate its expression and/or the operon might be carrying out function(s apart from the utilization of aromatic β-glucosides. This is consistent with the observations that the activated operon confers a Growth Advantage in Stationary Phase (GASP phenotype to Bgl+ cells and exerts its regulation on at least twelve downstream target genes.

  19. Regulation of gene expression: cryptic β-glucoside (bgl) operon of Escherichia coli as a paradigm.

    Science.gov (United States)

    Harwani, Dharmesh

    2014-01-01

    Bacteria have evolved various mechanisms to extract utilizable substrates from available resources and consequently acquire fitness advantage over competitors. One of the strategies is the exploitation of cryptic cellular functions encoded by genetic systems that are silent under laboratory conditions, such as the bgl (β-glucoside) operon of E. coli. The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to regulate its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. This is consistent with the observations that the activated operon confers a Growth Advantage in Stationary Phase (GASP) phenotype to Bgl(+) cells and exerts its regulation on at least twelve downstream target genes.

  20. Cloning and Expression of Cyclophilin from Platanus orientalis Pollens in Escherichia coli

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    Mojtaba Sankian

    2012-10-01

    Full Text Available Background: Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis trees are planted in many countries and their pollen causes allergic reactions. Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis. Methods: RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+ vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method. Results: The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen. Conclusion: The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen.

  1. Improved ethanol production from cheese whey, whey powder, and sugar beet molasses by "Vitreoscilla hemoglobin expressing" Escherichia coli.

    Science.gov (United States)

    Akbas, Meltem Yesilcimen; Sar, Taner; Ozcelik, Busra

    2014-01-01

    This work investigated the improvement of ethanol production by engineered ethanologenic Escherichia coli to express the hemoglobin from the bacterium Vitreoscilla (VHb). Ethanologenic E. coli strain FBR5 and FBR5 transformed with the VHb gene in two constructs (strains TS3 and TS4) were grown in cheese whey (CW) medium at small and large scales, at both high and low aeration, or with whey powder (WP) or sugar beet molasses hydrolysate (SBMH) media at large scale and low aeration. Culture pH, cell growth, VHb levels, and ethanol production were evaluated after 48 h. VHb expression in TS3 and TS4 enhanced their ethanol production in CW (21-419%), in WP (17-362%), or in SBMH (48-118%) media. This work extends the findings that "VHb technology" may be useful for improving the production of ethanol from waste and byproducts of various sources.

  2. Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12.

    OpenAIRE

    Stewart, V; Yanofsky, C

    1986-01-01

    We used site-directed mutagenesis to replace the Escherichia coli tryptophanase (tna) operon leader peptide start codon with AUC. This change greatly decreased the uninduced rate of tna operon expression, and it also lowered the response to inducer. We conclude that leader peptide synthesis plays an essential role in tna operon expression.

  3. Bet-hedging in bacteriocin producing Escherichia coli populations: the single cell perspective

    Science.gov (United States)

    Bayramoglu, Bihter; Toubiana, David; van Vliet, Simon; Inglis, R. Fredrik; Shnerb, Nadav; Gillor, Osnat

    2017-02-01

    Production of public goods in biological systems is often a collaborative effort that may be detrimental to the producers. It is therefore sustainable only if a small fraction of the population shoulders the cost while the majority reap the benefits. We modelled this scenario using Escherichia coli populations producing colicins, an antibiotic that kills producer cells’ close relatives. Colicin expression is a costly trait, and it has been proposed that only a small fraction of the population actively expresses the antibiotic. Colicinogenic populations were followed at the single-cell level using time-lapse microscopy, and showed two distinct, albeit dynamic, subpopulations: the majority silenced colicin expression, while a small fraction of elongated, slow-growing cells formed colicin-expressing hotspots, placing a significant burden on expressers. Moreover, monitoring lineages of individual colicinogenic cells showed stochastic switching between expressers and non-expressers. Hence, colicin expressers may be engaged in risk-reducing strategies—or bet-hedging—as they balance the cost of colicin production with the need to repel competitors. To test the bet-hedging strategy in colicin-mediated interactions, competitions between colicin-sensitive and producer cells were simulated using a numerical model, demonstrating a finely balanced expression range that is essential to sustaining the colicinogenic population.

  4. Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli.

    Science.gov (United States)

    Kim, Ha-Kun; Chun, Dae-Sik; Kim, Joon-Sik; Yun, Cheol-Ho; Lee, Ju-Hoon; Hong, Soon-Kwang; Kang, Dae-Kyung

    2006-09-01

    Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide-lactoferricin fusion gene. The monomeric acidic peptide-lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-beta-D-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.

  5. Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells

    DEFF Research Database (Denmark)

    Freiesleben, Ulrik Von

    1996-01-01

    Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome....... The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact...

  6. An enzyme-immunobinding assay for fast screening of expression of tissue plasminogen activator cDNA in E. coli

    International Nuclear Information System (INIS)

    Tang, J.C.T.; Li, S.H.

    1984-01-01

    Tissue plasminogen activator (TPA) has been isolated from normal human tissues and certain human cell lines in culture. The enzyme is a serine protease which converts an inactive zymogen, plasminogen to plasmin, and causes lysis of fibrin clots. The high affinity of TPA for fibrin indicates that it is a potential thrombolytic agent and is superior to urokinase-like plasminogen activators. Recently, TPA has been cloned and expressed in E. coli. Using TPA as a model protein, the authors report here the development of a direct, sensitive enzyme-immunoassay for the screening of a cDNA expression library using specific antibodies and peroxidase-labeled second antibody

  7. E. coli Nissle 1917 Affects Salmonella adhesion to porcine intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Peter Schierack

    Full Text Available BACKGROUND: The probiotic Escherichia coli strain Nissle 1917 (EcN has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. CONCLUSIONS: We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion.

  8. Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC.

    Directory of Open Access Journals (Sweden)

    Thorsten Kuczius

    Full Text Available Shiga toxin (Stx-producing Escherichia coli (STEC carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG. EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.

  9. Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12

    International Nuclear Information System (INIS)

    Thoms, B.; Wackernagel, W.

    1983-01-01

    Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage lambda in cells irradiated with ultraviolet prior to infection. The authors demonstrate that restriction of lambda is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of lambda. At this time more than 60% of the lambda DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of lambda DNA from infected cells and a mild detergent treatment removing adsorbed phages from the cellular surface showed that only a small specific fraction of all lambda infections is destined to escape restriction due to restriction alleviation. This fraction (10-20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of lambda phages explains why the SOS function restriction alleviation could initially be discovered. The authors show that the retarded mode of DNA injection is not required for another SOS function acting on lambda DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation). (Auth.)

  10. 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...

  11. Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli.

    Science.gov (United States)

    Kawano, Mitsuoki; Oshima, Taku; Kasai, Hiroaki; Mori, Hirotada

    2002-07-01

    Genome sequence analyses of Escherichia coli K-12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR-A, -B, -C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR-D), 450 bp in length and nearly identical to LDR-A, -B and -C, is located at 79.7 min, a position that is directly opposite the position of LDR-A, -B and -C. In this study, we demonstrate that LDR-D encodes a 35-amino-acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (approximately 370 nucleotides) encoding LdrD and an unstable cis-encoded antisense RNA (approximately 60 nucleotides), which functions as a trans-acting regulator of ldrD translation. We propose that LDR encodes a toxin-antitoxin module. LDR-homologous sequences are not pre-sent on any known plasmids but are conserved in Salmonella and other enterobacterial species.

  12. Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B

    Directory of Open Access Journals (Sweden)

    Riley Laura M

    2012-03-01

    Full Text Available Abstract Background Shigatoxigenic E. coli are a global and emerging health concern. Shiga toxin, Stx, is encoded on the genome of temperate, lambdoid Stx phages. Genes essential for phage maintenance and replication are encoded on approximately 50% of the genome, while most of the remaining genes are of unknown function nor is it known if these annotated hypothetical genes are even expressed. It is hypothesized that many of the latter have been maintained due to positive selection pressure, and that some, expressed in the lysogen host, have a role in pathogenicity. This study used Change Mediated Antigen Technology (CMAT™ and 2D-PAGE, in combination with RT-qPCR, to identify Stx phage genes that are expressed in E. coli during the lysogenic cycle. Results Lysogen cultures propagated for 5-6 hours produced a high cell density with a low proportion of spontaneous prophage induction events. The expression of 26 phage genes was detected in these cultures by differential 2D-PAGE of expressed proteins and CMAT. Detailed analyses of 10 of these genes revealed that three were unequivocally expressed in the lysogen, two expressed from a known lysogenic cycle promoter and one uncoupled from the phage regulatory network. Conclusion Propagation of a lysogen culture in which no cells at all are undergoing spontaneous lysis is impossible. To overcome this, RT-qPCR was used to determine gene expression profiles associated with the growth phase of lysogens. This enabled the definitive identification of three lambdoid Stx phage genes that are expressed in the lysogen and seven that are expressed during lysis. Conservation of these genes in this phage genome, and other Stx phages where they have been identified as present, indicates their importance in the phage/lysogen life cycle, with possible implications for the biology and pathogenicity of the bacterial host.

  13. Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness to Escherichia coli Shiga Toxin 1

    Science.gov (United States)

    Cells in the depth of the crypts in the bovine colon express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization 25 of cattle with human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used f...

  14. Efficient Extracellular Expression of Phospholipase D in Escherichia Coli with an Optimized Signal Peptide

    Science.gov (United States)

    Yang, Leyun; Xu, Yu; Chen, Yong; Ying, Hanjie

    2018-01-01

    New secretion vectors containing the synthetic signal sequence (OmpA’) was constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli Phospholipase D structural gene (Accession number:NC_018658) fused to various signal sequence were expressed from the Lac promoter in E. coli Rosetta strains by induction with 0.4mM IPTG at 28°C for 48h. SDS-PaGe analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of Phospholipase D (PLD) not only to the medium but also remained in periplasm of E. coli with OmpA’ signal sequence at the N-terminus of PLD. Thus the study on the effects of various surfactants on PLD extracellular production in Escherichia coli in shake flasks revealed that optimal PLD extracellular production could be achieved by adding 0.4% Triton X-100 into the medium. The maximal extracellular PLD production and extracellular enzyme activity were 0.23mg ml-1 and 16U ml-1, respectively. These results demonstrate the possibility of efficient secretory production of recombinant PLD in E. coli should be a potential industrial applications.

  15. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Woon, J. S. K., E-mail: jameswoon@siswa.ukm.edu.my; Murad, A. M. A., E-mail: munir@ukm.edu.my; Abu Bakar, F. D., E-mail: fabyff@ukm.edu.my [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor (Malaysia)

    2015-09-25

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

  16. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    Science.gov (United States)

    Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

    2015-09-01

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

  17. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

    International Nuclear Information System (INIS)

    Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

    2015-01-01

    A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli

  18. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the regulator AcrR from Escherichia coli

    International Nuclear Information System (INIS)

    Li, Ming; Qiu, Xi; Su, Chih-Chia; Long, Feng; Gu, Ruoyu; McDermott, Gerry; Yu, Edward W.

    2006-01-01

    The transcriptional regulator AcrR from Escherichia coli has been cloned, overexpressed, purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.5 Å. This paper describes the cloning, expression, purification and preliminary X-ray data analysis of the AcrR regulatory protein. The Escherichia coli AcrR is a member of the TetR family of transcriptional regulators. It regulates the expression of the AcrAB multidrug transporter. Recombinant AcrR with a 6×His tag at the C-terminus was expressed in E. coli and purified by metal-affinity chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted to 2.5 Å. The space group was determined to be P3 2 , with unit-cell parameters a = b = 46.61, c = 166.16 Å

  19. 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...

  20. Recombinant Escherichia coli Trx-JZTX-III represses the proliferation of mouse hepatocellular carcinoma cells through induction of cell cycle arrest.

    Science.gov (United States)

    Sun, Mei-Na; Zhao, Xue-Jiao; Zhao, Han-Dong; Zhang, Wei-Guang; Li, Feng-Lan; Chen, Ming-Zi; Li, Hui; Li, Guangchao

    2013-06-01

    The aim of the present study was to investigate the effects of recombinant Escherichia coli (E. coli) Trx-jingzhaotoxin (JZTX)-III on cell growth in the mouse hepatocellular carcinoma (HCC) cell line Hepa1-6. The JZTX-III gene sequence was synthesized and cloned into the pET-32a(+) vector to construct the recombinant fusion protein Trx-JZTX-III, which was subsequently purified. Hepa1-6 cells were treated with 0 to 1,000-µg/ml concentrations of Trx-JZTX-III; this was demonstrated to affect cell viability, as determined by the 3-(4,5-dimethylthiazol‑2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. The expression of the proliferating cell nuclear antigen (PCNA) protein was investigated using western blot analysis. A colony formation assay was used to determine Hepa1-6 cell proliferation, and the migration ability of cells was determined using a wound‑healing assay. Additionally, flow cytometry was employed to observe changes in the cell cycle. The MTT assay and quantification of PCNA expression indicated that recombinant E. coli Trx-JZTX-III significantly repressed the proliferation of Hepa1-6 cells. Colony formation and the migration of malignant cells was inhibited following treatment with recombinant E. coli Trx-JZTX-III. Flow cytometry showed that recombinant E. coli Trx-JZTX-III induced G0/G1 cell cycle arrest. In conclusion, recombinant E. coli Trx-JZTX-III functions as a tumor suppressor drug in mouse HCC and its underlying mechanism may involve the induction of G0/G1 cell cycle arrest.

  1. Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.

    Science.gov (United States)

    Gul, Nadia; Linares, Daniel M; Ho, Franz Y; Poolman, Bert

    2014-01-09

    The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins. © 2013.

  2. Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

    Science.gov (United States)

    Ellis, Mark; Patel, Pareshkumar; Edon, Marjory; Ramage, Walter; Dickinson, Robert; Humphreys, David P

    2017-01-01

    Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD 600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017. © 2016 American Institute of Chemical Engineers.

  3. Gene sequencing, cloning, and expression of the recombinant L- Asparaginase of Pseudomonas aeruginosa SN4 strain in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Arastoo Badoei-dalfard

    2016-03-01

    Full Text Available Introduction: L- asparaginase is in an excessive demand in medical applications and in food treating industries, the request for this therapeutic enzyme is growing several folds every year. Materials and methods: In this study, a L- asparaginase gene from Pseudomonas aeruginosa strain SN4 was sequenced and cloned in E. coli. Primers were designed based on L- asparaginase from P. aeruginosa DSM 50071, which show high similarity to SN4 strain, according to 16S rRNA sequence. The L- asparaginase gene was exposed to restriction digestion with NdeI and XhoI enzymes and then ligated into pET21a plasmid. The ligated sample was transformed into competent E. coli (DE3 pLysS DH5a cells, according to CaCl2 method. The transformed E. coli cells were grown into LB agar plate containing 100 µg/ml ampicillin, IPTG (1 mM. Results: Recombinant L- asparaginase from E. coli BL21 induced after 9 h of incubation and showed high L- asparaginase activity about 93.4 IU/ml. Recombinant L- asparaginase sequencing and alignments showed that the presumed amino acid sequence composed of 350 amino acid residues showed high similarity with P. aeruginosa L- asparaginases about 99%. The results also indicated that SN4 L- asparaginase has the catalytic residues and conserve region similar to other L- asparaginases. Discussion and conclusion: This is the first report on cloning and expression of P. aeruginosa L- asparaginases in Escherichia coli. These results indicated a potent source of L- asparaginase for in vitro and in vivio anticancer consideration. 

  4. Damage-induced DNA repair processes in Escherichia coli cells

    International Nuclear Information System (INIS)

    Slezarikova, V.

    1986-01-01

    The existing knowledge is summed up of the response of Escherichia coli cells to DNA damage due to various factors including ultraviolet radiation. So far, three inducible mechanisms caused by DNA damage are known, viz., SOS induction, adaptation and thermal shock induction. Greatest attention is devoted to SOS induction. Its mechanism is described and the importance of the lexA recA proteins is shown. In addition, direct or indirect role is played by other proteins, such as the ssb protein binding the single-strand DNA sections. The results are reported of a study of induced repair processes in Escherichia coli cells repeatedly irradiated with UV radiation. A model of induction by repeated cell irradiation discovered a new role of induced proteins, i.e., the elimination of alkali-labile points in the daughter DNA synthetized on a damaged model. The nature of the alkali-labile points has so far been unclear. In the adaptation process, regulation proteins are synthetized whose production is induced by the presence of alkylation agents. In the thermal shock induction, new proteins synthetize in cells, whose function has not yet been clarified. (E.S.)

  5. Reductone effect on UV-irradiated starved E. coli cells

    International Nuclear Information System (INIS)

    Felzenszwalb, I.; Gomes, R.A.

    1982-01-01

    A starvation-induced resistence enhancement (SIRE) to UV and reductone treatments was observed in repair-profient E. coli cells. The UV-reductone positive interaction, which is possibly related to excision repair mechanisms, was not modified by prestarvation when all cells in culture had completed their round of DNA replication. In irradiated prestarved reductone-treated cells, a decrease in the DNA degradation rate was detected after the removal of reductone and the induction of a lower number of DNA single-strand breaks. The induction kinectics of DNA single-strand breaks in prestarved UV-irradiated and the repair kinetics of these lesions are slower than in non-starved cells. The resistance enhancement demonstrated under these conditions could be justified either by the generation of fewer doubles strand breaks during repair or by the possibility of repair of these lesions. (Author) [pt

  6. Molecular cloning and expression of bovine kappa-casein in Escherichia coli

    International Nuclear Information System (INIS)

    Kang, Y.C.; Richardson, T.

    1988-01-01

    A cDNA library was constructed using poly(A) + RNA from bovine mammary gland. This cDNA library of 6000 clones was screened employing colony hybridization using 32 P-labelled oligonucleotide probes and restriction endonuclease mapping. The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method. The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein. The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76. The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively. Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein

  7. Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli.

    Science.gov (United States)

    Cao, Wei; Zhou, Yuxun; Ma, Yushu; Luo, Qingping; Wei, Dongzhi

    2005-04-01

    Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.

  8. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    Science.gov (United States)

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

    Science.gov (United States)

    Wurm, David Johannes; Veiter, Lukas; Ulonska, Sophia; Eggenreich, Britta; Herwig, Christoph; Spadiut, Oliver

    2016-10-01

    Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-D-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (q s,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (q s,lac) and q s,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying q s,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between q s,lac and q s,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.

  10. Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in Escherichia coli.

    Science.gov (United States)

    Chen, Weiwei; Yu, Hongwei; Ye, Lidan

    2016-07-01

    The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.

  11. Factors affecting expression of the recF gene of Escherichia coli K-12.

    Science.gov (United States)

    Sandler, S J; Clark, A J

    1990-01-31

    This report describes four factors which affect expression of the recF gene from strong upstream lambda promoters under temperature-sensitive cIAt2-encoded repressor control. The first factor was the long mRNA leader sequence consisting of the Escherichia coli dnaN gene and 95% of the dnaA gene and lambda bet, N (double amber) and 40% of the exo gene. When most of this DNA was deleted, RecF became detectable in maxicells. The second factor was the vector, pBEU28, a runaway replication plasmid. When we substituted pUC118 for pBEU28, RecF became detectable in whole cells by the Coomassie blue staining technique. The third factor was the efficiency of initiation of translation. We used site-directed mutagenesis to change the mRNA leader, ribosome-binding site and the 3 bp before and after the translational start codon. Monitoring the effect of these mutational changes by translational fusion to lacZ, we discovered that the efficiency of initiation of translation was increased 30-fold. Only an estimated two- or threefold increase in accumulated levels of RecF occurred, however. This led us to discover the fourth factor, namely sequences in the recF gene itself. These sequences reduce expression of the recF-lacZ fusion genes 100-fold. The sequences responsible for this decrease in expression occur in four regions in the N-terminal half of recF. Expression is reduced by some sequences at the transcriptional level and by others at the translational level.

  12. Cloning and expression of antibacterial goat lactoferricin from Escherichia coli AD494(DE3)pLysS expression system.

    Science.gov (United States)

    Chen, Gen-Hung; Yin, Li-Jung; Chiang, I-Hua; Jiang, Shann-Tzong

    2008-12-01

    Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His*Tag and GLfcin S-His*Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His*Tag and GLfcin S-His*Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His*Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His*Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100 degrees C.

  13. 'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

    Science.gov (United States)

    Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

    2016-04-01

    A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Evolved Escherichia coli Strains for Amplified, Functional Expression of Membrane Proteins

    NARCIS (Netherlands)

    Gul, Nadia; Linares, Daniel M.; Ho, Franz Y.; Poolman, Bert

    2014-01-01

    The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several

  15. 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...

  16. 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.

  17. Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli.

    NARCIS (Netherlands)

    Voordouw, G.; Hagen, W.R.; Kruse-Wolters, M.; Berkel-Arts, van A.; Veeger, C.

    1987-01-01

    Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa. Its two structural genes have been cloned on a 4664-base-pair fragment of known sequence in the vector pUC9. Expression of hydrogenase polypeptides in Escherichia coli transformed with

  18. 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...

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

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... expressed GST-fusion human vascular endothelial growth factors ... prognosis, monitoring of therapy and diagnosis. ... Alternative splicing of a single ..... Distamycin-A derivatives potentiate tumor–necrosis-factor activity via.

  20. Tunable recombinant protein expression with E. coli in a mixed-feed environment.

    Science.gov (United States)

    Sagmeister, Patrick; Schimek, Clemens; Meitz, Andrea; Herwig, Christoph; Spadiut, Oliver

    2014-04-01

    Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both D-glucose and L-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of L-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.

  1. Expression of human DNA polymerase β in Escherichia coli and characterization of the recombinant enzyme

    International Nuclear Information System (INIS)

    Abbotts, J.; SenGupta, D.N.; Zmudzka, B.; Widen, S.G.; Notario, V.; Wilson, S.H.

    1988-01-01

    The coding region of a human β-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with β-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as β-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural β-polymerases. The purified enzyme was free of nuclease activity. The authors studied detailed catalytic properties of the recombinant β-polymerase using defined template-primer systems. The results indicate that this β-polymerase is essentially identical with natural β-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and a double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N 3 -methyl-dT or O 6 -methyl-dG

  2. Recombinant protein production data after expression in the bacterium Escherichia coli

    Directory of Open Access Journals (Sweden)

    J. Enrique Cantu-Bustos

    2016-06-01

    Full Text Available Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]. Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP tagged with CusF, using Ag(I metal affinity chromatography.

  3. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    Science.gov (United States)

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  4. The study of preparation for immobilized cells membranes of E. Coli. by radiation technique

    International Nuclear Information System (INIS)

    Cao Jin; Chen Pin; Yu Yi

    1991-01-01

    The paper described the preparation of immobilized cells membranes with E. Coli by radiation technique. The nylon 6 was grafted with HEMA, which as a matrix to prepare immobilized cells membranes with E. Coli. by radiation entrapment at low temperature. The results showed that the retentive activity possessed a maximum value for membranes with E. Coli. when the irradiation dose was at 10-12 kGy, the entrapped cells has 2.3 g/ml at 50% HEMA concentration, the optimum pH and optimum temperature for membranes with E. Coli. are as same the original cells

  5. Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

    Science.gov (United States)

    Mittal, Rahul; Sukumaran, Sunil K; Selvaraj, Suresh K; Wooster, David G; Babu, M Madan; Schreiber, Alan D; Verbeek, J Sjef; Prasadarao, Nemani V

    2010-11-18

    Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

  6. Genome-wide analysis of E. coli cell-gene interactions.

    Science.gov (United States)

    Cardinale, S; Cambray, G

    2017-11-23

    The pursuit of standardization and reliability in synthetic biology has achieved, in recent years, a number of advances in the design of more predictable genetic parts for biological circuits. However, even with the development of high-throughput screening methods and whole-cell models, it is still not possible to predict reliably how a synthetic genetic construct interacts with all cellular endogenous systems. This study presents a genome-wide analysis of how the expression of synthetic genes is affected by systematic perturbations of cellular functions. We found that most perturbations modulate expression indirectly through an effect on cell size, putting forward the existence of a generic Size-Expression interaction in the model prokaryote Escherichia coli. The Size-Expression interaction was quantified by inserting a dual fluorescent reporter gene construct into each of the 3822 single-gene deletion strains comprised in the KEIO collection. Cellular size was measured for single cells via flow cytometry. Regression analyses were used to discriminate between expression-specific and gene-specific effects. Functions of the deleted genes broadly mapped onto three systems with distinct primary influence on the Size-Expression map. Perturbations in the Division and Biosynthesis (DB) system led to a large-cell and high-expression phenotype. In contrast, disruptions of the Membrane and Motility (MM) system caused small-cell and low-expression phenotypes. The Energy, Protein synthesis and Ribosome (EPR) system was predominantly associated with smaller cells and positive feedback on ribosome function. Feedback between cell growth and gene expression is widespread across cell systems. Even though most gene disruptions proximally affect one component of the Size-Expression interaction, the effect therefore ultimately propagates to both. More specifically, we describe the dual impact of growth on cell size and gene expression through cell division and ribosomal content

  7. Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

    Directory of Open Access Journals (Sweden)

    Kim Jaoon YH

    2012-01-01

    Full Text Available Abstract Background Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2 production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions. Results Recombinant E. coli BL21(DE3 co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s. We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s led to an increase (~1.8-fold in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%. Conclusion Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3 in a light intensity-dependent manner. These results demonstrate that E. coli

  8. The Dermatophagoides farinae group 22 allergen: cloning and expression in Escherichia coli.

    Science.gov (United States)

    Cui, Yu-bao; Cai, Hong-xing; Zhou, Ying; Wang, Nan; Yu, Li-li; Yang, Li; Zhang, Cheng-bo

    2015-09-01

    Dermatophagoides farinae (Hughes) (Acari: Pyroglyphidae) and other domestic mites produce allergens that affect people worldwide. Here, the complementary DNA (cDNA) coding for group 22 allergen of D. farinae (Der f 22) from China was cloned, sequenced, and expressed successfully. The cDNA encoding Der f 22 was synthesized by reverse transcription polymerase chain reaction (RT-PCR), then ligated to the pCold-TF for expression in Escherichia coli BL21 cells. The purified recombinant fusion protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western-blotting, and tandem matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF). The full-length cDNA comprised 468 nucleotides and was 99.57% (466/468) identical with the reference sequence (GenBank: DQ643992). After the plasmid pCold-TF-Der f 22 was transformed into E. coli BL21 and expressed with the induction of IPTG, SDS-PAGE showed a specific band for the recombinant fusion protein. The recombinant fusion protein, which was purified by chromatography, bound with a His-tagged antibody by Western blotting. MALDI-TOF/TOF mass spectrometry revealed that the structure of the recombinant protein was identical to the predicted Der f 22 structure. The hydrophilic protein contains a signal peptide of 20 amino acids, and the mature Der f 22 consists of 135 amino acid residues with a molecular weight of 14.7 kDa and theoretical isoelectric points (pI) of 6.38. Its secondary structure comprises an alpha helix (38.5%), beta-sheet (45.9%), random coils (11.85%), and beta-turns (11.1%). This work represents the first reported full-length sequence and successful cloning of Der f 22 from D. farinae in China; bioinformatics analysis can be used to further study the allergenicity and clinical utility of the recombinant Der f 22. © 2015 ARS-AAOA, LLC.

  9. Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.

    Directory of Open Access Journals (Sweden)

    Rika Etchuuya

    Full Text Available Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain. In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5-10(-6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.

  10. Cloning and expression of cellulase XF-818 of Xylella fastidiosa in Escherichia Coli Clonagem e expressão da celulase Xf-818 de Xylella Fastidiosa em Escherichia Coli

    Directory of Open Access Journals (Sweden)

    Nelson Arno Wulff

    2003-12-01

    Full Text Available Xylella fastidiosa's genome was the first of a plant pathogen to be completely sequenced. Through comparative sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. However, the biological role of each gene should be assigned experimentally. On this regard, heterologous protein expression is a powerful tool to produce proteins from such genes, allowing their characterization. X. fastidiosa lives inside xylem vessels and eventually would degrade pit membranes from xylem cells to move radialy into the host. The identification of several putative plant cell wall degrading enzymes on X. fastidiosa genome prompted the assession of the function of such proteins. The open reading frame (ORF Xf-818 was cloned into expression vector pET20b and E. coli cells harboring such plasmid exhibited cellulase activity. Using IPTG at 0.4 mmol L-1 with a 12 h incubation at 32°C are the best conditions to produce higher amounts of heterologous protein. The enzyme degrades cellulose confirming the endoglucanase activity of Xf-818.Xylella fastidiosa foi a primeira bactéria fitopatogênica que teve seu genoma completamente seqüenciado. A identificação de diversos genes, através de similaridade de seqüências, indicou os possíveis mecanismos de patogenicidade da bactéria. Entretanto, a determinação da função de um gene requer a confirmação experimental e, neste aspecto, a expressão heteróloga é uma poderosa ferramenta. X. fastidiosa coloniza somente o xilema das plantas hospedeiras e a identificação putativa de diversos genes semelhantes a enzimas que degradam a parede celular vegetal, estimularam o presente estudo de catacterização destas enzimas. A clonagem da ORF Xf-818 de X. fastidiosa no vetor de expressão pET20b possibilitou a produção da proteína heterologamente em E. coli. O emprego de IPTG a 0,4 mmol L-1 com 12 h a 32°C, possibilitou as melhores condições para E. coli

  11. New insights into enterocin CRL35: mechanism of action and immunity revealed by heterologous expression in Escherichia coli.

    Science.gov (United States)

    Barraza, Daniela E; Ríos Colombo, Natalia S; Galván, Adriana E; Acuña, Leonardo; Minahk, Carlos J; Bellomio, Augusto; Chalón, Miriam C

    2017-09-01

    The role of the class IIa bacteriocin membrane receptor protein remains unclear, and the following two different mechanisms have been proposed: the bacteriocin could interact with the receptor changing it to an open conformation or the receptor might act as an anchor allowing subsequent bacteriocin insertion and membrane disruption. Bacteriocin-producing cells synthesize an immunity protein that forms an inactive bacteriocin-receptor-immunity complex. To better understand the molecular mechanism of enterocin CRL35, the peptide was expressed as the suicidal probe EtpM-enterocin CRL35 in Escherichia coli, a naturally insensitive microorganism since it does not express the receptor. When the bacteriocin is anchored to the periplasmic face of the plasma membrane through the bitopic membrane protein, EtpM , E. coli cells depolarize and die. Moreover, co-expression of the immunity protein prevents the deleterious effect of EtpM-enterocin CRL35. The binding and anchoring of the bacteriocin to the membrane has demonstrated to be a sufficient condition for its membrane insertion. The final step of membrane disruption by EtpM-enterocin CRL35 is independent from the receptor, which means that the mannose PTS might not be involved in the pore structure. In addition, the immunity protein can protect even in the absence of the receptor. © 2017 John Wiley & Sons Ltd.

  12. Putative carotenoid genes expressed under the regulation of Shine-Dalgarno regions in Escherichia coli for efficient lycopene production.

    Science.gov (United States)

    Jin, Weiyue; Xu, Xian; Jiang, Ling; Zhang, Zhidong; Li, Shuang; Huang, He

    2015-11-01

    Putative genes crtE, crtB, and crtI from Deinococcus wulumiqiensis R12, a novel species, were identified by genome mining and were co-expressed using the optimized Shine-Dalgarno (SD) regions to improve lycopene yield. A lycopene biosynthesis pathway was constructed by co-expressing these three genes in Escherichia coli. After optimizing the upstream SD regions and the culture medium, the recombinant strain EDW11 produced 88 mg lycopene g(-1) dry cell wt (780 mg lycopene l(-1)) after 40 h fermentation without IPTG induction, while the strain EDW without optimized SD regions only produced 49 mg lycopene g(-1) dry cell wt (417 mg lycopene l(-1)). Based on the optimization of the upstream SD regions and culture medium, the yield of the strain EDW11 reached a high level during microbial lycopene production until now.

  13. Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

    Science.gov (United States)

    Funaro, Michael G; Nemani, Krishnamurthy V; Chen, Zhihang; Bhujwalla, Zaver M; Griswold, Karl E; Gimi, Barjor

    2016-02-01

    Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

  14. A Comparative Analysis of Recombinant Expression and Solubility Screening of Two Phytases in E. coli

    Directory of Open Access Journals (Sweden)

    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.

  15. Expression of human mag-1 gene in E. coli and preparation of its antibody

    International Nuclear Information System (INIS)

    Lin Huiyun; Xu Yuanji; Wang Yan; Chen Huihua; Du Zhiyan; Tan Xiaogang; Lu Yinglin

    2006-01-01

    Objective: To further investigate the new metastasis associated gene, mag-1 expressed in E. coli and its anti-body was prepared in rabbit. Methods: mag-1 was amplified by PCR from pcDNA3-mag-1 and directly cloned into pET-28a vector. The fusion protein was expressed in BL21 and identified by Western blot using anti-His monoclonal antibody. Rabbit was immunized with partially purified fusion protein subcutaneously. Results: Sequence analysis revealed identity of the sequence obtained to the previous report. The recombinant His-mag-1 could be expressed in E. coli as a fusion protein of 18 x 10 3 . The recombinant protein was mostly expressed in the inclusion bodies on the induction by 0.1 mmol/L IPTG at 37 degree C for 6 hours. Western blot analysis showed that the recombinant protein could be recognized by His monoclonal anti-body. The titer of polyclonal antibody against mag-1 was 1:160000. Conclusion: The mag-1 gene is expressed in E. coli highly and its antibody is prepared successfully. (authors)

  16. [Expression optimization and characterization of Tenebrio molitor antimicrobiol peptides TmAMP1m in Escherichia coli].

    Science.gov (United States)

    Alimu, Reyihanguli; Mao, Xinfang; Liu, Zhongyuan

    2013-06-01

    To improve the expression level of tmAMP1m gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMP1m by conditions, such as culture temperature, inducing time and the final concentration of inductor Isopropyl beta-D-thiogalactopyranoside (IPTG). We analyzed the optimum expression conditions by Tricine-SDS-PAGE electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. The results suggest that when inducing the recombinant plasmid with a final IPTG concentration of 0.1 mmol/L at 37 degrees C for 4 h, there was the highest expression level of fusion protein HIS-TmAMP1m in Escherichia coli. Under these conditions, the expression of fusion protein accounted for 40% of the total cell lysate with the best antibacterial activity. We purified the fusion protein HIS-TmAMPlm with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices. Western blotting analysis indicates that the His monoclonal antibody could be specifically bound to fusion protein HIS-TmAMPlm. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET30a-tmAMP1m. The fusion protein HIS-TmAMP1m had better stability and remained higher antibacterial activities when incubated at 100 degrees C for 10 h, repeated freeze thawing at -20 degrees C, dissolved in strong acid and alkali, or treated by organic solvents and protease. Moreover, the minimum inhibitory concentration results demonstrated that the fusion protein HIS-TmAMP1m has a good antibacterial activity against Staphylococcus aureus, Staphylococcus sp., Corynebacterium glutamicum, Bacillus thuringiensis, Corynebacterium sp. This study laid the foundation to promote the application of insect antimicrobial peptides and further research.

  17. Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.

    Science.gov (United States)

    Frothingham, R; Meeker-O'Connell, W A; Talbot, E A; George, J W; Kreuzer, K N

    1996-06-01

    Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which also has PZAase activity. We screened a transposon-generated E. coli insertion mutant library, using a qualitative PZAase assay. Two PZAase-negative mutants out of 4,000 colonies screened were identified. In each mutant, the transposon interrupted the same 639-bp open reading frame (ORF), ORF1. The expression of ORF1 on a multicopy plasmid complemented a PZAase-negative mutant, leading to PZAase activity levels approximately 10-fold greater than those of the wild type. PZA has a structure similar to that of nicotinamide, a pyridine nucleotide cycle intermediate, so we tested our strains for nicotinamidase activity (EC 3.5.1.19) (genetic locus pncA). The construct with multiple plasmid copies of ORF1 had an approximately 10-fold increase in levels of nicotinamidase activity. This overexpressing strain could utilize nicotinamide as a sole nitrogen source, through wild-type E. coli cannot. We conclude that a single E. coli enzyme accounts for both PZAase and nicotinamidase activities and that ORF1 is the E.coli PZAase and nicotinamidase gene, pncA.

  18. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.

    Science.gov (United States)

    Zheng, Wenjun; Wang, Qingsong; Bi, Qun

    2016-04-01

    Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni(2+) chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.

  19. Temperature control of molecular circuit switch responsible for virulent phenotype expression in uropathogenic Escherichia coli

    Science.gov (United States)

    Samoilov, Michael

    2010-03-01

    The behavior and fate of biological organisms are to a large extent dictated by their environment, which can be often viewed as a collection of features and constraints governed by physics laws. Since biological systems comprise networks of molecular interactions, one such key physical property is temperature, whose variations directly affect the rates of biochemical reactions involved. For instance, temperature is known to control many gene regulatory circuits responsible for pathogenicity in bacteria. One such example is type 1 fimbriae (T1F) -- the foremost virulence factor in uropathogenic E. coli (UPEC), which accounts for 80-90% of all community-acquired urinary tract infections (UTIs). The expression of T1F is randomly `phase variable', i.e. individual cells switch between virulent/fimbriate and avirulent/afimbriate phenotypes, with rates regulated by temperature. Our computational investigation of this process, which is based on FimB/FimE recombinase-mediated inversion of fimS DNA element, offers new insights into its discrete-stochastic kinetics. In particular, it elucidates the logic of T1F control optimization to the host temperature and contributes further understanding toward the development of novel therapeutic approaches to UPEC-caused UTIs.

  20. [miR-182 promotes cell proliferation of cervical cancer cells by targeting adenomatous polyposis coli (APC) gene].

    Science.gov (United States)

    Li, Pei; Hu, Jing; Zhang, Ying; Li, Jianping; Dang, Yunzhi; Zhang, Rui; Wei, Lichun; Shi, Mei

    2018-02-01

    Objective To investigate the role and mechanism of microRNA-182 (miR-182) in the proliferation of cervical cancer cells. Methods With liposome-mediated transient transfection method, the level of miR-182 in HeLa and SiHa cells was increased or decreased. CCK-8 assay and colony formation assay were used to observe the effect of miR-182 on the proliferation of cervical cancer cells. Using bioinformatics predictions, real-time quantitative PCR, and dual luciferase reporter assay, we clarified the role of miR-182 in posttranscriptional regulation of adenomatous polyposis coli (APC) gene and its effect on the downstream molecules (c-Myc and cyclin D1) of Wnt singling pathway. Results Up-regulation of miR-182 significantly promoted the proliferation of cervical cancer cells, while down-regulation of miR-182 significantly inhibited the proliferation of cervical cancer cells. Over-expression of miR-182 inhibited the expression of APC gene in cervical cancer cells and the regulation of miR-182 affected the expression of canonical Wnt signaling pathway downstream molecules in cervical cancer cells. Conclusion The miR-182 stimulates canonical Wnt signaling pathway by targeting APC gene and enhances the proliferation of cervical cancer cells.

  1. Bovine milk fat globule membrane affects virulence expression in Escherichia coli O157:H7.

    Science.gov (United States)

    Tellez, A; Corredig, M; Guri, A; Zanabria, R; Griffiths, M W; Delcenserie, V

    2012-11-01

    The aim of this study was to examine the effect of the bovine milk fat globule membrane (MFGM) on the virulence of Escherichia coli O157:H7. The MFGM was extracted from raw or heat-treated milk, resulting in 2 preparations differing in protein composition. Both heated and raw MFGM exerted an inhibitory effect on Shiga toxin gene expression by E. coli O157:H7 (ratios of -7.69 and -5.96, respectively). Interestingly, the effect was stronger with heated MFGM, with a larger decrease in expression of the virulence gene fliC (ratio of -9.43). The difference in effect observed between heated and raw MFGM could be explained by the difference in protein composition between the 2 preparations. These results show, for the first time, a specific effect of MFGM on expressionof Shiga toxin genes as well as genes involved in the motility of E. coli O157:H7. This may offer a new approach to mitigate the adverse health effects caused by E. coli O157:H7 infections. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  2. 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.

  3. Functional expression of a valencene dioxygenase from Pleurotus sapidus in E. coli.

    Science.gov (United States)

    Zelena, Kateryna; Krings, Ulrich; Berger, Ralf G

    2012-03-01

    Valencene dioxygenase (ValOx) from the edible basidiomycete Pleurotus sapidus converted the sesquiterpene (+)-valencene to the valuable grapefruit flavour (+)-nootkatone and to nootkatols through intermediate hydroperoxides. Expression of the enzyme was carried out in the cytosol and periplasm of Escherichia coli. The heterologous production led to high yields of inclusion bodies. The poor yield of soluble recombinant protein was improved by various strategies including cold shock expression, chaperone co-expression, and employment of mutant E. coli strains. Up to 60 mg of the biologically active, soluble ValOx was produced by cold shock under control of the cspA promoter at 8 °C in the BL21(DE3)Star strain and co-expression of the E. coli trigger factor. The recombinant enzyme, purified using the N-terminal His tag, showed the catalytic properties of the wild-type enzyme, as was confirmed by the LC-MS analysis of hydroperoxide intermediates and GC-MS analysis of the volatile products. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. 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.

  5. Influence of medium components on the expression of recombinant lipoproteins in Escherichia coli.

    Science.gov (United States)

    Tseng, Chi-Ling; Leng, Chih-Hsiang

    2012-02-01

    Bacterial lipoproteins are crucial antigens for protective immunity against bacterial pathogens. Expression of exogenous lipoproteins in Escherichia coli at high levels is thought to be an extremely difficult endeavor because it frequently results in incomplete or absent lipid modification. Previously, we identified a fusion sequence (D1) from a Neisseria meningitidis lipoprotein that induced a non-lipidated protein, E3 (the domain III of the dengue virus envelope protein), to become lipidated. However, without optimizing the growth conditions, some of the D1-fusion proteins were not lipidated. Here, we report the influence of medium components on the expression of recombinant lipoproteins in E. coli. For high-level expression of mature lipoproteins in the C43 (DE3) strain, M9 medium was better than M63 and the rich medium. Furthermore, we analyzed the influence of other media factors (including nitrogen and carbon sources, phosphate, ferrous ions, calcium, magnesium, and pH) on the levels of lipoprotein expression. The results showed that excess nitrogen sources and phosphate in M9 medium could increase the amount of immature lipoproteins, and glucose was a better carbon source than glycerol for expressing mature lipoproteins. We also found that lipoproteins tended to be completely processed in the alkaline environment, even in the nutrient-rich medium. Additional constructs expressing different immunogens or lipid signal peptides as targets were also utilized, demonstrating that these targets could be expressed as completely mature lipoproteins in the M9 medium but not in the rich medium. Our results provide the useful information for expressing mature exogenous lipoproteins in E. coli.

  6. 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

  7. Induction of genetic recombination in the lambda bacteriophage by ultraviolet radiation of the Escherichia Coli cells; Induccion de recombinacion genetica en el bacteriofago lambda por irradiacion ultravioleta de las cellulas de Escherichia Coli

    Energy Technology Data Exchange (ETDEWEB)

    Alcantara D, D [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    1986-12-15

    In this work there are reported the results that show that although the stimulation of the recombination of the Lambda bacteriophage, by UV irradiation of the cells of Escherichia Coli, it looks to be the result of the high expression of the functions of the SOS system, doesn't keep some relationship with the high concentration of protein reached RecA. (Author)

  8. Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

    Science.gov (United States)

    He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

    2015-10-01

    An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. 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....

  10. Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Inflammation and Cell Damage via Attenuation of ASC-Independent NLRP3 Inflammasome Activation.

    Science.gov (United States)

    Wu, Qiong; Liu, Ming-Chao; Yang, Jun; Wang, Jiu-Feng; Zhu, Yao-Hong

    2016-02-15

    Escherichia coli is a major environmental pathogen causing bovine mastitis, which leads to mammary tissue damage and cell death. We explored the effects of the probiotic Lactobacillus rhamnosus GR-1 on ameliorating E. coli-induced inflammation and cell damage in primary bovine mammary epithelial cells (BMECs). Increased Toll-like receptor 4 (TLR4), NOD1, and NOD2 mRNA expression was observed following E. coli challenge, but this increase was attenuated by L. rhamnosus GR-1 pretreatment. Immunofluorescence and Western blot analyses revealed that L. rhamnosus GR-1 pretreatment decreased the E. coli-induced increases in the expression of the NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) and the serine protease caspase 1. However, expression of the adaptor protein apoptosis-associated speck-like protein (ASC, encoded by the Pycard gene) was decreased during E. coli infection, even with L. rhamnosus GR-1 pretreatment. Pretreatment with L. rhamnosus GR-1 counteracted the E. coli-induced increases in interleukin-1β (IL-1β), -6, -8, and -18 and tumor necrosis factor alpha mRNA expression but upregulated IL-10 mRNA expression. Our data indicate that L. rhamnosus GR-1 reduces the adhesion of E. coli to BMECs, subsequently ameliorating E. coli-induced disruption of cellular morphology and ultrastructure and limiting detrimental inflammatory responses, partly via promoting TLR2 and NOD1 synergism and attenuating ASC-independent NLRP3 inflammasome activation. Although the residual pathogenic activity of L. rhamnosus, the dosage regimen, and the means of probiotic supplementation in cattle remain undefined, our data enhance our understanding of the mechanism of action of this candidate probiotic, allowing for development of specific probiotic-based therapies and strategies for preventing pathogenic infection of the bovine mammary gland. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. 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.

  12. Regulation of crp gene expression by the catabolite repressor/activator, Cra, in Escherichia coli.

    Science.gov (United States)

    Zhang, Zhongge; Aboulwafa, Mohammad; Saier, Milton H

    2014-01-01

    Growth of E. coli on several carbon sources is dependent on the catabolite repressor/activator (Cra) protein although a Cra consensus DNA-binding site is not present in the control regions of the relevant catabolic operons. We show that Cra regulates growth by activating expression of the crp gene. It thereby mediates catabolite repression of catabolic operons by an indirect mechanism. © 2014 S. Karger AG, Basel.

  13. Mutagenic action of heavy ions on Escherichia coli cells

    International Nuclear Information System (INIS)

    Tokarova, B.; Amirtayev, K.G.; Kozubek, S.; Krasavin, E.A.

    1989-01-01

    Introduction of direct mutations in the lactose operon of E.coli cells by γ radiation and accelerated heavy ions with different LET was studied. The experiments were performed with the wide type polA and lexA strains. The quadratic dependence of the mutation rate on the dose of different radiation for the wild type strain and the polA mutant was observed. However different radiations showed different relative genetic effectiveness. The dependence of RGE on LET for the wild type and polA strain has a maximum. In the experiments with the lexA strain low mutation rates and linear dose-response dependences N m /N(D) were observed. The RGE falls down with increasing LET of ionizing radiation. 22 refs.; 5 figs.; 2 tabs

  14. Optical Detection of Paraoxon Using Single-Walled Carbon Nanotube Films with Attached Organophosphorus Hydrolase-Expressed Escherichia coli

    Directory of Open Access Journals (Sweden)

    Intae Kim

    2015-05-01

    Full Text Available In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.

  15. Expression of the phycoerythrin gene of Gracilaria lemaneiformis (Rhodophyta) in E. coli and evaluation of the bioactivity of recombinant PE

    Science.gov (United States)

    Wen, Ruobing; Sui, Zhenghong; Zhang, Xuecheng; Zhang, Shuang; Qin, Song

    2007-10-01

    Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E. coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) ( Pprotein content. For the heating samples, except for the 0.2 mg mL-1 BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This research showed that phycoerythrin protein

  16. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

    Directory of Open Access Journals (Sweden)

    Perera Rajika L

    2004-12-01

    Full Text Available Abstract Background In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44, kinases (EGFR-cytoplasmic domain, CDK2 and 4, proteases (MMP1, CASP2, signal transduction proteins (GRB2, RAF1, HRAS and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX. Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. Results Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY, or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx and maltose binding protein (MBP were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. Conclusions By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases

  17. Dissecting the hydrogenase expression and activity of transformed escherichia coli with the bidirectional NiFe-hydrogenase from synechocystis sp. PCC 6803

    International Nuclear Information System (INIS)

    Moon, Yu Ran; Lee, Min Hee; An, Byung Chull; Chung, Byung Yeoup; Kim, Jae Sung; Kim, Jin Hong; Park, Youn Il; Kim, Cha Soon

    2009-01-01

    Synechocystis bidirectional hydrogenase genes (hoxEFUYH) and their putative promoter regions and transformed into E. coli. The hox genes were transcribed in the E. coli cells carrying the vector construct of pCCIFOS::phox::hox or pCCIFOS::pT7::hox and translated into HoxEFUYH proteins, suggesting that the putative hox promoter can be constitutively activated in E. coli. Accordingly, the total hydrogenase activity was markedly increased up to 192% or 169% in the transformed cells, while the hydrogen uptake was decreased up to about 30% of the negative control. Although the gene expression of LexA, the only one transcription regulator proven for the hox genes, was substantially decreased in the pCCIFOS::phox::hox cells after γ-irradiation of 30 Gy, the expression levels of HoxEFUYH proteins were not altered significantly. Thus, it is also suggested that other transcription regulators as well as LexA might contribute to activation of the hox promoter in E. coli

  18. Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities.

    Science.gov (United States)

    Jheng, Yi-Jyun; Tsai, Wei-Yi; Chen, Kuo-Hsuan; Lin, Kuo-Wei; Chyan, Chia-Lin; Yang, Ching-Chi; Lin, Kuo-Chih

    2012-09-01

    Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors

    Directory of Open Access Journals (Sweden)

    Kawe Martin

    2009-01-01

    Full Text Available Abstract Background Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such "benign" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored. Results We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size – all antibiotic-resistant non-producers – was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented. Conclusion The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein.

  20. 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

  1. 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.

  2. Tuning of Recombinant Protein Expression in Escherichia coli by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids.

    Science.gov (United States)

    Schlesinger, Orr; Chemla, Yonatan; Heltberg, Mathias; Ozer, Eden; Marshall, Ryan; Noireaux, Vincent; Jensen, Mogens Høgh; Alfonta, Lital

    2017-06-16

    Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the Eschrichia coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. On the basis of the results, we developed a biophysical model, which suggests that the density of co-transcriptional-translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

  3. Differential requirements of two recA mutants for constitutive SOS expression in Escherichia coli K-12.

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    Jarukit Edward Long

    Full Text Available Repairing DNA damage begins with its detection and is often followed by elicitation of a cellular response. In E. coli, RecA polymerizes on ssDNA produced after DNA damage and induces the SOS Response. The RecA-DNA filament is an allosteric effector of LexA auto-proteolysis. LexA is the repressor of the SOS Response. Not all RecA-DNA filaments, however, lead to an SOS Response. Certain recA mutants express the SOS Response (recA(C in the absence of external DNA damage in log phase cells.Genetic analysis of two recA(C mutants was used to determine the mechanism of constitutive SOS (SOS(C expression in a population of log phase cells using fluorescence of single cells carrying an SOS reporter system (sulAp-gfp. SOS(C expression in recA4142 mutants was dependent on its initial level of transcription, recBCD, recFOR, recX, dinI, xthA and the type of medium in which the cells were grown. SOS(C expression in recA730 mutants was affected by none of the mutations or conditions tested above.It is concluded that not all recA(C alleles cause SOS(C expression by the same mechanism. It is hypothesized that RecA4142 is loaded on to a double-strand end of DNA and that the RecA filament is stabilized by the presence of DinI and destabilized by RecX. RecFOR regulate the activity of RecX to destabilize the RecA filament. RecA730 causes SOS(C expression by binding to ssDNA in a mechanism yet to be determined.

  4. Protective vaccination with a recombinant fragment of Clostridium botulinum neurotoxin serotype A expressed from a synthetic gene in Escherichia coli.

    OpenAIRE

    Clayton, M A; Clayton, J M; Brown, D R; Middlebrook, J L

    1995-01-01

    A completely synthetic gene encoding fragment C, a approximately 50-kDa fragment, of botulinum neurotoxin serotype A was constructed from oligonucleotides. The gene was expressed in Escherichia coli, and full-sized product was produced as judged by Western blot (immunoblot) analysis. Crude extracts of E. coli expressing the gene were used to vaccinate mice and evaluate their survival against challenge with active toxin. Mice given three subcutaneous vaccinations were protected against an intr...

  5. Efek ekstrak daun singkong (Manihot utilissima terhadap ekspresi COX-2 pada monosit yang dipapar LPS E.coli (The effect of Manihot utilissima extracts on COX-2 expression of monocytes induced by LPS E. coli

    Directory of Open Access Journals (Sweden)

    Zahara Meilawaty

    2013-12-01

    Full Text Available Background: Periodontal disease is a common and widespread disease in the community. Gram negative bacteria have a role inperiodontitis. These bacteria secrete a variety of products such as endotoxin lipopolysaccharide (LPS, which causes the occurrenceof inflammation or infection. The body defense responses are neutrophils and mononuclear cells (monocytes and macrophages. Inresponse to defense mechanism, the body will be expressed enzyme cyclooxygenase (COX which functions convert arachidonic acidto prostaglandins. Cassava leaf cells known to play a role in reducing inflammation, but the mechanism for inhibiting COX-2, is notknown. Purpose: The study was aimed to determine the effect of cassava leaf extract (Manihot utilissima on expression of enzyme COX-2 in monocytes which were exposed by LPS E. coli. Methods: This study was in vitro experimental studies with the design of posttestonly control group design. The sample was the cassava leaves extract (Manihot utilissima at concentration of 12.5 % and 25 %. Theexpression of COX-2 was determined by immunocytochemistry method. Isolated monocytes were incubated in cassava leaf extract, andthen exposed to LPS, after washing imunostaning procedure was performed using a monoclonal antibody (MAb anti-human COX-2.The research data was the number of monocytes that express COX-2. Results: Expression of COX-2 in the group cassava leaf extractwas higher than the group that induced by LPS E. coli only. Conclusion: Cassava leaf extract did not inhibit the expression of COX-2in monocytes which were exposed by LPS E. coli.Latar belakang: Penyakit periodontal merupakan penyakit umum dan tersebar luas di masyarakat. Bakteri yang banyak berperanpada periodontitis adalah Gram negatif. Bakteri ini mengeluarkan berbagai produk antara lain endotoksin lipopolisakarida (LPS yangmenyebabkan inflamasi atau infeksi. Respon pertahanan tubuh pertama adalah netrofil dan sel mononuklear (monosit dan makrofag.Pada respon

  6. The 4.5 S RNA gene of Escherichia coli is essential for cell growth

    DEFF Research Database (Denmark)

    Brown, S; Fournier, M J

    1984-01-01

    The Escherichia coli gene coding for the metabolically stable 4.5 S RNA (ffs) has been shown to be required for cell viability. Essentiality was demonstrated by examining the recombination behavior of substitution mutations of ffs generated in vitro. Substitution mutants of ffs are able to replace...... the chromosomal allele only in the presence of a second, intact copy of ffs. Independent evidence of essentiality and the finding that 4.5 S RNA is important for protein synthetic activity came from characterization of cells dependent on the lac operon inducer isopropyl-beta-D-thiogalactoside for ffs gene...... expression. Here, a strain dependent on isopropyl-beta-D-thiogalactoside for 4.5 S RNA synthesis was developed by inactivation of the chromosomal ffs allele and lysogenization by a lambda phage containing 4.5 S DNA fused to a hybrid trp-lac promoter. Withdrawal of the thiogalactoside leads to a deficiency...

  7. Metabolic Regulation of a Bacterial Cell System with Emphasis on Escherichia coli Metabolism

    Science.gov (United States)

    Shimizu, Kazuyuki

    2013-01-01

    It is quite important to understand the overall metabolic regulation mechanism of bacterial cells such as Escherichia coli from both science (such as biochemistry) and engineering (such as metabolic engineering) points of view. Here, an attempt was made to clarify the overall metabolic regulation mechanism by focusing on the roles of global regulators which detect the culture or growth condition and manipulate a set of metabolic pathways by modulating the related gene expressions. For this, it was considered how the cell responds to a variety of culture environments such as carbon (catabolite regulation), nitrogen, and phosphate limitations, as well as the effects of oxygen level, pH (acid shock), temperature (heat shock), and nutrient starvation. PMID:25937963

  8. Variant innate immune responses of mammary epithelial cells to challenge by Staphylococcus aureus, Escherichia coli and the regulating effect of taurine on these bioprocesses.

    Science.gov (United States)

    Zheng, Liuhai; Xu, Yuanyuan; Lu, Jinye; Liu, Ming; Bin Dai; Miao, Jinfeng; Yin, Yulong

    2016-07-01

    Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are important pathogens causing subclinical and clinical bovine mastitis, respectively. Taurine, an organic acid found in animal tissues, has been used for the treatment of various superficial infections and chronic inflammations. We challenged a bovine mammary epithelial cell (MEC) line (MAC-T) or a mouse mammary epithelial cell line (EpH4-Ev) with either E. coli or S. aureus and compared the responses of MECs to these 2 pathogens. We also examined the regulatory effects of taurine on these responses. Receptor analyses showed that both TLR2 and TLR4 are upregulated upon exposure to either E. coli or S. aureus. Taurine pre-treatment dampened upregulation to some extent. E. coli and S. aureus stimulated comparable levels of ROS, which could be inhibited by taurine pre-treatment. E. coli infection elicited a dramatic change in iNOS expression. Taurine significantly decreased iNOS expression in the S. aureus challenged group. Protein microarray demonstrated that 32/40 and 8/40 inflammatory molecules/mediators were increased after E. coli or S. aureus challenge, respectively. The fold changes of most molecules were higher in the E. coli infection group than that in the S. aureus infection group. Taurine negatively regulated the inflammatory profile in both bacterial infections. Pro-inflammatory cytokines (such as TNF-α) connected with TLR activation were down-regulated by taurine pre-treatment. The influence of TAK-242 and OxPAPC on cytokine/molecule expression profiles to E. coli challenge are different than to S. aureus. Some important factors (MyD88, TNF-α, IL-1β, iNOS and IL-6) mediated by TLR activation were suppressed either in protein microarray or special assay (PCR/kits) or both. TAK-242 restrained ROS production and NAGase activity similar to the effect of taurine in E. coli challenge groups. The detection of 3 indices (T-AOC, SOD and MDA) reflecting oxidative stress in vivo, showed that

  9. Elevated Expression of GlpT and UhpT via FNR Activation Contributes to Increased Fosfomycin Susceptibility in Escherichia coli under Anaerobic Conditions

    Science.gov (United States)

    Kurabayashi, Kumiko; Tanimoto, Koichi; Fueki, Shinobu; Tomita, Haruyoshi

    2015-01-01

    Because a shortage of new antimicrobial agents is a critical issue at present, and with the spread of multidrug-resistant (MDR) pathogens, the use of fosfomycin to treat infections is being revisited as a “last-resort option.” This drug offers a particular benefit in that it is more effective against bacteria growing under oxygen-limited conditions, unlike other commonly used antimicrobials, such as fluoroquinolones and aminoglycosides. In this study, we showed that Escherichia coli strains, including enterohemorrhagic E. coli (EHEC), were more susceptible to fosfomycin when grown anaerobically than when grown aerobically, and we investigated how the activity of this drug was enhanced during anaerobic growth of E. coli. Our quantitative PCR analysis and a transport assay showed that E. coli cells grown under anaerobic conditions had higher levels of expression of glpT and uhpT, encoding proteins that transport fosfomycin into cells with their native substrates, i.e., glycerol-3-phosphate and glucose-6-phosphate, and led to increased intracellular accumulation of the drug. Elevation of expression of these genes during anaerobic growth requires FNR, a global transcriptional regulator that is activated under anaerobic conditions. Purified FNR bound to DNA fragments from regions upstream of glpT and uhpT, suggesting that it is an activator of expression of glpT and uhpT during anaerobic growth. We concluded that the increased antibacterial activity of fosfomycin toward E. coli under anaerobic conditions can be attributed to elevated expression of GlpT and UhpT following activation of FNR, leading to increased uptake of the drug. PMID:26248376

  10. 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

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

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    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.

  12. Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

    Science.gov (United States)

    Arabski, Michał; Węgierek-Ciuk, Aneta; Czerwonka, Grzegorz; Lankoff, Anna; Kaca, Wiesław

    2012-01-01

    Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed. PMID:22500084

  13. Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

    Directory of Open Access Journals (Sweden)

    Michał Arabski

    2012-01-01

    Full Text Available Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed.

  14. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

    Science.gov (United States)

    Yue, Chang-Wu; Zhang, Yi-Zheng

    2009-03-01

    A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

  15. 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...

  16. Production of Brugia malayi BmSXP Recombinant Protein Expressed in Escherichia coli

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    Khoo, T. K.

    2010-01-01

    Full Text Available A rapid antibody detection test is very useful for detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. One such kit, panLF RapidTM (commercialized by Malaysian BioDiagnostic Research Sdn. Bhd. had been developed in our laboratory for the detection of all species of filarial infections. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant Brugia malayi antigens, BmR1 and BmSXP. In this study, the growth of recombinant bacteria that produce BmSXP was optimized under shake flask fermentation for high yield of the recombinant antigen. The optimizations involved selection of suitable growth medium, IPTG concentration and induction time. The medium that yielded the highest biomass as well as total protein was Terrific Broth (TB medium, which is an undefined medium. Initiation of induction of protein expression was found to be best at mid-log phase (OD600 = 1.5, with IPTG concentration of 1.0 mM, and harvest time at 9 h post-induction. This study showed that under the optimized conditions, the shake flask culture produced 4 g/L biomass (dry cell weight of recombinant Escherichia coli BmSXP/pPROEXHTa/TOP10F’, which yielded 2.42 mg/L of purified BmSXP recombinant antigen. The purified antigen was analyzed by SDS-PAGE and the antigenicity of protein was confirmed by Western blot.

  17. Synthesis of dopamine in E. coli using plasmid-based expression system and its marked effect on host growth profiles.

    Science.gov (United States)

    Das, Arunangshu; Verma, Anita; Mukherjee, Krishna J

    2017-09-14

    L-Dopa and dopamine are important pathway intermediates toward the synthesis of catecholamine such as epinephrine and norepinephrine from amino acid L-tyrosine. Dopamine, secreted from dopaminergic nerve cells, serves as an important neurotransmitter. We report the synthesis of dopamine by extending the aromatic amino acid pathway of Escherichia coli DH5α by the expression of 4-hydroxyphenylacetate-3-hydrolase (HpaBC) from E. coli and an engineered dopa decarboxylase (DDC) from pig kidney cell. The activity of HpaBC and DDC require 200 µM iron supplementation and 50 µM vitamin B6, respectively as additives to the growth media. The maximum concentration of L-dopa and dopamine obtained from the broth was around 26 and 27 mg/L after 24 hr of separate shake flask studies. We observed that in the presence of dopamine synthesized in vivo host growth was remarkably enhanced. These observations lead us to an interesting finding about the role of these catecholamines on bacterial growth. It is clear that synthesis of dopamine in vivo actually promotes growth much efficiently as compared to when dopamine is added to the system from outside. From HPLC and GC-MS data it was further observed that L-dopa was stable within the observable time of experiments whereas dopamine actually was subjected to degradation via oxidation and host consumption.

  18. [Expression of enterotoxigenic Bacteroides fragilis and polyketide synthase gene-expressing Escherichia coli in colorectal adenoma patients].

    Science.gov (United States)

    Xie, L L; Wu, N; Zhu, Y M; Qiu, X Y; Chen, G D; Zhang, L M; Liu, Y L

    2016-03-29

    To investigate the distribution of various bacteria in adenoma tissue of colorectal adenoma (T/CRA), normal colonic mucosa tissue adjacent to the adenoma (N/CRA), and healthy colonic mucosa tissue (N/H) by comparing the number of total bacteria, Bacteroides fragilis (BF), enterotoxigenic Bacteroides fragilis (ETBF), polyketide synthase (pks) gene-expressing Escherichia coli(E.coli)(pks(+) E. coli)among the above 3 types of tissues. A total of 36 patients diagnosed with colorectal adenoma by colonoscopy and pathology in Department of Gastroenterology, Peking University People's Hospital from September 2011 to September 2013 were selected into this study. T/CRA and N/CRA tissues from the 36 patients and N/H tissues from 18 healthy controls were collected for DNA extraction. The number of total bacteria, BF, ETBF, pks(+) E. coli was detected by quantitative real time PCR, and their correlation with colorectal adenoma was analyzed. (1) The number of total bacteria decreased gradually from N/H, N/CRA, to T/CRA, with the median values being 3.18×10(8,) 1.57×10(8,) and 7.91×10(7) copies/g, respectively, and with significant difference among the three groups and between each two groups (all PCRA, to T/CRA, the median values being 6.03×10(5,) 4.28×10(4,) and 5.48×10(3) copies/g, respectively, and with significant difference among the three groups and between each two groups (all PCRA, to T/CRA, the relative expression being 1.73±0.30, 6.15±1.52, and 8.54±1.80, respectively. Significant difference was found between the T/CRA and N/H tissue (P=0.003), but not between any other two groups. (4) The expression of clbB in pks(+) E.coli was highest in T/CRA colonic tissue (2.96±0.28), followed by the N/CRA (2.79±0.19) and N/H tissue (1.06±0.08). Significant difference was found between T/CRA and N/H tissues, as well as between N/CRA and N/H tissues (both PCRA and N/CRA tissues. The number of total bacteria is markedly reduced in the colonic mucosa of CRA patients

  19. 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

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

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    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

  1. Spaceflight Modifies Escherichia coli Gene Expression in Response to Antibiotic Exposure and Reveals Role of Oxidative Stress Response

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    Thomas R. Aunins

    2018-03-01

    Full Text Available Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under

  2. Spaceflight Modifies Escherichia coli Gene Expression in Response to Antibiotic Exposure and Reveals Role of Oxidative Stress Response.

    Science.gov (United States)

    Aunins, Thomas R; Erickson, Keesha E; Prasad, Nripesh; Levy, Shawn E; Jones, Angela; Shrestha, Shristi; Mastracchio, Rick; Stodieck, Louis; Klaus, David; Zea, Luis; Chatterjee, Anushree

    2018-01-01

    Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under extreme stress

  3. Spaceflight Modifies Escherichia coli Gene Expression in Response to Antibiotic Exposure and Reveals Role of Oxidative Stress Response

    Science.gov (United States)

    Aunins, Thomas R.; Erickson, Keesha E.; Prasad, Nripesh; Levy, Shawn E.; Jones, Angela; Shrestha, Shristi; Mastracchio, Rick; Stodieck, Louis; Klaus, David; Zea, Luis; Chatterjee, Anushree

    2018-01-01

    Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under extreme stress

  4. Stationary phase expression of the arginine biosynthetic operon argCBH in Escherichia coli

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    Sun Yuan

    2006-02-01

    Full Text Available Abstract Background Arginine biosynthesis in Escherichia coli is elevated in response to nutrient limitation, stress or arginine restriction. Though control of the pathway in response to arginine limitation is largely modulated by the ArgR repressor, other factors may be involved in increased stationary phase and stress expression. Results In this study, we report that expression of the argCBH operon is induced in stationary phase cultures and is reduced in strains possessing a mutation in rpoS, which encodes an alternative sigma factor. Using strains carrying defined argR, and rpoS mutations, we evaluated the relative contributions of these two regulators to the expression of argH using operon-lacZ fusions. While ArgR was the main factor responsible for modulating expression of argCBH, RpoS was also required for full expression of this biosynthetic operon at low arginine concentrations (below 60 μM L-arginine, a level at which growth of an arginine auxotroph was limited by arginine. When the argCBH operon was fully de-repressed (arginine limited, levels of expression were only one third of those observed in ΔargR mutants, indicating that the argCBH operon is partially repressed by ArgR even in the absence of arginine. In addition, argCBH expression was 30-fold higher in ΔargR mutants relative to levels found in wild type, fully-repressed strains, and this expression was independent of RpoS. Conclusion The results of this study indicate that both derepression and positive control by RpoS are required for full control of arginine biosynthesis in stationary phase cultures of E. coli.

  5. Cloning and expression of pab gene of M. tuberculosis isolated from pulmonary TB patient in E.coli DH5α

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    Tri Y. M. Raras

    2011-11-01

    Full Text Available Background: Mycobacterium tuberculosis antigen38 is a potent serodiagnostic agent containing two M. tuberculosisspecific B-cell epitopes. The high price of imported diagnostic agents hinders realization of fast clinical TB diagnosis in developing countries. Therefore, we produced recombinant antigen38 (recAg38M from M. tuberculosis local strain, which might be used to produce economical tuberculosis serodiagnostic kit.Methods: Pab gene that was isolated from pulmonary TB patient in Malang was cloned into a plasmid vector (pGEMTeasy to construct pMB38. The E.coli DH5α clone carrying pMb38 was selected on X-gal medium. The expression of pab was mediated using pPRoExHTc under the control of Trc promoter and E.coli DH5α as host.Results: Alignment of the pab sequence from the white E.coli DH5α clones with that of M. tuberculosis H37Rv showed 98% homology. The recombinant protein in which the signal peptide has been deleted to prevent the protein being secreted into medium was found in the cytoplasm.Conclusion: pab gene of M. tuberculosis isolated from a TB patient could be expressed in heterologous system in E.coliDH5α. (Med J Indones 2011; 20:247-54Keywords: Mycobacterium tuberculosis, Pab gene expression, recombinant antigen38

  6. Biocatalytic Production of Trehalose from Maltose by Using Whole Cells of Permeabilized Recombinant Escherichia coli.

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    Zhaojuan Zheng

    Full Text Available Trehalose is a non-reducing disaccharide, which can protect proteins, lipid membranes, and cells from desiccation, refrigeration, dehydration, and other harsh environments. Trehalose can be produced by different pathways and trehalose synthase pathway is a convenient, practical, and low-cost pathway for the industrial production of trehalose. In this study, 3 candidate treS genes were screened from genomic databases of Pseudomonas and expressed in Escherichia coli. One of them from P. stutzeri A1501 exhibited the best transformation ability from maltose into trehalose and the least byproduct. Thus, whole cells of this recombinant E. coli were used as biocatalyst for trehalose production. In order to improve the conversion rate of maltose to trehalose, optimization of the permeabilization and biotransformation were carried out. Under optimal conditions, 92.2 g/l trehalose was produced with a high productivity of 23.1 g/(l h. No increase of glucose was detected during the whole course. The biocatalytic process developed in this study might serve as a candidate for the large scale production of trehalose.

  7. A new strategy for full-length Ebola virus glycoprotein expression in E.coli.

    Science.gov (United States)

    Zai, Junjie; Yi, Yinhua; Xia, Han; Zhang, Bo; Yuan, Zhiming

    2016-12-01

    Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

  8. Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli

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    Neubauer Peter

    2003-04-01

    Full Text Available Abstract Background Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments. Results Shake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-β-D-thiogalactopyranoside or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction. Conclusion Whey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG.

  9. Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli.

    Science.gov (United States)

    Mirzaei, Maryam; Saffar, Behnaz; Shareghi, Behzad

    2016-06-01

    Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals' foods to hydrolyze phytate and increase absorption of phosphorus. Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized Y. intermedia phytase gene in E. coli . The Y. intermedia phytase gene was optimized according to the codon usage in E. coli . The sequence was synthesized and sub-cloned in pET-22b (+) vector and transformed into E. coli Bl21 (DE3). The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30°C. The purification of recombinant protein was performed by Ni 2+ affinity chromatography. Phytase activity and stability were determined in various pH and temperatures. The codon optimized Y. intermedia phytase gene was sub-cloned successfully.The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme (approximately 45 kDa) was purified. Specific activity of enzyme was 3849 (U.mg -1 ) with optimal pH 5 and optimal temperature of 55°C. Thermostability (80°C for 15 min) and pH stability (3-6) of the enzyme were 56 and more than 80%, respectively. The results of the expression and enzyme characterization revealed that the optimized Y. intermedia phytase gene has a good potential to be produced commercially andto be applied in animals' foodsindustry.

  10. A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks

    International Nuclear Information System (INIS)

    Cai, Mengli; Huang, Ying; Yang, Renbin; Craigie, Robert; Clore, G. M.

    2016-01-01

    We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D_2O usage tenfold and d_7-glucose usage by 30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD_6_0_0 of up to 10. Inducing the cells with isopropyl β-d-1-thiogalactopyranoside at an OD_6_0_0 of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD_6_0_0 between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E. coli from H_2O-based to D_2O-based medium is also key for ensuring high levels of protein expression in D_2O. We find that a simple three-step adaptation approach—Luria–Bertani (LB) medium in H_2O to LB in D_2O to modified-M9 medium in D_2O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.

  11. Role of cloned carotenoid genes expressed in Escherichia coli in protecting against inactivation by near-UV light and specific phototoxic molecules

    International Nuclear Information System (INIS)

    Tuveson, R.W.; Larson, R.A.; Kagan, J.

    1988-01-01

    Genes controlling carotenoid synthesis were cloned from Erwinia herbicola and expressed in an Escherichia coli strain. Carotenoids protect against high fluences of near-UV (NUV; 320 to 400 nm) but not against far-UV (200-300 nm). Protection of E. coli cells was not observed following treatment with either psoralen or 8-methoxypsoralen plus NUV. However, significant protection of cells producing carotenoids was observed with three photosensitizing molecules activated by NUV (alpha-terthienyl, harmine, and phenylheptatriyne) which are thought to have the membrane as an important lethal target. Protection of carotenoid-producing cells against inactivation was not observed with acridine orange plus visible light but was seen with toluidine blue O plus visible light

  12. Microarray-Based Screening of Differentially Expressed Genes of E. coli O157:H7 Sakai during Preharvest Survival on Butterhead Lettuce

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    Inge Van der Linden

    2016-01-01

    Full Text Available Numerous outbreaks of Escherichia coli O157:H7 have been linked to the consumption of leafy vegetables. However, up to the present, little has been known about E. coli O157:H7’s adaptive responses to survival on actively growing (and thus responsive plants. In this study, whole genome transcriptional profiles were generated from E. coli O157:H7 cells (isolate Sakai, stx- one hour and two days after inoculation on the leaves of growing butterhead lettuce, and compared with an inoculum control. A total of 273 genes of E. coli O157:H7 Sakai (5.04% of the whole genome were significantly induced or repressed by at least two-fold (p < 0.01 in at least one of the analyzed time points in comparison with the control. Several E. coli O157:H7 genes associated with oxidative stress and antimicrobial resistance were upregulated, including the iron-sulfur cluster and the multiple antibiotic resistance (mar operon, whereas the Shiga toxin virulence genes were downregulated. Nearly 40% of the genes with significantly different expression were poorly characterized genes or genes with unknown functions. These genes are of special interest for future research as they may play an important role in the pathogens’ adaptation to a lifestyle on plants. In conclusion, these findings suggest that the pathogen actively interacts with the plant environment by adapting its metabolism and responding to oxidative stress.

  13. Rare codons effect on expression of recombinant gene cassette in Escherichia coli BL21(DE3

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    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.

  14. Zinc oxide nanoparticle reduced biofilm formation and antigen 43 expressions in uropathogenic Escherichia coli

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    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

  15. Expression and purification of lacticin Q by small ubiquitin-related modifier fusion in Escherichia coli.

    Science.gov (United States)

    Ma, Qingshan; Yu, Zhanqiao; Han, Bing; Wang, Qing; Zhang, Rijun

    2012-04-01

    Lacticin Q is a broad-spectrum class II bacteriocin with potential as an alternative to conventional antibiotics. The objective of this study was to produce recombinant lacticin Q using a small ubiquitin-related modifier (SUMO) fusion protein expression system. The 168-bp lacticin Q gene was cloned into the expression vector pET SUMO and transformed into Escherichia coli BL21(DE3). The soluble fusion protein was recovered with a Ni-NTA Sepharose column (95% purity); 130 mg protein was obtained per liter of fermentation culture. The SUMO tag was then proteolytically cleaved from the protein, which was re-applied to the column. Finally, about 32 mg lacticin Q (≥96% purity) was obtained. The recombinant protein exhibited antimicrobial properties similar to that of the native protein, demonstrating that lacticin Q had been successfully expressed by the SUMO fusion system.

  16. Penicillin-binding site on the Escherichia coli cell envelope

    International Nuclear Information System (INIS)

    Amaral, L.; Lee, Y.; Schwarz, U.; Lorian, V.

    1986-01-01

    The binding of 35 S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Global transcriptional regulatory network for Escherichia coli robustly connects gene expression to transcription factor activities

    Science.gov (United States)

    Fang, Xin; Sastry, Anand; Mih, Nathan; Kim, Donghyuk; Tan, Justin; Lloyd, Colton J.; Gao, Ye; Yang, Laurence; Palsson, Bernhard O.

    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 gene expression using this TRN; and (iii) how robust is our understanding of the TRN? First, we reconstructed a high-confidence TRN (hiTRN) consisting of 147 transcription factors (TFs) regulating 1,538 transcription units (TUs) encoding 1,764 genes. The 3,797 high-confidence regulatory interactions 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 algorithms to predict the expression of 1,364 TUs given TF activities using 441 samples. The algorithms accurately predicted condition-specific expression for 86% (1,174 of 1,364) of the TUs, while 193 TUs (14%) were predicted better than random TRNs. Third, we identified 10 regulatory modules whose definitions were robust against changes to the TRN or expression compendium. Using surrogate variable analysis, we also identified three unmodeled factors that systematically influenced gene expression. Our computational workflow comprehensively characterizes the predictive capabilities and systems-level functions of an organism’s TRN from disparate data types. PMID:28874552

  20. Aberrant expression of sonic hedgehog pathway in colon cancer and melanosis coli.

    Science.gov (United States)

    Wang, Zhong Chuan; Gao, Jun; Zi, Shu Ming; Yang, Ming; Du, Peng; Cui, Long

    2013-08-01

    To determine the hedgehog (Hh) signaling pathway correlated with the development of colon cancer and melanosis coli. Protein and mRNA levels of Hh signaling pathway components (sonic hedgehog [Shh], protein patched homolog 1 [Ptch 1], GLI family zinc finger 1 [Gli 1] and suppressor of fused homolog [Drosophila] [Sufu]) in 127 patients with colon cancer, 36 with melanosis coli and 20 adjacent normal mucosal tissues taken from surgical specimens were evaluated using antibody staining and quantitative real-time polymerase chain reaction. In adjacent normal tissue Shh and Ptch1, but not Gli1 or Sufu, were weakly expressed and mainly in the lining epithelium of the colonic mucosa. In cancerous tissues Shh and Gli1 were uniformly strong while Ptch1 was patchy and weak, and Sufu uniformly weak, which paralleled their levels of corresponding mRNA. Elevated protein levels of Shh and Ptch were significantly associated with mucinous colonic tissues. Elevated Sufu protein levels were positively correlated with the diameter and invasion of the tumor. In patients with melanosis coli, mRNA levels of Shh, Ptch1, Gli1 and Sufu were very low, which was similar to those of adjacent normal tissues; but protein levels of Shh, Ptch1 and Gli1, but not Sufu, were high, which was similar to those of cancerous tissues. The mRNA and protein levels of Hh pathway components are aberrantly elevated in colon cancer, which may be the potential molecular classification markers. Further studies are required to determine the role of melanosis coli in the colon tumorigenesis. © 2013 The Authors. Journal of Digestive Diseases © 2013 Wiley Publishing Asia Pty Ltd and Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine.

  1. Radiometric study of the metabolic processes in cell cultures inoculated with E.coli 0111

    International Nuclear Information System (INIS)

    Stankova-Shindarova, I.

    1977-01-01

    The penetration and propagation of bacteria in tissue cells is accompanied by changes in the metabolic processes. A group of strains, belonging to one serologic type comprises invasive and noninvasive variants. Twenty two E.coli 0111 strains were studied. By labelling strains with 3 H-thymidine, 3 H-uridine and 14C-leucine it was demonstrated that the amino acid and protein synthesis of RC 3 cells inoculated with invasive E.coli 0111 variants becomes more intensive. Amino acid and protein synthesis in noninvasive E.coli 0111 following previous high incorporation of the three labelled compounds is rapidly reduced and remains within control limits. (author)

  2. Uropathogenic Escherichia coli Express Type 1 Fimbriae Only in Surface Adherent Populations Under Physiological Growth Conditions

    DEFF Research Database (Denmark)

    Stærk, Kristian; Kolmos, Hans Jørn; Khandige, Surabhi

    2016-01-01

    were correlated with the ability to adhere to and invade cultured human bladder cells. RESULTS:  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...... 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....... 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. CONCLUSION:  Our results demonstrate that T1F expression is strictly regulated under physiological growth conditions...

  3. Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

    International Nuclear Information System (INIS)

    Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

    2006-01-01

    The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials

  4. Comparative method of protein expression and isolation of EBV epitope in E.coli DH5α

    Science.gov (United States)

    Anyndita, Nadya V. M.; Dluha, Nurul; Himmah, Karimatul; Rifa'i, Muhaimin; Widodo

    2017-11-01

    Epstein-Barr Virus (EBV) or human herpes virus 4 (HHV-4) is a virus that infects human B cell and leads to nasopharyngeal carcinoma (NPC). The prevention of this disease remains unsuccessful since the vaccine has not been discovered. The objective of this study is to over-produce EBV gp350/220 epitope using several methods in E.coli DH5α. EBV epitope sequences were inserted into pMAL-p5x vector, then transformed into DH5α E.coli and over-produced using 0.3, 1 and 2 mM IPTG. Plasmid transformation was validated using AflIII restriction enzyme in 0.8% agarose. Periplasmic protein was isolated using 2 comparative methods and then analyzed using SDS-PAGE. Method A produced a protein band around 50 kDa and appeared only at transformant. Method B failed to isolate the protein, indicated by no protein band appearing. In addition, any variations in IPTG concentration didn't give a different result. Thus it can be concluded that even the lowest IPTG concentration is able to induce protein expression.

  5. Cloning expression and analysis of phytochelatin synthase (pcs) gene from Anabaena sp. PCC 7120 offering multiple stress tolerance in Escherichia coli

    International Nuclear Information System (INIS)

    Chaurasia, Neha; Mishra, Yogesh; Rai, Lal Chand

    2008-01-01

    Phytochelatin synthase (PCS) is involved in the synthesis of phytochelatins (PCs), plays role in heavy metal detoxification. The present study describes for first time the functional expression and characterization of pcs gene of Anabaena sp. PCC 7120 in Escherichia coli in terms of offering protection against heat, salt, carbofuron (pesticide), cadmium, copper, and UV-B stress. The involvement of pcs gene in tolerance to above abiotic stresses was investigated by cloning of pcs gene in expression vector pGEX-5X-2 and its transformation in E. coli BL21 (DE3). The E. coli cells transformed with pGEX-5X-pcs showed better growth than control cells (pGEX-5X-2) under temperature (47 deg. C), NaCl (6% w/v), carbofuron (0.025 mg ml -1 ), CdCl 2 (4 mM), CuCl 2 (1 mM), and UV-B (10 min) exposure. The enhanced expression of pcs gene revealed by RT-PCR analysis under above stresses at different time intervals further advocates its role in tolerance against above abiotic stresses

  6. Strategies for Protein Overproduction in Escherichia coli.

    Science.gov (United States)

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  7. [Expression and functions of adaptive response genes in Escherichia coli treated with mono- and bifunctional alkylating agents. Interference with SOS response].

    Science.gov (United States)

    Vasil'eva, S V; Makhova, E V; Moshkovskaia, E Iu

    1999-04-01

    The expression of genes belonging to the Ada regulon of Escherichia coli under the action of mono- and bifunctional alkylating agents--high-efficiency antitumor HMM, ACNU, and BCNU preparations--was studied. The functional specificity of the alkA, alkB, and aidB1 genes concerning both the structure and volume of DNA alkylation and the specificity of cell preadaptation was revealed. Additional experimental evidence for the role of the aidB1 gene as a unique "hazard gene", a component of the E. coli ada operon, was obtained. A phenomenon of positive interference between alternative SOS and Ada responses was observed for the first time upon gene expression.

  8. Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.

    Science.gov (United States)

    Cui, Yanbing; Meng, Yiwei; Zhang, Juan; Cheng, Bin; Yin, Huijia; Gao, Chao; Xu, Ping; Yang, Chunyu

    2017-01-01

    In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli.

    Science.gov (United States)

    Kulmala, Antti; Huovinen, Tuomas; Lamminmäki, Urpo

    2017-06-19

    Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.

  10. Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by Recombinant E. coli

    OpenAIRE

    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 s...

  11. Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

    OpenAIRE

    Rossi, Luciana; Di Giancamillo, Alessia; Reggi, Serena; Domeneghini, Cinzia; Baldi, Antonella; Sala, Vittorio; Dell'Orto, Vittorio; Coddens, Annelies; Cox, Eric; Fogher, Corrado

    2013-01-01

    Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and ...

  12. TROUBLESHOOTING IN EXPRESSION AND PURIFICATION OF RECOMBINANT SEVERE ACUTE RESPIRATORY SYNDROME-ASSOCIATED CORONAVIRUS NUCLEOCAPSID PROTEIN IN Escherichia coli BL21

    Directory of Open Access Journals (Sweden)

    Budiman Bela

    2010-11-01

    Full Text Available Considering importance of N protein for study of viral pathogenesis or development of immunodiagnostic assay, wereported effects of several conditions on purity and homogeneity of recombinant SARS-CoV N protein expressed in E.coli BL21. The SARS-CoV N gene was reverse transcribed and amplified by the reverse transcription-polymerase chainreaction (RT-PCR technique. The amplicons were cloned into pGEX-6P1 and followed by subcloning of the targetgene into pQE-80L. After inserting the recombinant plasmid (pQE80-N into E. coli, the recombinant protein (6 x Histag-N protein fusion was expressed by inducing the bacterial cells with 0.1-0.5 mM isopropyl-1-thio-Dgalactopyranoside(IPTG for 1-5 h. The protein recombinant were extracted from the bacterial cells by NTT buffercontaining 0-20 mM imidazol, and followed by Ni-NTA affinity resin purification. The results showed that induction ofE. coli BL21 with 0.2 mM IPTG for 4 h and followed with lysis of bacterial cells in NTT buffer containing 10 mMimidazol were optimal conditions to obtain the pure recombinant SARS-CoV N protein.

  13. A empiric expression to interpret the approximation of λ cI phages to E. coli C600 bacteria

    International Nuclear Information System (INIS)

    Garces, F.; Vidania, R. de

    1984-01-01

    In general the process of adsorption of phages to bacteria is considered in the bibliography as an statistical process. In this work we use an empiric expression which allows to interpret the approximation of λcI pages to E. coli C 6 00 bacteria. This expression introduces some changes respect to a pure statistical description of the approximation process. (Author) 26 refs

  14. 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.

  15. The approaches to mathematical modeling of recA, umuD genes expression in bacteria Escherichia coli after UV-irradiation

    International Nuclear Information System (INIS)

    Belov, O.V.

    2006-01-01

    The modern data of recA, umuD genes expression of the system of SOS-repair at classical object of radiation genetic researches - bacteria Escherichia coli, after ultraviolet irradiation are presented. Essentially a new method of analysis of SOS-genes expression is considered. It was shown that using this method it is possible to determine the character of induction of some SOS-genes more precisely. The possible approach to the mathematical description of SOS-response of cells by construction of the system of the differential equations is presented

  16. Differential gene expression and adherence of Escherichia coli O157:H7 in vitro and in ligated pig intestines.

    Directory of Open Access Journals (Sweden)

    Xianhua Yin

    Full Text Available BACKGROUND: Escherichia coli O157:H7 strain 86-24 grown in MacConkey broth (MB shows almost no adherence to cultured epithelial cells but adheres well in pig ligated intestines. This study investigated the mechanisms associated with the difference between in-vitro and in-vivo adherence of the MB culture. METHODOLOGY/PRINCIPAL FINDINGS: It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR. Expression of selected virulence-related genes associated with adherence and CCR was then examined by quantitative PCR. When bacteria were grown in MB and Brain Heart Infusion with NaHCO(3 (BHIN plus lactose, pH was reduced to 5.5-5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP, and two negative regulators of the LEE, gadE and hfq. Putative virulence genes stcE, hlyA, ent and nleA were also decreased in vitro. Reversal of these changes was noted for bacteria recovered from the intestine, where transcripts for qseF and fis and putative virulence factors AidA(15, TerC and Ent/EspL2 were significantly increased, and transcripts for AIDA(48, Iha, UreC, Efa1A, Efa1B, ToxB, EhxA, StcE, NleA and NleB were expressed at high levels. CONCLUSIONS/SIGNIFICANCE: Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo. CCR and/or acidic pH may have played a role in repression of the LEE genes. Bacterial pathogens need to integrate their nutritional metabolism with expression of virulence genes but little is known of how this is done in E. coli O157:H7. This study indicates one aspect of the subject that should be investigated further.

  17. Identification and quantification of N alpha-acetylated Y. pestis fusion protein F1-V expressed in Escherichia coli using LCMS E.

    Science.gov (United States)

    Bariola, Pauline A; Russell, Brett A; Monahan, Steven J; Stroop, Steven D

    2007-05-31

    N-terminal acetylation in E coli is a rare event catalyzed by three known N-acetyl-transferases (NATs), each having a specific ribosomal protein substrate. Multiple, gram-scale lots of recombinant F1-V, a fusion protein constructed from Y. Pestis antigens, were expressed and purified from a single stably transformed E. coli cell bank. A variant form of F1-V with mass increased by 42-43 Da was detected in all purified lots by electrospray orthogonal acceleration time-of-flight mass spectrometry (MS). Peptide mapping LCMS localized the increased mass to an N-terminal Lys-C peptide, residues 1-24, and defined it as +42.0308+/-0.0231 Da using a LockSpray exact mass feature and a leucine enkaphalin mass standard. Sequencing of the variant 1-24 peptide by LCMS and high-energy collision induced dissociation (LCMS(E)) further localized the modification to the amino terminal tri-peptide ADL and identified the modification as N(alpha)-acetylation. The average content of N(alpha)-acetylated F1-V in five lots was 24.7+/-2.6% indicating that a stable acetylation activity for F1-V was established in the E. coli expression system. Alignment of the F1-V N-terminal sequence with those of other known N(alpha)-acetylated ectopic proteins expressed in E. coli reveals a substrate motif analogous to the eukaryote NatA' acetylation pathway and distinct from endogenous E. coli NAT substrates.

  18. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Kenyon, C.J.; Walker, G.C.

    1988-05-01

    Operon fusions in Escherichia coli were obtained that showed increased beta-galactosidase expression in response to treatment with the DNA-damaging agent mitomycin C. These fusions were generated by using the Mud(ApR, lac) vector to insert the lactose structural genes randomly into the bacterial chromosome. Induction of beta-galactosidase in these strains, which carried fusions of lac to these din (damage-inducible) loci, was (i) triggered by UV light as well as by mitomycin C and (ii) abolished by either a recA- or a lexA- mutation. Similar characteristics of induction were observed when the lactose genes were fused to a prophage lambda promoter by using Mud(ApR, lac). These results indicate that E. coli contains a set of genes that, like prophage lambda genes, are expressed in response to DNA-damaging agents and regulated by the recA and lexA gene products. These din genes map at five bacterial loci. One din::Mud(ApR, lac) insertion results in a UV-sensitive phenotype and may be within the uvrA transcriptional unit.

  19. 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.

  20. Vaccine development against the Taenia solium parasite: the role of recombinant protein expression in Escherichia coli.

    Science.gov (United States)

    Gauci, Charles; Jayashi, César; Lightowlers, Marshall W

    2013-01-01

    Taenia solium is a zoonotic parasite that causes cysticercosis. The parasite is a major cause of human disease in impoverished communities where it is transmitted to humans from pigs which act as intermediate hosts. Vaccination of pigs to prevent transmission of T. solium to humans is an approach that has been investigated to control the disease. A recombinant vaccine antigen, TSOL18, has been remarkably successful at reducing infection of pigs with T. solium in several experimental challenge trials. The vaccine has been shown to eliminate transmission of naturally acquired T. solium in a field trial conducted in Africa. We recently reported that the vaccine was also effective in a field trial conducted in Peru. The TSOL18 recombinant antigen for each of these trials has been produced by expression in Escherichia coli. Here we discuss research that has been undertaken on the TSOL18 antigen and related antigens with a focus on improved methods of preparation of recombinant TSOL18 and optimized expression in Escherichia coli.

  1. Cloning and expression in Escherichia coli of cellulases genes from Clostridium IBUN 22A

    Directory of Open Access Journals (Sweden)

    Lucy Carolina Vargas Pabón

    2002-01-01

    Full Text Available Genomic library of the native strain Clostridium IBUN 22A was constructed, using plasmid pBluescriptlI® KS+/ - as cloning vector and its expression in Escherichia coli was evaluated. Eight recombination clones with enzymatic activity were detected by enzymatic screening and using the red-Congo test with three substrates: cellobiose, carboxymethyl cellulose (CMC and cellulose powder (native. Restriction analysis of three recombination plasmids, representative of each enzymatic activity showed the inserted size (1600, 13000 and 11000bp approximately for pBS68, pBS25 and pBS57 respectively. More studies of protein expression and enzymatic characterization will allow theses enzymes and other typical parameters to be defined. In the same way the fragment sequence cloned will lead to a more detailed analysis and definition of the biotechnological potential of this strain regarding solvent production using cellulosic substrates for fermentation.

  2. A Structural Study of Escherichia coli Cells Using an In Situ Liquid Chamber TEM Technology

    Directory of Open Access Journals (Sweden)

    Yibing Wang

    2015-01-01

    Full Text Available Studying cell microstructures and their behaviors under living conditions has been a challenging subject in microbiology. In this work, in situ liquid chamber TEM was used to study structures of Escherichia coli cells in aqueous solutions at a nanometer-scale resolution. Most of the cells remained intact under electron beam irradiation, and nanoscale structures were observed during the TEM imaging. The analysis revealed structures of pili surrounding the E. coli cells; the movements of the pili in the liquid were also observed during the in situ tests. This technology also allowed the observation of features of the nucleoid in the E. coli cells. Overall, in situ TEM can be applied as a valuable tool to study real-time microscopic structures and processes in microbial cells residing in native aqueous solutions.

  3. Following Drug Uptake and Reactions inside Escherichia coli Cells by Raman Microspectroscopy

    Science.gov (United States)

    2015-01-01

    Raman microspectroscopy combined with Raman difference spectroscopy reveals the details of chemical reactions within bacterial cells. The method provides direct quantitative data on penetration of druglike molecules into Escherichia coli cells in situ along with the details of drug–target reactions. With this label-free technique, clavulanic acid and tazobactam can be observed as they penetrate into E. coli cells and subsequently inhibit β-lactamase enzymes produced within these cells. When E. coli cells contain a β-lactamase that forms a stable complex with an inhibitor, the Raman signature of the known enamine acyl–enzyme complex is detected. From Raman intensities it is facile to measure semiquantitatively the number of clavulanic acid molecules taken up by the lactamase-free cells during growth. PMID:24901294

  4. Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

    Directory of Open Access Journals (Sweden)

    Nannenga Brent L

    2011-12-01

    Full Text Available Abstract Background Membrane proteins (MPs populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. Results Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells, toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host. Conclusions Our system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

  5. 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.

  6. 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.

  7. Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli.

    Science.gov (United States)

    Wu, Hong; Chen, Jinchun; Chen, Guo-Qiang

    2016-12-01

    E. coli JM109∆envC∆nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109∆minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109∆envC∆nlpD and E. coli JM109∆minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109∆minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109∆envC∆nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.

  8. Oxygen-Dependent Cell-to-Cell Variability in the Output of the Escherichia coli Tor Phosphorelay.

    Science.gov (United States)

    Roggiani, Manuela; Goulian, Mark

    2015-06-15

    Escherichia coli senses and responds to trimethylamine-N-oxide (TMAO) in the environment through the TorT-TorS-TorR signal transduction system. The periplasmic protein TorT binds TMAO and stimulates the hybrid kinase TorS to phosphorylate the response regulator TorR through a phosphorelay. Phosphorylated TorR, in turn, activates transcription of the torCAD operon, which encodes the proteins required for anaerobic respiration via reduction of TMAO to trimethylamine. Interestingly, E. coli respires TMAO in both the presence and absence of oxygen, a behavior that is markedly different from the utilization of other alternative electron acceptors by this bacterium. Here we describe an unusual form of regulation by oxygen for this system. While the average level of torCAD transcription is the same for aerobic and anaerobic cultures containing TMAO, the behavior across the population of cells is strikingly different under the two growth conditions. Cellular levels of torCAD transcription in aerobic cultures are highly heterogeneous, in contrast to the relatively homogeneous distribution in anaerobic cultures. Thus, oxygen regulates the variance of the output but not the mean for the Tor system. We further show that this oxygen-dependent variability stems from the phosphorelay. Trimethylamine-N-oxide (TMAO) is utilized by numerous bacteria as an electron acceptor for anaerobic respiration. In E. coli, expression of the proteins required for TMAO respiration is tightly regulated by a signal transduction system that is activated by TMAO. Curiously, although oxygen is the energetically preferred electron acceptor, TMAO is respired even in the presence of oxygen. Here we describe an interesting and unexpected form of regulation for this system in which oxygen produces highly variable expression of the TMAO utilization proteins across a population of cells without affecting the mean expression of these proteins. To our knowledge, this is the first reported example of a stimulus

  9. [Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

    Science.gov (United States)

    Yue, Chen-Li; Shi, Jie-Ran; Shi, Chang-Hong; Zhang, Hai; Zhao, Lei; Zhang, Ting-Fen; Zhao, Yong; Xi, Li

    2008-10-01

    To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

  10. 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

  11. Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3

    Energy Technology Data Exchange (ETDEWEB)

    Scalise, Mariafrancesca; Galluccio, Michele; Pochini, Lorena [Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende (Italy); Indiveri, Cesare, E-mail: indiveri@unical.it [Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende (Italy)

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer mOCTN3 transport protein has been cloned in pET-21a(+) and over-expressed in Escherichia coli. Black-Right-Pointing-Pointer The expressed mOCTN3 has been purified to homogeneity by Ni-chelating chromatography. Black-Right-Pointing-Pointer The protein solubilised in Triton X-100 has been reconstituted in liposomes. Black-Right-Pointing-Pointer Recombinant mOCTN3 catalyses transport of carnitine by a uniport mode. -- Abstract: pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni{sup 2+}-chelating chromatography with a yield of 2 mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [{sup 3}H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K{sub m} for carnitine was 36 {mu}M. [{sup 3}H]carnitine transport was inhibited by carnitine analogues and some Cys and NH{sub 2} reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.

  12. Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3

    International Nuclear Information System (INIS)

    Scalise, Mariafrancesca; Galluccio, Michele; Pochini, Lorena; Indiveri, Cesare

    2012-01-01

    Highlights: ► mOCTN3 transport protein has been cloned in pET-21a(+) and over-expressed in Escherichia coli. ► The expressed mOCTN3 has been purified to homogeneity by Ni-chelating chromatography. ► The protein solubilised in Triton X-100 has been reconstituted in liposomes. ► Recombinant mOCTN3 catalyses transport of carnitine by a uniport mode. -- Abstract: pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni 2+ -chelating chromatography with a yield of 2 mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [ 3 H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K m for carnitine was 36 μM. [ 3 H]carnitine transport was inhibited by carnitine analogues and some Cys and NH 2 reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.

  13. 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.

  14. Enhancement of crystallinity of cellulose produced by Escherichia coli through heterologous expression of bcsD gene from Gluconacetobacter xylinus.

    Science.gov (United States)

    Sajadi, Elaheh; Babaipour, Valiollah; Deldar, Ali Asghar; Yakhchali, Bagher; Fatemi, Seyed Safa-Ali

    2017-09-01

    To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001. The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD. The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

  15. Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity

    Science.gov (United States)

    Kacena, M. A.; Smith, E. E.; Todd, P.

    1999-01-01

    The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

  16. Effect of radiation doses rate on SOS response induction in irradiated Escherichia coli Cells

    International Nuclear Information System (INIS)

    Cuetara Lugo, Elizabeth B.; Fuentes Lorenzo, Jorge L.; Almeida Varela, Eliseo; Prieto Miranda, Enrique F.; Sanchez Lamar, Angel; Llagostera Casal, Montserrat

    2005-01-01

    The present work is aimed to study the effect of radiation dose rate on the induction of SOS response in Escherichia coli cells. We measured the induction of sul A reporter gene in PQ-37 (SOS Chromotest) cells. Lead devises were built with different diameter and these were used for diminishing the dose rate of PX- -30M irradiator. Our results show that radiation doses rate significantly modifies the induction of SOS response. Induction factor increases proportionally to doses rate in Escherichia coli cells defective to nucleotide excision repair (uvrA), but not in wild type cells. We conclude that the dose rate affects the level of induction of SOS response

  17. Photoreactivable sector of lethal damage in ultraviolet-irradiated Escherichia coli cells

    International Nuclear Information System (INIS)

    Balgavy, P.

    1976-01-01

    The photoreactivable sector of lethal damage in Escherichia coli Bsub(s-1), Escherichia coli B/r Hcr - and Escherichia coli B/r Hcr + cells after ultraviolet irradiation at 254 nm is 0.823 +- 0.004, 0.70 +- 0.01 and 0.53 +- 0.06, respectively, at 99% confidence limits. For the low values of the photoreactivable sector in the B/r Hcr - and B/r Hcr + strains are likely to be responsible dark repair processes which eliminate lethal damage, brought about by pyrimidine dimers, preferably in comparison with lethal damage caused by photoproducts of another type. (author)

  18. Gene expression of Escherichia coli in continuous culture during adaptation to artificial sunlight.

    Science.gov (United States)

    Berney, Michael; Weilenmann, Hans-Ulrich; Egli, Thomas

    2006-09-01

    Escherichia coli growing in continuous culture under continuous UVA irradiation exhibits growth inhibition with a subsequent adaptation to the stress. Transcriptome analysis was performed during transient growth inhibition and in the UVA light-adapted growth state. The results indicate that UVA light induces stringent response and an additional response that includes the upregulation of the synthesis of valine, isoleucine, leucine, phenylalanine, histidine and glutamate. The induction of several SOS response-genes strongly points to DNA damage as a result of UVA exposure. The involvement of oxidative stress was observed with the induction of ahpCF. Taken together it supports the hypothesis of the production of reactive oxygen species by UVA light. In the UVA-adapted cell population strong repression of the acid tolerance response was found. We identified the enzyme chorismate mutase as a possible chromophore for UVA light-inactivation and found strong repression of the pyrBI operon and the gene mgtA encoding for an ATP-dependent Mg2+ transporter. Furthermore, our results indicate that the role of RpoS may not be as important in the adaptation of E. coli to UVA light as it was implicated by previous results with starved cells, but that RpoS might be of crucial importance for the resistance under transient light exposure.

  19. Induction of genetic recombination in the lambda bacteriophage by ultraviolet radiation of the Escherichia Coli cells; Induccion de recombinacion genetica en el bacteriofago lambda por irradiacion ultravioleta de las cellulas de Escherichia Coli

    Energy Technology Data Exchange (ETDEWEB)

    Alcantara D, D. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    1986-12-15

    In this work there are reported the results that show that although the stimulation of the recombination of the Lambda bacteriophage, by UV irradiation of the cells of Escherichia Coli, it looks to be the result of the high expression of the functions of the SOS system, doesn't keep some relationship with the high concentration of protein reached RecA. (Author)

  20. Epithelial Cell Adherence Mediated by the Enterotoxigenic Escherichia coli Tia Protein

    OpenAIRE

    Mammarappallil, Joseph G.; Elsinghorst, Eric A.

    2000-01-01

    In vitro studies have shown that enterotoxigenic Escherichia coli (ETEC) strains are capable of invading cultured epithelial cells derived from the human ileum and colon. Two separate invasion loci (tia and tib) have previously been isolated from the classical ETEC strain H10407. The tia locus has been shown to direct the synthesis of Tia, a 25-kDa outer membrane protein. Tia is sufficient to confer the adherence and invasion phenotypes on laboratory stains of E. coli, suggesting that this pr...

  1. Adherence of Enterohemorrhagic Escherichia coli to Human Epithelial Cells: The Role of Intimin

    Science.gov (United States)

    1995-04-28

    mucosa (e.g., enterotoxigenic E. coli, Vibrio cholerae , and Boroetella pertussis); ii) damage to the epithelial cell microvilli induced by the...diarrhea in Mayan childm in Mexico . J. Infect. Dis. 163, 507-513. G6mez-Ouarte, O.G. and Kaper, J.B. (1995). A plasmid-encoded regulartory region...de la Cabaca, F., and Garibay, E.V. (1987). Enteroadherent Escherichia coli as a cause of diarrhea among children in Mexico . J . Clin. Microbiol. 25

  2. A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli.

    Science.gov (United States)

    Xu, Li; Song, Xiaoyu; Jia, Lingyun

    2017-11-01

    Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  3. Controlling Citrate Synthase Expression by CRISPR/Cas9 Genome Editing for n-Butanol Production in Escherichia coli

    DEFF Research Database (Denmark)

    Heo, Min-Ji; Jung, Hwi-Min; Um, Jaeyong

    2017-01-01

    Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively produce n-butanol in a defined medium under microaerobic condition. The butanol synthetic pathway genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in metabolically...... prediction program, UTR designer, and modified using the CRISPR/Cas9 genome editing method to reduce its expression level. E. coli strains with decreased citrate synthase expression produced more butanol and the citrate synthase activity was correlated with butanol production. These results demonstrate...

  4. Global gene expression profiling of the asymptomatic bacteriuria Escherichia coli strain 83972 in the human urinary tract

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) are an important health problem worldwide, with many million cases each year. Escherichia coli is the most common organism causing UTIs in humans. The asymptomatic bacteriuria E. coli strain 83972 is an excellent colonizer of the human urinary tract, where it causes...... long-term bladder colonization. The strain has been used for prophylactic purposes in patients prone to more severe and recurrent UTIs. For this study, we used DNA microarrays to monitor the expression profile of strain 83972 in the human urinary tract. Significant differences in expression levels were...

  5. Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Hon-Yeung Cheung

    Full Text Available Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of gram-positive and gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.

  6. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Choi Yoo

    2012-10-01

    Full Text Available Abstract Background In nature, mussel adhesive proteins (MAPs show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate

  7. Vascular Endothelial Growth Factor Receptor 1 Contributes to Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway▿ †

    OpenAIRE

    Zhao, Wei-Dong; Liu, Wei; Fang, Wen-Gang; Kim, Kwang Sik; Chen, Yu-Hua

    2010-01-01

    Escherichia coli is the most common Gram-negative organism causing neonatal meningitis. Previous studies demonstrated that E. coli K1 invasion of brain microvascular endothelial cells (BMEC) is required for penetration into the central nervous system, but the microbe-host interactions that are involved in this process remain incompletely understood. Here we report the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) expressed on human brain microvascular endothelial cells...

  8. Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

    Directory of Open Access Journals (Sweden)

    Muyrers Joep PP

    2003-01-01

    Full Text Available Abstract Background The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. Results Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα but only requires a phage annealing protein (RecT or Redβ. Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. Conclusion Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

  9. Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis.

    Science.gov (United States)

    Zhao, Xiao-wei; Yang, Yong-xin; Huang, Dong-wei; Cheng, Guang-long; Zhao, Hui-ling

    2015-01-01

    Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and a-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.

  10. Influence of bromouracil density labelling on viability of UV irradiated Escherichia coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Brozmanova, J [Slovenska Akademia Vied, Bratislava (Czechoslovakia). Vyskumny Ustav Onkologicky

    1976-01-01

    Influence of 5-bromouracil cultivation on cell viability and DNA synthesis in the Escherichia coli B/r thy/sup -/ trp/sup -/ Hcr/sup +/ and Escherichia coli C thy-321 strains was followed. It was found that a 120 min cultivation in the bromouracil medium (unirradiated cells) does not essentially influence the viability of the two investigated strains but has an inhibitory effect on DNA synthesis in cells of the E. coli B/r Hcr/sup +/ strain. However, cultivation with bromouracil after ultraviolet irradiation leads to a decreased surviving ability of the irradiated cells of both investigated strains. Repair of damage induced by ultraviolet radiation probably exhausts a considerable proportion of repair activity, so that additional injury produced by bromouracil cultivation cannot be liquidated immediately.

  11. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, L.D., E-mail: yuld@thep-center.org [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Sangwijit, K. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Chiang Mai 50290 (Thailand); Phanchaisri, B. [Institute of Science and Technology Research, Chiang Mai University, Chiang Mai 50200 (Thailand); Thongkumkoon, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thopan, P. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Singkarat, S. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Anuntalabhochai, S. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2014-05-01

    Highlights: • Ion bombardment could induce DNA transfer into E. coli cells. • The DNA transfer induction depended on ion energy and fluence. • The mechanism was associated with the bacterial cell envelope structure. • A mechanism phase diagram was proposed to summarize the mechanism. - Abstract: As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10–20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence.

  12. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    International Nuclear Information System (INIS)

    Yu, L.D.; Sangwijit, K.; Prakrajang, K.; Phanchaisri, B.; Thongkumkoon, P.; Thopan, P.; Singkarat, S.; Anuntalabhochai, S.

    2014-01-01

    Highlights: • Ion bombardment could induce DNA transfer into E. coli cells. • The DNA transfer induction depended on ion energy and fluence. • The mechanism was associated with the bacterial cell envelope structure. • A mechanism phase diagram was proposed to summarize the mechanism. - Abstract: As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10–20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence

  13. EXPRESSION OF A BEE-VENOM PHOSPHOLIPASE A2 FROM APIS CERANA CERANA IN E,.qCHERICHIA COLI

    Institute of Scientific and Technical Information of China (English)

    Li-rongShen; Jia-anCheng; Chuan-xiZhang

    2004-01-01

    The venomous phospholipase A2 (AcPLA2) coding reading region of the Chinese honeybee (Apis cerana cerana), which is composed of 405 bp encoding a mature glycosylated peptide with 134 amino residues was transformed into the expression vector pETblue-1. Then the recombinant vector was introduced into Escherichia coli Tuner (DE3) plac I for expression. Analysis result of SDS-PAGE showed that the expression products had a protein band of about 15 kD. Detection of western blot using ant-European honeybee (Apis mellifera) phospholipase A2 (AmPLA2) polyclonal serum as the first antibody showed that the expression products appeared a special blot same as the native AmPLA2.The result demonstrated that the AcPLA2 peptide had been expressed in E. coli and the AcPLA2 has the similar antigenicity as the AmPLA2.

  14. Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3).

    Science.gov (United States)

    Chen, Hui; Huang, Rui; Zhang, Y-H Percival

    2017-06-01

    The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).

  15. Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Pohl Martina

    2010-10-01

    Full Text Available Abstract Background The benzoylformate decarboxylase (BFD from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS. Results Although the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished. Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro had typical OTR curves, the second type (clones expressing the wild type BFD, Ser181Thr or His281Ala showed an early drop of OTR in LB and TB medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone expressing Leu476Gln behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor binding strength of the different BFD-variants correlated with the differences

  16. Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

    International Nuclear Information System (INIS)

    Castro, Miguel E.; Molina, Roberto C.; Diaz, Waldo A.; Pradenas, Gonzalo A.; Vasquez, Claudio C.

    2009-01-01

    Potassium tellurite (K 2 TeO 3 ) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

  17. 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...... to CK2 from human. Kinetic measurements of the recombinant maize holoenzyme (rmCK2) revealed k(cat) values for ATP and GTP of 4 and 2s(-1), respectively; whereas the recombinant maize catalytic subunit showed almost equal values for ATP and GTP, i.e., ca. 0.8s(-1). A comparison of the k(cat)/K(m) ratio...

  18. Effect of simulated microgravity on E. coli K12 MG1655 growth and gene expression

    Data.gov (United States)

    National Aeronautics and Space Administration — This study demonstrates simulated microgravity effects on E. coli K 12 MG1655 when grown on LB medium supplemented with glycerol. The results imply that E. coli...

  19. Experimental validation of the predicted binding site of Escherichia coli K1 outer membrane protein A to human brain microvascular endothelial cells: identification of critical mutations that prevent E. coli meningitis.

    Science.gov (United States)

    Pascal, Tod A; Abrol, Ravinder; Mittal, Rahul; Wang, Ying; Prasadarao, Nemani V; Goddard, William A

    2010-11-26

    Escherichia coli K1, the most common cause of meningitis in neonates, has been shown to interact with GlcNAc1-4GlcNAc epitopes of Ecgp96 on human brain microvascular endothelial cells (HBMECs) via OmpA (outer membrane protein A). However, the precise domains of extracellular loops of OmpA interacting with the chitobiose epitopes have not been elucidated. We report the loop-barrel model of these OmpA interactions with the carbohydrate moieties of Ecgp96 predicted from molecular modeling. To test this model experimentally, we generated E. coli K1 strains expressing OmpA with mutations of residues predicted to be critical for interaction with the HBMEC and tested E. coli invasion efficiency. For these same mutations, we predicted the interaction free energies (including explicit calculation of the entropy) from molecular dynamics (MD), finding excellent correlation (R(2) = 90%) with experimental invasion efficiency. Particularly important is that mutating specific residues in loops 1, 2, and 4 to alanines resulted in significant inhibition of E. coli K1 invasion in HBMECs, which is consistent with the complete lack of binding found in the MD simulations for these two cases. These studies suggest that inhibition of the interactions of these residues of Loop 1, 2, and 4 with Ecgp96 could provide a therapeutic strategy to prevent neonatal meningitis due to E. coli K1.

  20. Mutations at Several Loci Cause Increased Expression of Ribonucleotide Reductase in Escherichia coli

    Science.gov (United States)

    Feeney, Morgan Anne; Ke, Na

    2012-01-01

    Production of deoxyribonucleotides for DNA synthesis is an essential and tightly regulated process. The class Ia ribonucleotide reductase (RNR), the product of the nrdAB genes, is required for aerobic growth of Escherichia coli. In catalyzing the reduction of ribonucleotides, two of the cysteines of RNR become oxidized, forming a disulfide bond. To regenerate active RNR, the cell uses thioredoxins and glutaredoxins to reduce the disulfide bond. Strains that lack thioredoxins 1 and 2 and glutaredoxin 1 do not grow because RNR remains in its oxidized, inactive form. However, suppressor mutations that lead to RNR overproduction allow glutaredoxin 3 to reduce sufficient RNR for growth of these mutant strains. We previously described suppressor mutations in the dnaA and dnaN genes that had such effects. Here we report the isolation of new mutations that lead to increased levels of RNR. These include mutations that were not known to influence production of RNR previously, such as a mutation in the hda gene and insertions in the nrdAB promoter region of insertion elements IS1 and IS5. Bioinformatic analysis raises the possibility that IS element insertion in this region represents an adaptive mechanism in nrdAB regulation in E. coli and closely related species. We also characterize mutations altering different amino acids in DnaA and DnaN from those isolated before. PMID:22247510

  1. Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip

    DEFF Research Database (Denmark)

    Matos, T.; Senkbeil, Silja; Mendonça, A.

    2013-01-01

    technique has been developed which is based on exposing E. coli cells to low voltages to allow extraction of nucleic acids and proteins. The flow-through electropermeability chip used consists of a microfluidic channel with integrated gold electrodes that promote cell envelope channel formation at low...

  2. Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells

    NARCIS (Netherlands)

    Roubos-van den Hil, P.J.; Nout, M.J.R.; Beumer, R.R.; Meulen, van der J.; Zwietering, M.H.

    2009-01-01

    Aims: This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and

  3. Colocalization and interaction between elongasome and divisome during a preparative cell division phase in Escherichia coli

    NARCIS (Netherlands)

    Ploeg, van der R.; Verheul, J.; Vischer, N.O.E.; Alexeeva, S.V.; Hoogendoorn, E.; Postma, M.; Banzhaf, M.; Vollmer, W.; Blaauwen, den T.

    2013-01-01

    The rod-shaped bacterium Escherichia coli grows by insertion of peptidoglycan into the lateral wall during cell elongation and synthesis of new poles during cell division. The monofunctional transpeptidases PBP2 and PBP3 are part of specialized protein complexes called elongasome and divisome,

  4. Induction of sos response in Escherichia Coli cells by gamma rays

    International Nuclear Information System (INIS)

    Fuentes Lorenzo, J.L.; Padron Soler, E.; Martin Hernandez, G.; Perez Tamayo, N.; del Sol Abascal, E.R.; Almeida Varela, E.

    1996-01-01

    The kinetics of sos response induction in Escherichia Coli cells was studied by means of the gene fusion SfiA:LacZ. In these cells, the specific beta galactosidase activity and the cellular growth rate showed an exponential behaviour. The sensitivity of the GC 2181 starin to gamma irradiation is equal to Do -1= 0.00088/Gy. The beta galactosidase activity

  5. Cell extracts of propionic acid bacteria reactivate cells of Escherichia coli inactivated by ultraviolet radiation

    International Nuclear Information System (INIS)

    Vorob'eva, L.I.; Nikitenko, G.V.; Khodzhaev, E.Yu.; Ponomareva, G.M.

    1994-01-01

    Cell extracts of three Propionibacterium shermanii strains were shown to exert a reactivating effect on cells of E. coli AB 1157 inactivated by ultraviolet radiation. The reactivating effect was revealed after both preincubation and postincubation of the irradiated cells with the extracts. The effect increased with a decrease of the survival rate within the range of 1.8-0.006%. The protective factor (or factors) is dialyzable and thermolabile; it was detected both in the fraction of soluble proteins and in the fraction of nucleoproteins and nucleic acids. The protective properties of dialyzate disappear after incubation with proteinase K and trypsin, decrease after incubation with α-amylase, deoxyribonuclease-1, or ribonuclease, and do not change under the influence of lipase. The reactivating factor is believed to be of a polypeptide nature

  6. Repeated batch fermentation of immobilized E. coli expressing Vitreoscilla hemoglobin for long-term use.

    Science.gov (United States)

    Sar, Taner; Seker, Gamze; Erman, Ayse Gokce; Stark, Benjamin C; Yesilcimen Akbas, Meltem

    2017-09-03

    This study describes an efficient and reusable process for ethanol production from medium containing whey powder, using alginate immobilized ethanologenic E. coli strains either expressing (TS3) or not expressing (FBR5) Vitreoscilla hemoglobin. Reuseabilities of the FBR5 and TS3 strains were investigated regarding their ethanol production capacities over the course of 15 successive 96-h batch fermentations. The ethanol production was fairly stable over the entire duration of the experiment, with strain TS3 maintaining a substantial advantage over strain FBR5. Storage of both strains in 2 different solutions for up to 60 d resulted in only a modest loss of ethanol production, with strain TS3 consistently outperforming strain FBR5 by a substantial amount. Strains stored for 15 or 30 d maintained their abilities to produce ethanol without dimunition over the course of 8 successive batch fermentations; again strain TS3 maintained a substantial advantage over strain FBR5 throughout the entire experiment. Thus, immobilization is a useful strategy to maintain the advantage in ethanol productivity afforded by expression of Vitreoscilla hemoglobin over long periods of time and large numbers of repeated batch fermentations, including, as in this case, using media with food processing wastes as the carbon source.

  7. High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.

    Directory of Open Access Journals (Sweden)

    Yao Nie

    Full Text Available Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3/pET-20b(+-pul and E. coli BL21(DE3/pET-22b(+-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3/pET-20b(+-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3/pET-22b(+-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3/pET-22b(+-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The

  8. Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

    International Nuclear Information System (INIS)

    Sedliakova, M.; Slezarikova, V.; Brozmanova, J.; Masek, F.; Bayerova, V.

    1980-01-01

    3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7, proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival. We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP + ). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells given with potentially lethal doses of UV irradiation. (orig.)

  9. Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

    Science.gov (United States)

    Gąciarz, Anna; Khatri, Narendar Kumar; Velez-Suberbie, M Lourdes; Saaranen, Mirva J; Uchida, Yuko; Keshavarz-Moore, Eli; Ruddock, Lloyd W

    2017-06-15

    The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

  10. Reactivity of some mammalian sera with the bovine leukaemia virus env gene polypeptide expressed in Escherichia coli

    International Nuclear Information System (INIS)

    Slavikova, K.; Zajac, V.

    1989-01-01

    Sera from bovine leukaemia virus (BLV)-infected cattle and sheep were tested by radioimmunoassay and Western blot for their reactivity with 60,000 protein coded by the env gene of BLV and expressed in Escherichia coli. This protein, antigenically similar to BLV protein, reacted with antibodies against BLV antigens in the sera tested. (author). 3 figs., 1 tab., 13 refs

  11. Quantification of contamination of lettuce by GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium

    NARCIS (Netherlands)

    Franz, Eelco; Visser, Anna A; Van Diepeningen, Anne D; Klerks, Michel M; Termorshuizen, Aad J; van Bruggen, Ariena H C

    The primary objective of this study was to determine the possibility of internalization of GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. Typhimurium) strains MAE 110 (multi-cellular morphology) and 119 (wild type morphology) into lettuce seedlings (Lactuca

  12. Improving the expression of recombinant proteins in E. coli BL21 (DE3) under acetate stress: an alkaline pH shift approach.

    Science.gov (United States)

    Wang, Hengwei; Wang, Fengqing; Wang, Wei; Yao, Xueling; Wei, Dongzhi; Cheng, Hairong; Deng, Zixin

    2014-01-01

    Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5-8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0 ± 0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

  13. A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Mengli [National Institutes of Health, Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Huang, Ying; Yang, Renbin; Craigie, Robert, E-mail: robertc@niddk.nih.gov [National Institutes of Health, Laboratories of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Clore, G. M., E-mail: mariusc@mail.nih.gov [National Institutes of Health, Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2016-10-15

    We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D{sub 2}O usage tenfold and d{sub 7}-glucose usage by 30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD{sub 600} of up to 10. Inducing the cells with isopropyl β-d-1-thiogalactopyranoside at an OD{sub 600} of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD{sub 600} between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E. coli from H{sub 2}O-based to D{sub 2}O-based medium is also key for ensuring high levels of protein expression in D{sub 2}O. We find that a simple three-step adaptation approach—Luria–Bertani (LB) medium in H{sub 2}O to LB in D{sub 2}O to modified-M9 medium in D{sub 2}O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.

  14. Comparison of mutagenic efficiency of decay of 32P incorporated in E.Coli WP-2 and E.Coli WP-2S cells

    International Nuclear Information System (INIS)

    Pluciennik, H.

    1975-01-01

    32 P-labelled Escherichia coli WP-2 and Escherichia coli WP-2S cells were stored at -196 0 . The lethal effect induced by 32 P decay was equal in both strains. Lethal efficiency of 32 P→ 32 S transmutation in DNA amounted to 0.046. Reversion try→try + were induced with a ten times higher efficiency in UV-sensitive strain WP-2S, as compared with strain WP-2. (author)

  15. Comparison of mutagenic efficiency of decay of /sup 32/P incorporated in E. Coli WP-2 and E. Coli WP-2S cells

    Energy Technology Data Exchange (ETDEWEB)

    Pluciennik, H [Warsaw Univ. (Poland). Instytut Podstawowych Problemow Chemii

    1975-01-01

    Phosphorous-32 labelled Escherichia coli WP-2 and Escherichia coli WP-2S cells were stored at -196/sup 0/. The lethal effect induced by /sup 32/P decay was equal in both strains. Lethal efficiency of /sup 32/P..-->../sup 32/S transmutation in DNA amounted to 0.046. Reversion try..-->..try/sup +/ were induced with a ten times higher efficiency in uv-sensitive strain WP-2S, as compared with strain WP-2.

  16. DamX Controls Reversible Cell Morphology Switching in Uropathogenic Escherichia coli

    DEFF Research Database (Denmark)

    Khandige, Surabhi; Antoinette Asferg, Cecilie; Rasmussen, Karina Juhl

    2016-01-01

    undertaking targeted investigations that are challenging to perform in animal infection models. IMPORTANCE: Urinary tract infections (UTIs) are most often caused by uropathogenic Escherichia coli (UPEC) and account for a considerable health care burden. UPEC exhibits a dynamic lifestyle in the course....... In aiming to uncover genes underlying the phenomenon of UPEC morphotype switching, this study identifies damX, a cell division gene, as a mediator of reversible filamentation during UTI. DamX-mediated filamentation represents an additional pathway for bacterial cell shape control, an alternative to Sul......A-mediated FtsZ sequestration during E. coli uropathogenesis, and hence represents a potential target for combating UTI....

  17. “Inclonals”: IgGs and IgG-enzyme fusion proteins produced in an E. coli expression-refolding system

    OpenAIRE

    Hakim, Rahely; Benhar, Itai

    2009-01-01

    Full-length antibodies and antibodies that ferry a cargo to target cells are desired biopharmaceuticals. We describe the production of full-length IgGs and IgG-toxin fusion proteins in E. coli. In the presented examples of anti CD30 and anti EGF-receptor antibodies, the antibody heavy and light chains or toxin fusions thereof were expressed in separate bacterial cultures, where they accumulated as insoluble inclusion bodies. Following refolding and purification, high yields (up to 50 mg/L of ...

  18. Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria.

    Science.gov (United States)

    Klocke, Michael; Mundt, Kerstin; Idler, Frank; Jung, Sabrina; Backhausen, Jan E

    2005-06-01

    The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD(cenA)) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.

  19. Expression of an Acid Urease with Urethanase Activity in E. coli and Analysis of Urease Gene.

    Science.gov (United States)

    Liu, Xiaofeng; Zhang, Qian; Zhou, Nandi; Tian, Yaping

    2017-03-01

    Urea in alcoholic beverage is a precursor of ethyl carbamate (EC), which is carcinogenic. Enzymatic elimination of urea has attracted much research interest. Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications. In the present work, the structural genes of urease from Providencia rettgeri JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) in an active form (apourease) exhibiting both urease and urethanase (hydrolyze EC) activities. The specific activities of the purified apourease were comparatively low, which were 2.1 U/mg for urease and 0.6 U/mg for urethanase, respectively. However, apourease exhibited good resistance toward ethanol and acidic conditions. The relative activities of urease and urethanase remained over 80% in the buffers within pH 4-7. And the recoveries of both urease and urethanase activities were more than 50% in 5-25% ethanol solution. Apourease was utilized to eliminate urea in wine, and the residual urea in model wine was less than 50% after treatment with apourease for 30 h. Then 3D structure of UreC was predicted, and it was docked with urea and EC, respectively. The docking result revealed that three hydrogen bonds were formed between urea and amino acid residues in the active site of urease, whereas only one hydrogen bond can be formed between EC and the active center. Moreover, EC exhibited greater steric hindrance than urea when combined with the active site. Due to the low specific activities of apourease, both structural genes and accessory genes of urease were co-expressed in E. coli BL21(DE3). The holoenzyme was expressed as inclusion body. After renaturation and purification, the specific activities of urease and urethanase reached 10.7 and 3.8 U/mg, which were 5.62-fold and 6.33-fold of those of apourease, respectively. Therefore, accessory subunits of urease play an important role in enhancing urease and urethanase activities.

  20. Expression optimization and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Escherichia coli novablue.

    Science.gov (United States)

    Yao, Ya-Feng; Weng, Yih-Ming; Hu, Hui-Yu; Ku, Kuo-Lung; Lin, Long-Liu

    2006-09-01

    A truncated Escherichia coli Novablue gamma-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His(6)-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 degrees C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 degrees C and 9, respectively. The apparent K (m) and V (max) values for gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor in the transpeptidation reaction were 37.9 microM and 53.7 x 10(-3) mM min(-1), respectively. The synthesis of L -theanine was performed in a reaction mixture containing 10 mM L -Gln, 40 mM ethylamine, and 1.04 U His(6)-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.

  1. Laccase of Cyathus bulleri: structural, catalytic characterization and expression in Escherichia coli.

    Science.gov (United States)

    Salony; Garg, N; Baranwal, R; Chhabra, M; Mishra, S; Chaudhuri, T K; Bisaria, V S

    2008-02-01

    Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% alpha-helical, 26% beta-sheet and 38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher beta-sheet structures. The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.

  2. 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.

  3. Rho GTPase expression in human myeloid cells.

    Directory of Open Access Journals (Sweden)

    Suzanne F G van Helden

    Full Text Available Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.

  4. Production of chlorphenesin galactoside by whole cells of β-galactosidase-containing Escherichia coli.

    Science.gov (United States)

    Lee, Sang-Eun; Lee, Hyang-Yeol; Jung, Kyung-Hwan

    2013-06-28

    We investigated the transgalactosylation reaction of chlorphenesin (CPN) using β-galactosidase (β-gal)-containing Escherichia coli (E. coli) cells, in which galactose from lactose was transferred to CPN. The optimal CPN concentration for CPN galactoside (CPN-G) synthesis was observed at 40 mM under the conditions that lactose and β-gal (as E. coli cells) were 400 g/l and 4.8 U/ml, respectively, and the pH and temperature were 7.0 and 40oC, respectively. The time-course profile of CPN-G synthesis under these optimal conditions showed that CPN-G synthesis from 40 mM CPN reached a maximum of about 27 mM at 12 h. This value corresponded to an about 67% conversion of CPN to CPN-G, which was 4.47-5.36-fold higher than values in previous reports. In addition, we demonstrated by thin-layer chromatography to detect the sugar moiety that galactose was mainly transferred from lactose to CPN. Liquid chromatography-mass spectrometry revealed that CPN-G and CPN-GG (CPN galactoside, which accepted two galactose molecules) were definitively identified as the synthesized products using β-gal-containing E. coli cells. In particular, because we did not use purified β-gal, our β-gal-containing E. coli cells might be practical and cost-effective for enzymatically synthesizing CPN-G. It is expected that the use of β-gal-containing E. coli will be extended to galactose derivatization of other drugs to improve their functionality.

  5. High-level expression and characterization of two chitinases, ChiCH and ChiCW, of Bacillus cereus 28-9 in Escherichia coli

    International Nuclear Information System (INIS)

    Huang, C.-J.; Chen, C.-Y.

    2005-01-01

    Many chitinase genes have been cloned and sequenced from prokaryotes and eukaryotes but overexpression of chitinases in Escherichia coli cells was less reported. ChiCH and ChiCW of Bacillus cereus 28-9 belong to two distinct groups based on their amino acid sequences of catalytic domains, and in addition, domain structures of two enzymes are different. In this study, we established an ideal method for high-level expression of chitinases in E. coli as glutathione-S-transferase fusion proteins using pGEX-6P-1 vector. Both ChiCH and ChiCW were successfully highly expressed in E. coli cells as soluble GST-chitinase fusion proteins, and recombinant native ChiCH and ChiCW could be purified after cleavage with PreScission protease to remove GST tag. Purified chitinases were used for biochemical characterization of kinetics, hydrolysis products, and binding activities. The results indicate that ChiCW is an endo-chitinase and effectively hydrolyzes chitin and chito-multimers to chito-oligomers and the end product chitobiose, and ChiCH is an exo-chitinase and degrades chito-oligomers to produce chitobiose. Furthermore, due to higher affinity of ChiCW toward colloidal chitin than Avicel, C-terminal domain of ChiCW should be classified as a chitin-binding domain not a cellulose-binding domain although that was revealed as a cellulose-binding domain by conserved domain analysis. Therefore, the method of high-level expression of chitinases is helpful to studies and applications of chitinases

  6. Sodium nitroprusside may modulate Escherichia coli antioxidant enzyme expression by interacting with the ferric uptake regulator.

    Science.gov (United States)

    Bertrand, R; Danielson, D; Gong, V; Olynik, B; Eze, M O

    2012-01-01

    Efforts to explore possible relationships between nitric oxide (NO) and antioxidant enzymes in an Escherichia coli model have uncovered a possible interaction between sodium nitroprusside (SNP), a potent, NO-donating drug, and the ferric uptake regulator (Fur), an iron(II)--dependent regulator of antioxidant and iron acquisition proteins present in Gram-negative bacteria. The enzymatic profiles of superoxide dismutase and hydroperoxidase during logarithmic phase of growth were studied via non-denaturing polyacrylamide gel electrophoresis and activity staining specific to each enzyme. Though NO is known to induce transcription of the manganese-bearing isozyme of SOD (MnSOD), treatment with SNP paradoxically suppressed MnSOD expression and greatly enhanced the activity of the iron-containing equivalent (FeSOD). Fur, one of six global regulators of MnSOD transcription, is uniquely capable of suppressing MnSOD while enhancing FeSOD expression through distinct mechanisms. We thus hypothesize that Fur is complacent in causing this behaviour and that the iron(II) component of SNP is activating Fur. E. coli was also treated with the SNP structural analogues, potassium ferricyanide (PFi) and potassium ferrocyanide (PFo). Remarkably, the ferrous PFo was capable of mimicking the SNP-related pattern, whereas the ferric PFi was not. As Fur depends upon ferrous iron for activation, we submit this observation of redox-specificity as preliminary supporting evidence for the hypothesized Fur-SNP interaction. Iron is an essential metal that the human innate immune system sequesters to prevent its use by invading pathogens. As NO is known to inhibit iron-bound Fur, and as activated Fur regulates iron uptake through feedback inhibition, we speculate that the administration of this drug may disrupt this strategic management of iron in favour of residing Gram-negative species by providing a source of iron in an otherwise iron-scarce environment capable of encouraging its own uptake

  7. Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197

    CSIR Research Space (South Africa)

    Roth, Robyn L

    2017-04-01

    Full Text Available The aim of this article is to investigate the production of soluble cross-reacting material 197 (CRM(sub197)) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application...

  8. Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa - their role in the development of resistance.

    Science.gov (United States)

    Dhar, Supurna; Kumari, Hansi; Balasubramanian, Deepak; Mathee, Kalai

    2018-01-01

    The bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus - a tightly cross-linked peptidoglycan mesh unique to bacteria. Cell-wall synthesis and recycling are critical cellular processes essential for cell growth, elongation and division. Both de novo synthesis and recycling involve an array of enzymes across all cellular compartments, namely the outer membrane, periplasm, inner membrane and cytoplasm. Due to the exclusivity of peptidoglycan in the bacterial cell-wall, these players are the target of choice for many antibacterial agents. Our current understanding of cell-wall biochemistry and biogenesis in Gram-negative organisms stems mostly from studies of Escherichia coli. An incomplete knowledge on these processes exists for the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. In this review, cell-wall synthesis and recycling in the various cellular compartments are compared and contrasted between E. coli and P. aeruginosa. Despite the fact that there is a remarkable similarity of these processes between the two bacterial species, crucial differences alter their resistance to β-lactams, fluoroquinolones and aminoglycosides. One of the common mediators underlying resistance is the amp system whose mechanism of action is closely associated with the cell-wall recycling pathway. The activation of amp genes results in expression of AmpC β-lactamase through its cognate regulator AmpR which further regulates multi-drug resistance. In addition, other cell-wall recycling enzymes also contribute to antibiotic resistance. This comprehensive summary of the information should spawn new ideas on how to effectively target cell-wall processes to combat the growing resistance to existing antibiotics.

  9. DFI-seq identification of environment-specific gene expression in uropathogenic Escherichia coli

    DEFF Research Database (Denmark)

    Madelung, Michelle; Kronborg, Tina; Doktor, Thomas Koed

    2017-01-01

    response. We combined differential fluorescence induction (DFI) with next-generation sequencing, collectively termed DFI-seq, to identify differentially expressed genes in UPEC strain UTI89 during growth in human urine and bladder cells. RESULTS: DFI-seq eliminates the need for iterative cell sorting...... hypothetical proteins. One such gene UTI89_C5139, displayed increased adhesion and invasion of J82 cells when deleted from UPEC strain UTI89. CONCLUSIONS: We demonstrate the usefulness of DFI-seq for identification of genes required for optimal growth of UPEC in human urine, as well as potential virulence...

  10. Escherichia coli producing colibactin triggers premature and transmissible senescence in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Thomas Secher

    Full Text Available Cellular senescence is an irreversible state of proliferation arrest evoked by a myriad of stresses including oncogene activation, telomere shortening/dysfunction and genotoxic insults. It has been associated with tumor activation, immune suppression and aging, owing to the secretion of proinflammatory mediators. The bacterial genotoxin colibactin, encoded by the pks genomic island is frequently harboured by Escherichia coli strains of the B2 phylogenetic group. Mammalian cells exposed to live pks+ bacteria exhibit DNA-double strand breaks (DSB and undergo cell-cycle arrest and death. Here we show that cells that survive the acute bacterial infection with pks+ E. coli display hallmarks of cellular senescence: chronic DSB, prolonged cell-cycle arrest, enhanced senescence-associated β-galactosidase (SA-β-Gal activity, expansion of promyelocytic leukemia nuclear foci and senescence-associated heterochromatin foci. This was accompanied by reactive oxygen species production and pro-inflammatory cytokines, chemokines and proteases secretion. These mediators were able to trigger DSB and enhanced SA-β-Gal activity in bystander recipient cells treated with conditioned medium from senescent cells. Furthermore, these senescent cells promoted the growth of human tumor cells. In conclusion, the present data demonstrated that the E. coli genotoxin colibactin induces cellular senescence and subsequently propel bystander genotoxic and oncogenic effects.

  11. High-yield expression in Escherichia coli, purification and application of budding yeast K2 killer protein.

    Science.gov (United States)

    Podoliankaitė, Monika; Lukša, Juliana; Vyšniauskis, Gintautas; Sereikaitė, Jolanta; Melvydas, Vytautas; Serva, Saulius; Servienė, Elena

    2014-07-01

    Saccharomyces cerevisiae K2 toxin is a highly active extracellular protein, important as a biocontrol agent for biotechnological applications in the wine industry. This protein is produced at negligible levels in yeast, making difficult to isolate it in amounts sufficient for investigation and generation of analysis tools. In this work, we demonstrate the use of a bacterial system for expression of the recombinant K2 protein, suitable for generation of antibodies specific for toxin of the yeast origin. Synthesis of the full-length S. cerevisiae K2 preprotoxin in Escherichia coli was found to be toxic to the host cell, resulting in diminished growth. Such effect was abolished by the introduction of the C-terminal truncation into K2 protein, directing it into non-toxic inclusion body fraction. The obtained protein is of limited solubility thus, facilitating the purification by simple and efficient chromatography-free procedure. The protein aggregates were successfully refolded into a soluble form yielding sufficient amounts of a tag-less truncated K2 protein suitable for polyclonal antibody production. Antibodies were raised in rabbit and found to be specific for detection of both antigen and native S. cerevisiae K2 toxin.

  12. Preparation of α-deuterated L-amino acids using E.coli cells containing tryptophanase

    International Nuclear Information System (INIS)

    Faleev, N.G.; Ruvinov, S.B.; Saporovskaya, M.B.; Belikov, V.M.; Zakomyrdina, L.N.; Sakharova, I.S.; Torchinskij, Yu.M.

    1989-01-01

    Method for preparation of a series of α-deuterated L-amino acids of high optical purity with quantitative chemica yield, suing stereospecific isotopic exchange in D 2 O under the effect of E.coli cells with high tryptophanase activity was developed

  13. Problem-Solving Test: RNA and Protein Synthesis in Bacteriophage-Infected "E. coli" Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2008-01-01

    The classic experiment presented in this problem-solving test was designed to identify the template molecules of translation by analyzing the synthesis of phage proteins in "Escherichia coli" cells infected with bacteriophage T4. The work described in this test led to one of the most seminal discoveries of early molecular biology: it dealt a…

  14. Ingestion of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli by human peritoneal mesothelial cells

    NARCIS (Netherlands)

    Visser, C. E.; Brouwer-Steenbergen, J. J.; Schadee-Eestermans, I. L.; Meijer, S.; Krediet, R. T.; Beelen, R. H.

    1996-01-01

    In the present study we examined whether mesothelial cells can ingest and digest bacteria. The results showed that all strains were ingested. Ingested staphylococci proliferated abundantly, and only a few were digested. Escherichia coli, however, was digested during the first 8 h, whereafter the

  15. Immunocytochemical localization of the elongation factor Tu in E. coli cells

    NARCIS (Netherlands)

    Slot, J.W.; Schilstra, M.J.; Meide, P.H. van der; Posthuma, G.; Cremers, A.F.M.; Bosch, L.

    1984-01-01

    The localization of the elongation factor Tu (EF-Tu) in ultrathin cryosections of E. coli cells was determined with the electron microscope using a highly specific immunological labellin technique. EF-Tu is distributed almost homogeneously throughout the cytoplasm. Although it has often been

  16. Restriction alleviation of phage λ in Escherichia Coli K-12 cells after γ-irradiation

    International Nuclear Information System (INIS)

    Rabinkova, E.V.; Torosyan, M.V.; Fradkin, G.E.

    1987-01-01

    In γ-irradiated cells of Escherichia coli K-12 restriction allevation of an unmodified phage λ is only observed in AB1157 strain. No restriction allevation by γ-rays is registered in AB1157 mutants (rec A and ssb-1)

  17. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

    Directory of Open Access Journals (Sweden)

    Lobstein Julie

    2012-05-01

    Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

  18. Modified Vero cell induced by Bifidobacterium bifidum inhibits enterohemorrhagic Escherichia coli O157:H7 cytopathic effect

    Directory of Open Access Journals (Sweden)

    Tahamtan, Y.

    2014-11-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC, such as E. coli O157:H7, are emerging food-borne pathogens worldwide. This micro-organism can damage the epithelial tissue of the large intestine. The cytotoxic effects can be neutralized by probiotics such as Bifidobacterium bifidum. Probiotics are viable cells that have beneficial effects on the health of the host. The preventing activity of B. bifidum against E. coli O157 was studied using a Vero cell model. Vero cell was pretreated with viable B. bifidum and incubated for either 3 h to 24 h and then collected from the cell to make modified Vero cell (MVC. Indirect antibacterial effects of B. bifidum were demonstrated by reduction of attachment of E. coli O157:H7 to MVC. The maximum reduction was resulted in pretreatment of Vero cell with B. bifidum for 24 h before infection. B. bifidum attenuated E. coli O157:H7 attachment to MVC up to 10 days of incubation. To our knowledge, MCV prevented Vero cell line injury induced by E. coli O157:H7. Therefore, B. bifidum can be used for inhibition of E. coli O157:H7 cytopathic effect (CPE in Vero cell model, even as pretreatment of the cell line.

  19. An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

    International Nuclear Information System (INIS)

    Xu, H. Howard; Real, Lilian; Bailey, Melissa Wu

    2006-01-01

    With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats

  20. Modification of radiation response of E. coli B/r cells by phenothiazines

    International Nuclear Information System (INIS)

    Maniar, H.S.; Singh, B.B.

    1983-01-01

    Promethazine and trimeprazine sensitized anoxic E. coli B/r cells to 60 Co gamma-rays, but both drugs showed a radioprotective effect under euoxic conditions. Their radiosensitizing effect was found to be due to the reaction of radiolytically induced hydroxyl radicals with the sensitizers. The radioprotective effect of these drugs is attributed to changes in the membrane structure conducive with chemical repair of the damaged sites in the gel region of the cellular membrane by intracellular sulphydryl compounds. Pre-irradiation depletion of sulphydryls from E. coli B/r by treatment with N-ethyl maleimide abolished the radioprotective effect of these drugs under euoxic conditions. (author)

  1. Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli

    International Nuclear Information System (INIS)

    Plotch, S.J.; Palant, O.; Gluzman, Y.

    1989-01-01

    A cDNA clone encoding the RNA polymerase of poliovirus has been expressed in Escherichia coli under the transcriptional control of a T7 bacteriophage promoter. This poliovirus enzyme was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant enzyme has been purified to near homogeneity, and polyclonal antibodies have been prepared against it. The enzyme exhibits poly(A)-dependent oligo(U)-primed ply(U) polymerase activity as well as RNA polymerase activity. In the presence of an oligo(U) primer, the enzyme catalyzes the synthesis of a full-length copy of either poliovirus or globin RNA templates. In the absence of added primer, RNA products up to twice the length of the template are synthesized. When incubated in the presence of a single nucleoside triphosphate, [α- 32 P]UTP, the enzyme catalyzes the incorporation of radioactive label into template RNA. These results are discussed in light of previously proposed models of poliovirus RNA synthesis in vitro

  2. Soluble multimer of recombinant endostatin expressed in E. coli has anti-angiogenesis activity

    International Nuclear Information System (INIS)

    Wei Dongmei; Gao Yan; Cao Xiangrong; Zhu Nianchun; Liang Jianfu; Xie Weiping; Zhen Mingying; Zhu Minsheng

    2006-01-01

    The bioactivity, refolding, and multimer formation of endostatin, particularly of recombinant endostatin produced from bacteria, are proved challenging for clinical application. In order to determine the biological activity of recombinant endostatin multimer, first, we expressed endostatin in Escherichia coli and purified it with ion-exchange chromatography. The purified active protein could elicit multimer formation spontaneously, but still has comparable activity. Aim to determine the anti-angiogenic activity of multimer endostatin, by use of RP-HPLC, we then successfully separated endostatin monomer and multimer for subjecting to anti-angiogenesis assay. The results from CAM (chorioallantoic membrane) inhibition assay showed that both monomer and multimer suppressed CAM vascularization significantly. At the dosage of 0.8 μg, inhibition rates of multimeric and monomeric proteins were about 58% and 38%, respectively. Multimeric endostatin exerted a higher activity than monomeric endostatin (p 0.05), although both of them show a high inhibition effect in contrast to control. The results from HUVEC proliferation assay also showed similar effects at dosages of 0.6 and 1.6 μg/ml, multimer exerted a higher activity on inhibition of HUVEC proliferation comparing with monomer (p < 0.05). In conclusion, our results suggest that endostatin multimer has a comparable or higher bioactivity and multimerization will not affect its bioactivity, implying that endostatin activity is insensitive to structure conformation contributed by disulfide bonds

  3. Expression, purification and characterization of soluble red rooster laforin as a fusion protein in Escherichia coli.

    Science.gov (United States)

    Brewer, M Kathryn; Husodo, Satrio; Dukhande, Vikas V; Johnson, Mary Beth; Gentry, Matthew S

    2014-04-02

    The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue. In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin. Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease.

  4. Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli.

    Science.gov (United States)

    Ko, Chun-Han; Tsai, Chung-Hung; Lin, Po-Heng; Chang, Ko-Cheng; Tu, Jenn; Wang, Ya-Nang; Yang, Chien-Ying

    2010-10-01

    The Cel-BL11 gene from Paenibacillus campinasensis BL11 was cloned and expressed in Escherichia coli as a His-tag fusion protein. Zymographic analysis of the recombinant protein revealed cellulase activity corresponding to a protein with a 38-kDa molecular weight. The optimum temperature and pH for purified cellulase were 60 °C and pH 7.0, respectively. The enzyme retained more than 80% activity after 8h at 60 °C at pH 6 and 7. The cellulase has a Km of 11.25 mg/ml and a Vmax of 1250 μmol/min/mg with carboxylmethyl cellulose (CMC). Then enzyme was active on Avicel, swollen Avicel, CMC, barley β-glucan, laminarin in the presence of 100 mM acetate buffer. It was inhibited by Hg²⁺, Cu²⁺ and Zn²⁺. Significant kraft pulp refining energy saving, 10%, was exhibited by the pretreatment of this cellulase applied at 2 IU per gram of oven-dried pulp. Broad pH and temperature stability render this cellulase a convenient applicability toward current mainstream biomass conversion and other industrial processes. Copyright © 2010 Elsevier Ltd. All rights reserved.

  5. In vitro assembly of Ebola virus nucleocapsid-like complex expressed in E. coli

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    Ruchao Peng

    2016-09-01

    Full Text Available Abstract Ebola virus (EBOV harbors an RNA genome encapsidated by nucleoprotein (NP along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451–739 alone is capable of forming a helical nucleocapsid-like complex (NLC. However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451–739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.

  6. 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 predi...

  7. Expression and purification of PprI protein from D.radiodurans R1 in escherichia coli

    International Nuclear Information System (INIS)

    Zhang Yongqin; Zhou Hui; Chen Jie; Yang Zhanshan

    2011-01-01

    In order to express and purify PprI protein from D.radiodurans R1 in E. coli, the full length of pprI gene was gained by PCR amplification using pCMV-HA-pprI as a template. The gene segment was inserted into vector pET-28a after digested by two restriction endonucleases Nco I and EcoR I. Then the recombinant vector pET-28a-His-pprI was transfected into E. coli BL21(DE3) RP. The PprI protein expression was induced by IPTG and the fusion protein was confirmed by SDS-PAGE and Western blotting. The expressive conditions of the protein such as E. coli' A 600 , concentration of IPTG, time and temperature of culture, were optimized. Finally the fusion protein was purified by Ni-NTA His Bind Resins and molecule boult. The experimental results show the fusion protein confirmed by Western blotting is 6 x His-PprI and its molecular weight is 37 kDa. The ladders of PprI protein at molecular weight 37 kDa were different due to difference of the PprI protein expression conditions if E. coli. The PprI protein exists both in supernatant and precipitation. The concentration of purified protein is about 0.15 mg/mL which was measured by BCA method. It is concluded that the recombinant plasmid pET-28a-His-pprI is constructed and the PprI fusion protein is expressed and purified. The results lay a solid foundation for studying the radio-resistance and immunity of PprI protein. (authors)

  8. Cloning, Expression, and Immunogenicity of Fimbrial-F17A Subunit Vaccine against Escherichia coli Isolated from Bovine Mastitis

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    Wei Chen

    2017-01-01

    Full Text Available There is a need to identify and select new promising immunodominant antigens that have the ability to provide protective immunity against E. coli causing bovine mastitis. Recently we showed that f17a was found to be the most prevalent and crucial virulent factor among the pathogenic E. coli isolated from bovine mastitis. Here, in this report, the recombinant F17A based subunit vaccine adjuvant with MF59 was tested for immunogenicity against E. coli in a murine model. The vaccinated mice did not show any abnormal behavioral changes and histopathological lesions after vaccination. The specific antibody level against F17A was significantly higher in MF59-adjuvant-group, and also lasted for longer duration with a significant (P<0.01 production level of IgG1 and IgG2a. Moreover, we noted higher survival rate in mice injected with F17A-MF59-adjuvant group after challenging with the clinical E. coli strain. Our findings of bacterial clearance test revealed that elimination rate from liver, spleen, and kidney in MF59-adjuvant-group was significantly higher than the control group. Finally, the proportion of CD4+T cells was increased, while CD8+ was decreased in MF59-adjuvant group. In conclusion, the current study reveals the capability of F17A-MF59 as a potential vaccine candidate against pathogenic E. coli causing mastitis in dairy animals.

  9. Foxp3 expression in human cancer cells

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    Gourgoulianis Konstantinos I

    2008-04-01

    Full Text Available Abstract Objective Transcription factor forkhead box protein 3 (Foxp3 specifically characterizes the thymically derived naturally occurring regulatory T cells (Tregs. Limited evidence indicates that it is also expressed, albeit to a lesser extent, in tissues other than thymus and spleen, while, very recently, it was shown that Foxp3 is expressed by pancreatic carcinoma. This study was scheduled to investigate whether expression of Foxp3 transcripts and mature protein occurs constitutively in various tumor types. Materials and methods Twenty five tumor cell lines of different tissue origins (lung cancer, colon cancer, breast cancer, melanoma, erythroid leukemia, acute T-cell leukemia were studied. Detection of Foxp3 mRNA was performed using both conventional RT-PCR and quantitative real-time PCR while protein expression was assessed by immunocytochemistry and flow cytometry, using different antibody clones. Results Foxp3 mRNA as well as Foxp3 protein was detected in all tumor cell lines, albeit in variable levels, not related to the tissue of origin. This expression correlated with the expression levels of IL-10 and TGFb1. Conclusion We offer evidence that Foxp3 expression, characterizes tumor cells of various tissue origins. The biological significance of these findings warrants further investigation in the context of tumor immune escape, and especially under the light of current anti-cancer efforts interfering with Foxp3 expression.

  10. Decorin expression in quiescent myogenic cells

    International Nuclear Information System (INIS)

    Nishimura, Takanori; Nozu, Kenjiro; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Hattori, Akihito

    2008-01-01

    Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells

  11. Insect cells are superior to Escherichia coli in producing malaria proteins inducing IgG targeting PfEMP1 on infected erythrocytes

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    Joergensen Louise

    2010-11-01

    Full Text Available Abstract Background The PFD1235w Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1 antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and Escherichia coli based system was used to express single and double domains encoded by the pfd1235w var gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w PfEMP1 antigen expressed on 3D7PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed. Methods The recombinant proteins were run on SDS-PAGE and Western blots for quantification and size estimation. Insect cell and E. coli-produced recombinant proteins were coupled to a bead-based Luminex assay to measure the plasma antibody reactivity of 180 samples collected from Tanzanian individuals. The recombinant proteins used for immunization of rats and antisera were also tested by flow cytometry for their ability to surface label 3D7PFD1235w-IE. Results All seven pAcGP67A constructs were successfully expressed as recombinant protein in baculovirus-infected insect cells and subsequently produced to a purity of 60-97% and a yield of 2-15 mg/L. By comparison, only three of seven pET101/D-TOPO constructs expressed in the E. coli system could be produced at all with purity and yield ranging from 3-95% and 6-11 mg/L. All seven insect cell, but only two of the E. coli produced proteins induced antibodies reactive with native PFD1235w expressed on 3D7PFD1235w-IE. The recombinant proteins were recognized in an age- and transmission intensity-dependent manner by antibodies from 180 Tanzanian individuals in a bead-based Luminex assay. Conclusions The baculovirus based insect cell

  12. A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

    Science.gov (United States)

    Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

    2015-01-01

    The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Heterologous expression of mannanase and developing a new Reporter gene system in Lactobacillus casei and Escherichia coli

    DEFF Research Database (Denmark)

    Lin, Jinzhong; Zou, Yexia; Ma, Chengjie

    2015-01-01

    Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were clone....... casei and E.coli....

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production.

    Science.gov (United States)

    Goers, Lisa; Ainsworth, Catherine; Goey, Cher Hui; Kontoravdi, Cleo; Freemont, Paul S; Polizzi, Karen M

    2017-06-01

    Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole-cell biosensors. Biotechnol. Bioeng. 2017;114: 1290-1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

  16. Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

    Science.gov (United States)

    Goers, Lisa; Ainsworth, Catherine; Goey, Cher Hui; Kontoravdi, Cleo; Freemont, Paul S.

    2017-01-01

    ABSTRACT Many high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:28112405

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Mechanism of reactivation of the UV-inactivated cells of Escherichia coli by cell extracts of propionic acid bacteria

    International Nuclear Information System (INIS)

    Vorob'eva, L.I.; Khodzhaev, E.Y.; Ponomareva, G.M.

    1995-01-01

    Two mechanisms of reactivation of UV-inactivated Escherichia coli cells - photoreactivation (PhR) and reactivation by the dialyzate of cell extract of propionic acid bacteria - are shown to be different but not completely additive. PhR displays an insignificant negative effect on the reactivaton by active substances (peptides) of the dialyzate, whereas reactivation by dialyzate inhibits PhR. The maximal reactivation can be attained under complete PhR followed by the protective action of dialyzate. The dialyzate protects UV-irradiated E. coli cells with PolA, UvrA, and RecA mutations and Salmonella typhimurium TA 100 (UvrB) cells, and also exerts an antimutagenic effect on S. typhimurium TA 100. Protection by dialyzate is suggested to be due to restoration of the cell division mechanism damaged by UV irradiation. 14 refs., 3 figs., 5 tabs

  19. Over expression of beta-1, 4-xylanase by auto-induction in E. coli

    International Nuclear Information System (INIS)

    Khan, M.I.K.; Sajjad, M.; Akhtar, W.

    2013-01-01

    Catalytic domain of β-1, 4-xylanase gene, (xynZ.CD) of Clostridium thermocellum was cloned in pET28a expression vector and over-expressed in Escherichia colt BL21 CodonPlus (RIL). The production of XynZ.CD in E. colt was optimized using different concentrations of lactose and induction of the enzyme at different stages of growth. The maximum growth of the cells and the enzyme activity were observed when the cells were induced with 10mM lactose after 8 hours of incubation. The enzyme was found to constitute >40% of the total cell proteins in the supernatant of the lysed cells transformed with recombinant pET28a/xynZ.CD. It was purified by heating the cell lysate at 65 degree C for 30 m followed by fractionation through FPLC. Molecular weight of XynZ.CD was found to be approximately 38,524 D by MALDI-TOF analysis. The enzyme variant was quite stable within broad pH range of 5.5 - 8.0 and it retained >85% of xylanase activity after 2 h incubation at 70 degre C. (author)

  20. Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli

    International Nuclear Information System (INIS)

    Billen, D.

    1987-01-01

    When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed

  1. How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

    Directory of Open Access Journals (Sweden)

    Barth Sandra

    2005-04-01

    Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

  2. Efficient Production of γ-GABA Using Recombinant E. coli Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote Saccharomyces cerevisiae.

    Science.gov (United States)

    Xiong, Qiang; Xu, Zheng; Xu, Lu; Yao, Zhong; Li, Sha; Xu, Hong

    2017-12-01

    γ-Aminobutyric acid (γ-GABA) is a non-proteinogenic amino acid, which acts as a major regulator in the central nervous system. Glutamate decarboxylase (namely GAD, EC 4.1.1.15) is known to be an ideal enzyme for γ-GABA production using L-glutamic acid as substrate. In this study, we cloned and expressed GAD gene from eukaryote Saccharomyces cerevisiae (ScGAD) in E. coli BL21(DE3). This enzyme was further purified and its optimal reaction temperature and pH were 37 °C and pH 4.2, respectively. The cofactor of ScGAD was verified to be either pyridoxal 5'-phosphate (PLP) or pyridoxal hydrochloride. The optimal concentration of either cofactor was 50 mg/L. The optimal medium for E. coli-ScGAD cultivation and expression were 10 g/L lactose, 5 g/L glycerol, 20 g/L yeast extract, and 10 g/L sodium chloride, resulting in an activity of 55 U/mL medium, three times higher than that of using Luria-Bertani (LB) medium. The maximal concentration of γ-GABA was 245 g/L whereas L-glutamic acid was near completely converted. These findings provided us a good example for bio-production of γ-GABA using recombinant E. coli expressing a GAD enzyme derived from eukaryote.

  3. Effect of aqueous media on the copper-ion-mediated phototoxicity of CuO nanoparticles toward green fluorescent protein-expressing Escherichia coli.

    Science.gov (United States)

    Shang, Enxiang; Li, Yang; Niu, Junfeng; Guo, Huiyuan; Zhou, Yijing; Liu, Han; Zhang, Xinqi

    2015-12-01

    Quantitative comparison of different aqueous media on the phototoxicity of copper oxide nanoparticles (CuO NPs) is crucial for understanding their ecological effects. In this study, the phototoxicity of CuO NPs toward the green fluorescent protein-expressing Escherichia coli (GFP-E. coli) under UV irradiation (365 nm) was investigated in Luria-Bertani medium (LB), NaCl solution, deionized water (DI) and phosphate-buffered saline (PBS). The phototoxicity of CuO NPs toward GFP-E. coli decreased in the order of DI>NaCl>PBS>LB because of different released concentrations of Cu(2+). The 3h released Cu(2+) concentrations by 10mg/L CuO NPs in DI water, NaCl solution, LB medium, and PBS were 1946.3 ± 75.6, 1242.5 ± 47.6, 1023.4 ± 41.2, and 1162.1 ± 41.9 μg/L, respectively. Transmission electron microscope and laser scanning confocal microscope images of E. coli exposed to CuO NPs demonstrated that the released Cu(2+) resulted in fragmentation of bacterial cell walls, leakage of intracellular components, and finally death of bacteria in four media after UV light irradiation. In each medium, the bacterial mortality rate logarithmically increased with the releasing concentrations of Cu(2+) by CuO NPs (R(2)>0.90) exposed to 3h UV light. This study highlights the importance of taking into consideration of water chemistry when the phototoxicity of CuO NPs is assessed in nanotoxicity research. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Evidence for significantly enhancing reduction of Azo dyes in Escherichia coli by expressed cytoplasmic Azoreductase (AzoA) of Enterococcus faecalis.

    Science.gov (United States)

    Feng, J; Heinze, T M; Xu, H; Cerniglia, C E; Chen, H

    2010-05-01

    Although cytoplasmic azoreductases have been purified and characterized from various bacteria, little evidence demonstrating that these azoreductases are directly involved in azo dye reduction in vivo is known. In order to evaluate the contribution of the enzyme to azo dye reduction in vivo, experiments were conducted to determine the effect of a recombinant cytoplasmic azoreductase (AzoA) from Enterococcus faecalis expressed in Escherichia coli on the rate of metabolism of Methyl Red, Ponceau BS and Orange II. The intact cells that contained IPTG induced AzoA had a higher rate of dye reduction with increases of 2 (Methyl Red), 4 (Ponceau BS) and 2.6 (Orange II)-fold compared to noninduced cells, respectively. Metabolites of Methyl Red isolated from induced cultures were identified as N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid through liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analyses. In conclusion, our data demonstrate that AzoA from Ent. faecalis is capable of increasing the reduction of azo dyes in intact E. coli cells and that cytoplasmic azoreductase is involved in bacterial dye degradation in vivo.

  5. Effect of Light-Activated Hypocrellin B on the Growth and Membrane Permeability of Gram-Negative Escherichia coli Cells

    Directory of Open Access Journals (Sweden)

    Yuan Jiang

    2014-01-01

    Full Text Available Aim. To investigate the effect of light-activated hypocrellin B on the growth and membrane permeability of Gram-negative bacteria. Methods. Escherichia coli (E. coli as a model bacterium of Gram-negative bacteria was incubated with various concentrations of hypocrellin B for 60 min and was subsequently irradiated by blue light with wavelength of 470 nm at the dose of 12 J/cm2. Colony forming units were counted and the growth inhibition rate of E. coli cells was calculated after light-activated hypocrellin B. Membrane permeability was measured using flow cytometry and confocal laser scanning microscopy (CLSM with propidium iodide (PI staining. Bacterial morphology was observed using transmission electron microscopy (TEM. Reactive oxygen species in bacterial cells were measured using flow cytometry with DCFH-DA staining. Results. Significant growth inhibition rate of E. coli cells was observed after photodynamic action of hypocrellin B. Remarkable damage to the ultrastructure of E. coli was also observed by TEM. Flow cytometry and CLSM observation showed that light-activated hypocrellin B markedly increased membrane permeability of E. coli. Flow cytometry showed the intracellular ROS increase in E. coli treated by photodynamic action of hypocrellin B. Conclusion. Light-activated hypocrellin B caused intracellular ROS increase and structural damages and inhibited the growth of Gram-negative E. coli cells.

  6. Isolating E.Coli Bacteriophage from Raw Sewage and Determining its Selectivity to the Host Cell

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    SM Imeni

    2016-05-01

    Full Text Available Introduction: Bacteriophages are viruses that infect and destroy prokaryote cells, specifically the bacteria. They act too selective, so as each bacteriophage affects only on specific type of bacteria. Due to their specific features, bacteriophages can be used as an appropriate substitute for antibiotics in infectious diseases treatment. Therefore, this study aimed to isolate E. coli-specific bacteriophage from raw sewage. Methods: Eight samples of raw sewage, each containing approximately 50 ml of raw sewage with 10 minute gap, were prepared from Zargandeh wastewater treatment plant, Tehran, Iran. The sewages were mixed with Brain-heart infusion medium (BHI as a liquid culture medium in order to let the microorganisms grow. Incubation, purification and determination of bacteria were followed repeatedly to isolate the bacteriophage. Then it was tested on E.coli (ATCC 25922, Enterococcus faecalis (ATCC 19433, Staphylococcus aureus (ATCC 2392, and Yersinia enterocolitica (ATCC 9610 in order to determine the bacteriophage selectivity. Results: The E.coli bacteriophages were successfully isolated from all the eight samples, that were completely able to lyse and destroy E.coli bacterial cells, though no effect was observed on other types of bacteria. Conclusion: The study findings revealed that bacteriophages act selectively. Considering the raise of antibiotic resistance in the world, bacteriophages can serve as a good substitute for antibiotics in treating infectious diseases.

  7. Differential effect of immune cells on non-pathogenic Gram-negative bacteria-induced nuclear factor-kappaB activation and pro-inflammatory gene expression in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Haller, D.; Holt, L.; Parlesak, Alexandr

    2004-01-01

    stimulation, interleukin-8 (IL-8) mRNA accumulation is strongly induced in Escherichia coli- but not Bacteroides vulgatus-stimulated IEC cocultured with peripheral blood (PBMC) and lamina propria mononuclear cells (LPMC). The presence of PBMC triggered both E. coli- and B. vulgatus-induced mRNA expression...... in the presence of PBMC. Interestingly, B. vulgatus- and E. coli-derived lipopolysaccharide-induced similar IL-8 mRNA expression in epithelial cells after basolateral stimulation of HT-29/PBMC cocultures. Although luminal enteric bacteria have adjuvant and antigenic properties in chronic intestinal inflammation...

  8. Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-02-01

    Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.

    Science.gov (United States)

    Marshall, Ryan; Maxwell, Colin S; Collins, Scott P; Jacobsen, Thomas; Luo, Michelle L; Begemann, Matthew B; Gray, Benjamin N; January, Emma; Singer, Anna; He, Yonghua; Beisel, Chase L; Noireaux, Vincent

    2018-01-04

    CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.

    Science.gov (United States)

    Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

    2009-10-01

    Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

  11. Cloning, characterization, and expression of the dapE gene of Escherichia coli.

    Science.gov (United States)

    Bouvier, J; Richaud, C; Higgins, W; Bögler, O; Stragier, P

    1992-08-01

    The dapE gene of Escherichia coli encodes N-succinyl-L-diaminopimelic acid desuccinylase, an enzyme that catalyzes the synthesis of LL-diaminopimelic acid, one of the last steps in the diaminopimelic acid-lysine pathway. The dapE gene region was previously purified from a lambda bacteriophage transducing the neighboring purC gene (J. Parker, J. Bacteriol. 157:712-717, 1984). Various subcloning steps led to the identification of a 2.3-kb fragment that complemented several dapE mutants and allowed more than 400-fold overexpression of N-succinyl-L-diaminopimelic acid desuccinylase. Sequencing of this fragment revealed the presence of two closely linked open reading frames. The second one encodes a 375-residue, 41,129-M(r) polypeptide that was identified as N-succinyl-L-diaminopimelic acid desuccinylase. The first one encodes a 118-residue polypeptide that is not required for diaminopimelic acid biosynthesis, as judged by the wild-type phenotype of a strain in which this gene was disrupted. Expression of the dapE gene was studied by monitoring amylomaltase activity in strains in which the malPQ operon was under the control of various fragments located upstream of the dapE gene. The major promoter governing dapE transcription was found to be located in the adjacent orf118 gene, while a minor promoter allowed the transcription of both orf118 and dapE. Neither of these two promoters is regulated by the lysine concentration in the growth medium.

  12. Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

    International Nuclear Information System (INIS)

    Nickbarg, E.B.; Knowles, J.R.

    1988-01-01

    Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)- 3 H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase

  13. Expression of enterovirus 71 capsid protein VP1 in Escherichia coli and its clinical application

    Directory of Open Access Journals (Sweden)

    Mei Shi

    2013-12-01

    Full Text Available The VPl gene of enterovirus 71 (EV71 was synthesized, construct a recombinant plasmid pET15b/VP1 and expressed in E. coli BL21. The recombinant VP1 protein could specifically react with EV71-infected patient sera without the cross-reaction with serum antibodies of coxsackievirus A16 (CA16, A4, A5, B3 and B5 as well as echovirus 6. In acute and convalescent phases, IgM and IgG antibodies of 182 serum samples were detected by ELISA with recombinant VP1 protein as a coated antigen. The results showed that the sensitivity, specificity, positive predictive value (PPV and negative predictive value (NPV of IgM antibodies in serum samples for the diagnosis of EV71 infection were 90.1, 98.4, 98.8 and 88.7%, respectively; similarly, those of IgG antibodies in serum samples were 82.4, 89.1, 91.5 and 78.1%, respectively. Five of 80 samples (6.25% from CA16infected patients were detected positive by ELISA with recombinant VP1 protein in which indicated the cross reactions and 0 of 5 samples from patients infected with other enteroviruses including CA4, CA5, CB3, CB5 and echovirus 6. Therefore, the recombinant VP1 protein of EV7l may provide a theoretical reference for establishing an effective antibody screening of IgM for EV71-infected patients with clinically suspected hand, foot, and mouth disease (HFMD.

  14. Global transcriptional response of Escherichia coli MG1655 cells exposed to the oxygenated monoterpenes citral and carvacrol.

    Science.gov (United States)

    Chueca, Beatriz; Pérez-Sáez, Elisa; Pagán, Rafael; García-Gonzalo, Diego

    2017-09-18

    DNA microarrays were used to study the mechanism of bacterial inactivation by carvacrol and citral. After 10-min treatments of Escherichia coli MG1655 cells with 100 and 50ppm of carvacrol and citral, 76 and 156 genes demonstrated significant transcriptional differences (p≤0.05), respectively. Among the up-regulated genes after carvacrol treatment, we found gene coding for multidrug efflux pumps (acrA, mdtM), genes related to phage shock response (pspA, pspB, pspC, pspD, pspF and pspG), biosynthesis of arginine (argC, argG, artJ), and purine nucleotides (purC, purM). In citral-treated cells, transcription of purH and pyrB and pyrI was 2 times higher. Deletion of several differentially expressed genes confirmed the role of ygaV, yjbO, pspC, sdhA, yejG and ygaV in the mechanisms of E. coli inactivation by carvacrol and citral. These results would indicate that citral and carvacrol treatments cause membrane damage and activate metabolism through the production of nucleotides required for DNA and RNA synthesis and metabolic processes. Comparative transcriptomics of the response of E. coli to a heat treatment, which caused a significant change of the transcription of 1422 genes, revealed a much weaker response to both individual constituents of essential oils (ICs).·Thus, inactivation by citral or carvacrol was not multitarget in nature. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli.

    Science.gov (United States)

    Le Chevanton, L; Leblon, G

    1989-04-15

    We cloned the ura5 gene coding for the orotate phosphoribosyl transferase from the ascomycete Sordaria macrospora by heterologous probing of a Sordaria genomic DNA library with the corresponding Podospora anserina sequence. The Sordaria gene was expressed in an Escherichia coli pyrE mutant strain defective for the same enzyme, and expression was shown to be promoted by plasmid sequences. The nucleotide sequence of the 1246-bp DNA fragment encompassing the region of homology with the Podospora gene has been determined. This sequence contains an open reading frame of 699 nucleotides. The deduced amino acid sequence shows 72% similarity with the corresponding Podospora protein.

  16. Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA.

    Science.gov (United States)

    Zheng, Huabao; Wang, Xuan; Yomano, Lorraine P; Shanmugam, Keelnatham T; Ingram, Lonnie O

    2012-06-01

    Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886, Escherichia coli NC3, and Zymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing the thyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in the de novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression of thyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA in E. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA in E. coli would be expected to increase the cellular requirement for dTMP. Increased expression of thyA (E. coli, B. subtilis, or Z. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

  17. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Cai Xuepeng

    2010-07-01

    Full Text Available Abstract Background Porcine circovirus 2 (PCV2 is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS. The capsid (Cap protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli , because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO. The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection.

  18. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

    Science.gov (United States)

    2010-01-01

    Background Porcine circovirus 2 (PCV2) is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS). The capsid (Cap) protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs) in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli ), because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO). The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection. PMID:20646322

  19. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    by recombinant technics to examine the relation between the TKl gene and the TKl protein. In the second part of this investigation a direct expression system for human TKl in E.coli was developed to produce a source of high amounts of TKl, to be able to examine the structure of TKl. The resulting recombinant TKl...... cells and that this modification can not be performed in E.coli....... infections. In the first part of the present investigation a sensitive test for quantitating TKl mRNA (competitive PCR) is developed and the results show that PHA stimulated lymphocytes reveal the same pattern concerning expression of TKl mRNA and TKl enzyme activity as serum-stimulated cells. This pattern...

  20. A general procedure for small-scale purification of fimbriae expressed by porcine enterotoxigenic Escherichia coli strains

    Directory of Open Access Journals (Sweden)

    Ana Cristina Campal Espinosa

    2008-01-01

    Full Text Available Fimbriae expression by enterotoxigenic Escherichia coli strains is a complex process which is controlled by global and local transcriptional regulators and post-transcriptional control. It is influenced by factors such as bacterial growth rate, culture medium composition, pH and temperature. Fimbrial expression could thus frequently become lost. Bacterial culture procedures favouring fimbrial expression are thus needed. The fimbriated bacterial population was therefore enriched by static culture in Mueller–Hinton broth. Fimbrial expression was then maintained by making it grow consecutively in agar CFA and Minca or minimal broth according to the fimbrial serotype. Maximum fimbrial expression was reached after 4h or 5h in culture. The fimbriae were extracted by heat -shock treatment and precipitated with 40% ammonium sulphate. Further purification was carried out by molecular exclusion and sodium deoxycholate treatment. This methodology integrates known procedures in a simple and reproducible process for obtaining F4, F5, F6 and F41 fimbriae in sufficient quantities for their subsequent use in producing antibodies, immunoassays and other studies (at laboratory level requiring high-purity preparations (80% to maintain their native structure. Key words: Enterotoxigenic Escherichia coli; fimbriae; Minca; minimal medium; CFA.

  1. Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

    Science.gov (United States)

    Sharma, Prachi; Sharma, Susheel; Singh, Jasvir; Saha, Swati; Baranwal, V K

    2016-04-01

    Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms.

    Science.gov (United States)

    Prada Medina, Cesar Augusto; Aristizabal Tessmer, Elke Tatjana; Quintero Ruiz, Nathalia; Serment-Guerrero, Jorge; Fuentes, Jorge Luis

    2016-06-01

    Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280-315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed.

  3. Expression, crystallization and preliminary X-ray analysis of the periplasmic stress sensory protein RseB from Escherichia coli

    International Nuclear Information System (INIS)

    Wollmann, Petra; Zeth, Kornelius

    2006-01-01

    The periplasmic stress protein RseB from E. coli was cloned, expressed and crystallized. Crystallographic data are presented and structure solution using the multiple isomorphous replacement approach (MIR) is in progress. Sensing external stress in the bacterial periplasm and signal transduction to the cytoplasm are important functions of the CpxAR, Bae and σ E signalling pathways. In Escherichia coli, the σ E pathway can be activated through degradation of the antisigma factor RseA by DegS and YaeL. The periplasmic protein RseB plays an important role in this pathway by exerting a direct or indirect negative effect on YaeL cleavage efficiency. RseB from E. coli, missing the periplasmic signal sequence (RseB ΔN ), was cloned, expressed, purified and crystallized. Crystals were obtained in two different forms belonging to space group P42 1 2 (form I) and C222 1 (form II) and diffracted to 2.8 and 2.4 Å resolution, respectively. In crystal form I two copies of the protein were located in the asymmetric unit according to heavy-atom analysis, while crystal form II contained three copies

  4. Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

    Science.gov (United States)

    Kawano, Susumu; Iyaguchi, Daisuke; Okada, Chiaki; Sasaki, Yusuke; Toyota, Eiko

    2013-06-01

    Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

  5. Relevance of DNA repair pathways on ascorbic acid effects on Echerichia Coli K-12 cells

    International Nuclear Information System (INIS)

    Slyus, M.A. van; Oliveira, R.L.B. da C.; Felzenszwalb, I.; Gomes, R.A.; Menck, C.F.

    1985-01-01

    Inactivation kinetics were performed with repair proficient and deficient Escherichia coli K-12 cells treated with oxidized solutions of ascorbic acid. The repair pathways controlled by the recA and uvrA gene products are essential for cell survival to the treatment. However, SOS chromotest result indicates that the SOS functions are only induced at high and toxic concentrations of the drug. Moreover, single strand breaks in DNA from treated cells are detected, demonstrating genome damage promoted by oxidized solutions of ascorbate. (M.A.C.) [pt

  6. Hyperthermia studies using inductive and ultrasound methods on E. coli bacteria and mouse glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Cabral–Prieto, A., E-mail: agustin.cabral@inin.gob.mx; López-Callejas, R., E-mail: regulo.lopez@inin.gob.mx; Rodríguez-Méndez, B. G., E-mail: benjamin.rodriguez@inin.gob.mx; Santos-Cuevas, C. L., E-mail: clara.cuevas@inin.gob.mx [Carretera México-Toluca s/n, La Marquesa, Instituto Nacional de Investigaciones Nucleares (Mexico); Celis-Almazán, J., E-mail: jony-jac-5@hotmail.com; Olea-Mejía, O., E-mail: oleaoscar@yahoo.com.mx [Universidad Autónoma del Estado de México, Centro Conjunto de Investigación en Química Sustentable (Mexico); Gómez-Morales, J. L. [Universidad Autónoma del Estado de México, Campus El Cerrillo, Facultad de Ciencias (Mexico); Peña-Eguiluz, R., E-mail: rosendo.eguiluz@inin.gob.mx; Valencia-Alvarado, R., E-mail: raul.valencia@inin.gob.mx; Mercado-Cabrera, A., E-mail: antonio.mercado@inin.gob.mx; Muñoz-Castro, A. E., E-mail: arturo.munoz@inin.gob.mx [Carretera México-Toluca s/n, La Marquesa, Instituto Nacional de Investigaciones Nucleares (Mexico); García-Santibañez, F., E-mail: fegasa2@yahoo.com.mx [Universidad Autónoma del Estado de México, Campus El Cerrillo, Facultad de Ciencias (Mexico)

    2017-11-15

    The survival of Escherichia coli bacteria and mouse glioma cells were studied under different temperatures using direct heating in water, ultrasound, and magnetic fluid hyperthermia. The survival of these microorganisms depended on whether the heating mode was continuous or discontinuous, surviving more in the former than in the discontinuous heating mode. Whereas Escherichia coli bacteria did not survive at temperatures ≥50{sup ∘}C, the mouse glioma cells did not survive at temperatures ≥48{sup ∘}C. The survival of both these microorganisms was independent of the presence or absence of the magnetic nanoparticles of magnetite, suggesting that these, having mean particle sizes of 9.5, 8.5 and 5, did not show any apparent cytotoxicity effect. Present results also showed that the inductive heating system which used a radiofrequency of 13.56 MHz, providing a maximum magnetic field strength of 160 A/m, the electric rather than magnetic heating predominated.

  7. Hyperthermia studies using inductive and ultrasound methods on E. coli bacteria and mouse glioma cells

    International Nuclear Information System (INIS)

    Cabral–Prieto, A.; López-Callejas, R.; Rodríguez-Méndez, B. G.; Santos-Cuevas, C. L.; Celis-Almazán, J.; Olea-Mejía, O.; Gómez-Morales, J. L.; Peña-Eguiluz, R.; Valencia-Alvarado, R.; Mercado-Cabrera, A.; Muñoz-Castro, A. E.; García-Santibañez, F.

    2017-01-01

    The survival of Escherichia coli bacteria and mouse glioma cells were studied under different temperatures using direct heating in water, ultrasound, and magnetic fluid hyperthermia. The survival of these microorganisms depended on whether the heating mode was continuous or discontinuous, surviving more in the former than in the discontinuous heating mode. Whereas Escherichia coli bacteria did not survive at temperatures ≥50"∘C, the mouse glioma cells did not survive at temperatures ≥48"∘C. The survival of both these microorganisms was independent of the presence or absence of the magnetic nanoparticles of magnetite, suggesting that these, having mean particle sizes of 9.5, 8.5 and 5, did not show any apparent cytotoxicity effect. Present results also showed that the inductive heating system which used a radiofrequency of 13.56 MHz, providing a maximum magnetic field strength of 160 A/m, the electric rather than magnetic heating predominated.

  8. CNPase Expression in Olfactory Ensheathing Cells

    Directory of Open Access Journals (Sweden)

    Christine Radtke

    2011-01-01

    Full Text Available A large body of work supports the proposal that transplantation of olfactory ensheathing cells (OECs into nerve or spinal cord injuries can promote axonal regeneration and remyelination. Yet, some investigators have questioned whether the transplanted OECs associate with axons and form peripheral myelin, or if they recruit endogenous Schwann cells that form myelin. Olfactory bulbs from transgenic mice expressing the enhanced green fluorescent protein (eGFP under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase promoter were studied. CNPase is expressed in myelin-forming cells throughout their lineage. We examined CNPase expression in both in situ in the olfactory bulb and in vitro to determine if OECs express CNPase commensurate with their myelination potential. eGFP was observed in the outer nerve layer of the olfactory bulb. Dissociated OECs maintained in culture had both intense eGFP expression and CNPase immunostaining. Transplantation of OECs into transected peripheral nerve longitudinally associated with the regenerated axons. These data indicate that OECs in the outer nerve layer of the olfactory bulb of CNPase transgenic mice express CNPase. Thus, while OECs do not normally form myelin on olfactory nerve axons, their expression of CNPase is commensurate with their potential to form myelin when transplanted into injured peripheral nerve.

  9. Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase

    Directory of Open Access Journals (Sweden)

    Zheng Yanning

    2012-10-01

    Full Text Available Abstract Background Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs, releasing them as free fatty acids (FFAs, which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in Escherichia coli. Therefore, exploring more promising thioesterases will contribute to the development of industrial microbial lipids production. Results We cloned and expressed a cytosolic Acinetobacter baylyi thioesterase (‘AcTesA in E. coli by deleting its leader sequence. Protein sequence alignment, structure modeling and site-directed mutagenesis demonstrated that Ser10, Gly48, Asn77, Asp158 and His161 residues composed the active centre of ‘AcTesA. The engineered strain that overexpressed ‘AcTesA achieved a FFAs titer of up to 501.2 mg/L in shake flask, in contrast to only 20.5 mg/L obtained in wild-type E. coli, demonstrating that the expression of ‘AcTesA indeed boosted the synthesis of FFAs. The ‘AcTesA exhibited a substrate preference towards the C8-C16 acyl groups, with C14:0, C16:1, C12:0 and C8:0 FFAs being the top four components. Optimization of expression level of ‘AcTesA made the FFAs production increase to 551.3 mg/L. The FFAs production further increased to 716.1 mg/L by optimization of the culture medium. Fed-batch fermentation was also carried out to evaluate the FFAs production in a scaleable process. Finally, 3.6 g/L FFAs were accumulated within 48 h, and a maximal FFAs yield of 6.1% was achieved in 12–16 h post induction. Conclusions For the first time, an A. baylyi thioesterase was cloned and solubly expressed in the cytosol of E. coli. This leaderless thioesterase (‘AcTesA was found to be capable of enhancing the FFAs production of E. coli. Without detailed optimization of the strain and fermentation, the finally achieved 3.6 g/L FFAs is encouraging. In

  10. Melibiose permease and alpha-galactosidase of Escherichia coli: Identification by selective labeling using a T7 RNA polymerase/promoter expression system

    International Nuclear Information System (INIS)

    Pourcher, T.; Bassilana, M.; Sarkar, H.K.; Kaback, H.R.; Leblanc, G.

    1990-01-01

    Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested

  11. Translational regulation of gene expression by an anaerobically induced small non-coding RNA in Escherichia coli

    DEFF Research Database (Denmark)

    Boysen, Anders; Møller-Jensen, Jakob; Kallipolitis, Birgitte H.

    2010-01-01

    Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post...... that adaptation to anaerobic growth involves the action of a small regulatory RNA....... of at least one sRNA regulator. Here, we extend this view by the identification and characterization of a highly conserved, anaerobically induced small sRNA in E. coli, whose expression is strictly dependent on the anaerobic transcriptional fumarate and nitrate reductase regulator (FNR). The sRNA, named Fnr...

  12. λ-prophage induction in E.coli cells by radiation with different LET

    International Nuclear Information System (INIS)

    Bonev, M.N.; Collev, S.D.

    1997-01-01

    λ-prophage induction in E.coli H fr H (λ) strain after irradiation with α-particles, accelerated helium ions, boron and carbon ions, as well as deuterons is investigated. The dose dependence of the fraction of induced cells is measured and its initial slope (λ-induction potency - λ i p) is determined. It is shown that the dependence of λ i p on LET is a curve with a maximum

  13. Conditional expression of the vesicular stomatitis virus glycoprotein gene in Escherichia coli.

    OpenAIRE

    Rose, J K; Shafferman, A

    1981-01-01

    Bacterial plasmids that directed expression of the vesicular stomatitis virus glycoprotein (G-protein) gene under control of the tryptophan operon regulatory region were constructed. A plasmid directing the synthesis of a G-protein-like protein (containing the NH2-terminal segment of seven amino acids encoded by the trpE gene fused to the complete G-protein sequence lacking only its NH2-terminal methionine) could be transformed into trpR+ (repressed) but not into trpR- (derepressed) cells. Th...

  14. Insect cells are superior to Escherichia coli in producing malaria proteins inducing IgG targeting PfEMP1 on infected erythrocytes

    DEFF Research Database (Denmark)

    Victor, Michala E; Bengtsson, Anja; Andersen, Gorm

    2010-01-01

    -exposed epitopes are unknown. An insect cell and Escherichia coli based system was used to express single and double domains encoded by the pfd1235w var gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w...... PfEMP1 antigen expressed on 3D7PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed....

  15. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo.

    Science.gov (United States)

    Sankova, Tatiana P; Orlov, Iurii A; Saveliev, Andrey N; Kirilenko, Demid A; Babich, Polina S; Brunkov, Pavel N; Puchkova, Ludmila V

    2017-11-03

    There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST) and the N-terminal domain (ectodomain) of human high affinity copper transporter CTR1 (hNdCTR1), which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell's copper metabolism and its chelating properties are discussed.

  16. Characterization of substances that restore impaired cell division of UV-irradiated E. coli B

    International Nuclear Information System (INIS)

    Yoshiyama, Y.; Shimoii, H.; Tamura, G.

    1981-01-01

    Substances which restore impaired cell division in UV-irradiated E. coli B were surveyed among various bacteria. The active substance was found only in several genera of Gram-negative bacteria, i.e., Escherichia, Enterobacter, Salmonella and some species of Pseudomonas. The activity in the dialyzed cell extract of E. coli B/r was observed in the presence of β-NAD and was enhanced by Mg 2+ and Mn 2+ . The active substance was very labile, but the activity was protected by 1 mM dithiothreitol in the process of purification. The activity of a fraction recovered through DEAE-cellulose column chromatography was stimulated by the presence of membrane fraction. Upon treatment with lipid-degrading enzymes and proteases, the division-stimulating activity was lost or reduced. It appears that the inactivation by lipase and phospholipase A2 was due to the formation of lysophospholipids and that a proteinous substance participated in the recovery of impaired cell division of UV-irradiated E. coli B

  17. 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.

  18. Cloning and Expression of 31kDa Outer Membrane Protein of Brucella melitansis in E.coli

    Directory of Open Access Journals (Sweden)

    Sayeneh Khodadadi

    2012-04-01

    Full Text Available Background & Objectives: The identification of Brucella spp. antigens with the capacity to elicit a protective immune response is of the great interest for the researchers. So, characterization and assessment of diverse antigens of Brucella need to be evaluated. In this study, we report the cloning and expression of the gene coding for 31 KDa OMP (OMP31 of Brucella melitensis 16M.   Methods: Brucella melitensis Omp31 gene was amplified with specific primers, cloned into pJET1/2 and subsequently subcloned in pET28a (+ vector. Both these recombinant plasmids were sequenced and then after, expression of recombinant protein was induced by 1mM IPTG. Western blot analysis was also performed by polyclonal rabbit antiserum.   Results: Omp31 successfully was cloned in both plasmid vectors. The recombinant Omp31 was expressed in E.coli host and purified with significant yield. Western blot results along with those of sequencing ensured accurate production of recombinant omp31 and retaining of its partial epitopes.   Conclusion: Our results show that, an expression host such as E. coli is suitable for omp31 production.

  19. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...... in cell adhesion and the cytoskeleton. If the proteins involved in tethering cells to the extracellular matrix are important in conferring drug resistance, it may be possible to improve chemotherapy by designing drugs that target these proteins....

  20. Dynamic single-cell NAD(P)H measurement reveals oscillatory metabolism throughout the E. coli cell division cycle.

    Science.gov (United States)

    Zhang, Zheng; Milias-Argeitis, Andreas; Heinemann, Matthias

    2018-02-01

    Recent work has shown that metabolism between individual bacterial cells in an otherwise isogenetic population can be different. To investigate such heterogeneity, experimental methods to zoom into the metabolism of individual cells are required. To this end, the autofluoresence of the redox cofactors NADH and NADPH offers great potential for single-cell dynamic NAD(P)H measurements. However, NAD(P)H excitation requires UV light, which can cause cell damage. In this work, we developed a method for time-lapse NAD(P)H imaging in single E. coli cells. Our method combines a setup with reduced background emission, UV-enhanced microscopy equipment and optimized exposure settings, overall generating acceptable NAD(P)H signals from single cells, with minimal negative effect on cell growth. Through different experiments, in which we perturb E. coli's redox metabolism, we demonstrated that the acquired fluorescence signal indeed corresponds to NAD(P)H. Using this new method, for the first time, we report that intracellular NAD(P)H levels oscillate along the bacterial cell division cycle. The developed method for dynamic measurement of NAD(P)H in single bacterial cells will be an important tool to zoom into metabolism of individual cells.

  1. Inhibitory effect of membrane-specific drugs on liquid-holding recovery in U.V.-irradiated E. coli cells

    International Nuclear Information System (INIS)

    Yonei, S.

    1980-01-01

    Liquid-holding recovery (LHR), as been shown to be dependent on the polA + -dependent DNA repair pathways. The experiment described attempted to examine whether the membrane-specific drugs, procaine and chlorpromazine, can inhibit the LHR in U.V.-irradiated cells of E. coli B. Results show that cell membranes may influence DNA repair and ultimate survival of E. coli. (author)

  2. Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells

    International Nuclear Information System (INIS)

    Canuto, K.S.; Guimaraes, O.R.; Geller, M.; Sergio, L.P.S.; Paoli, F.; Fonseca, A.S.

    2015-01-01

    Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia coli cells that were exposed to ultraviolet C (UVC) radiation. Exponential and stationary wild-type and uvrA-deficient E. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC). Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out. (author)

  3. Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Canuto, K.S.; Guimaraes, O.R.; Geller, M. [Centro Universitario Serra dos Orgaos, Teresopolis, RJ (Brazil). Centro de Ciencias da Saude; Sergio, L.P.S. [Instituto de Biologia Roberto Alcantara Gomes, Rio de Janeiro, RJ (Brazil). Departamento de Biofisica e Biometria; Paoli, F. [Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, MG (Brazil). Departamento de Morfologia; Fonseca, A.S., E-mail: adnfonseca@ig.com.br [Universidade Federal do Estado do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ (Brazil). Departamento de Ciencias Fisiologicas

    2015-10-15

    Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia coli cells that were exposed to ultraviolet C (UVC) radiation. Exponential and stationary wild-type and uvrA-deficient E. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC). Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out. (author)

  4. Network analysis of the transcriptional pattern of young and old cells of Escherichia coli during lag phase

    Directory of Open Access Journals (Sweden)

    Hinton Jay CD

    2009-11-01

    Full Text Available Abstract Background The aging process of bacteria in stationary phase is halted if cells are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells and 16 days (old cells. Results A genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186 than in young cells (467. Relatively, few genes (62 were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells. Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were up-regulated in both young

  5. Network analysis of the transcriptional pattern of young and old cells of Escherichia coli during lag phase

    LENUS (Irish Health Repository)

    Pin, Carmen

    2009-11-16

    Abstract Background The aging process of bacteria in stationary phase is halted if cells are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells) and 16 days (old cells). Results A genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186) than in young cells (467). Relatively, few genes (62) were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells. Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were up-regulated in both young and old cells

  6. 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

  7. Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

    International Nuclear Information System (INIS)

    Shigeno, Yuta; Uchiumi, Toshio; Nomura, Takaomi

    2016-01-01

    Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

  8. Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Shigeno, Yuta [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan); Uchiumi, Toshio [Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181 (Japan); Nomura, Takaomi, E-mail: nomurat@shinshu-u.ac.jp [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan)

    2016-04-22

    Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

  9. Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.

    Science.gov (United States)

    Arii, Yasuhiro; Yamaguchi, Hidenori; Fukuoka, Shin-Ichi

    2007-10-01

    The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.

  10. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore.......05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...... studies of vitamin D's effect on TCC cells in vitro are necessary before the efficacy of treatment with vitamin D analogues in TCC can be evaluated in patients....

  11. In Vivo Biochemistry: Single-Cell Dynamics of Cyclic Di-GMP in Escherichia coli in Response to Zinc Overload.

    Science.gov (United States)

    Yeo, Jongchan; Dippel, Andrew B; Wang, Xin C; Hammond, Ming C

    2018-01-09

    Intracellular signaling enzymes drive critical changes in cellular physiology and gene expression, but their endogenous activities in vivo remain highly challenging to study in real time and for individual cells. Here we show that flow cytometry can be performed in complex media to monitor single-cell population distributions and dynamics of cyclic di-GMP signaling, which controls the bacterial colonization program. These in vivo biochemistry experiments are enabled by our second-generation RNA-based fluorescent (RBF) biosensors, which exhibit high fluorescence turn-on in response to cyclic di-GMP. Specifically, we demonstrate that intracellular levels of cyclic di-GMP in Escherichia coli are repressed with excess zinc, but not with other divalent metals. Furthermore, in both flow cytometry and fluorescence microscopy setups, we monitor the dynamic increase in cellular cyclic di-GMP levels upon zinc depletion and show that this response is due to de-repression of the endogenous diguanylate cyclase DgcZ. In the presence of zinc, cells exhibit enhanced cell motility and increased sensitivity to antibiotics due to inhibited biofilm formation. Taken together, these results showcase the application of RBF biosensors in visualizing single-cell dynamic changes in cyclic di-GMP signaling in direct response to environmental cues such as zinc and highlight our ability to assess whether observed phenotypes are related to specific signaling enzymes and pathways.

  12. Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode.

    Science.gov (United States)

    Rezaie, F; Davami, F; Mansouri, K; Agha Amiri, S; Fazel, R; Mahdian, R; Davoudi, N; Enayati, S; Azizi, M; Khalaj, V

    2017-05-08

    The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase ® cultivation mode and a redox mutant strain in the E. coli expression system. A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle ® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase ® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis ® . This study demonstrated that the combination of SUMO fusion technology, EnBase ® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments. © 2017 The Society for Applied Microbiology.

  13. Green biosynthesis of biocompatible CdSe quantum dots in living Escherichia coli cells

    International Nuclear Information System (INIS)

    Yan, Zhengyu; Qian, Jing; Su, Yilong; Ai, Xiaoxia; Wu, Shengmei; Gu, Yueqing

    2014-01-01

    A green and efficient biosynthesis method to prepare fluorescence-tunable biocompatible cadmium selenide quantum dots using Escherichia coli cells as biological matrix was proposed. Decisive factors in biosynthesis of cadmium selenide quantum dots in a designed route in Escherichia coli cells were elaborately investigated, including the influence of the biological matrix growth stage, the working concentration of inorganic reactants, and the co-incubation duration of inorganic metals to biomatrix. Ultraviolet-visible, photoluminescence, and inverted fluorescence microscope analysis confirmed the unique optical properties of the biosynthesized cadmium selenide quantum dots. The size distribution of the nanocrystals extracted from cells and the location of nanocrystals foci in vivo were also detected seriously by transmission electron microscopy. A surface protein capping layer outside the nanocrystals was confirmed by Fourier transform infrared spectroscopy measurements, which were supposed to contribute to reducing cytotoxicity and maintain a high viability of cells when incubating with quantum dots at concentrations as high as 2 μM. Cell morphology observation indicated an effective labeling of living cells by the biosynthesized quantum dots after a 48 h co-incubation. The present work demonstrated an economical and environmentally friendly approach to fabricating highly fluorescent quantum dots which were expected to be an excellent fluorescent dye for broad bio-imaging and labeling. (papers)

  14. Expression, purification, crystallization and preliminary diffraction studies of the mammalian DAG kinase homologue YegS from Escherichia coli

    International Nuclear Information System (INIS)

    Bakali H, M. Amin; Nordlund, Pär; Hallberg, B. Martin

    2006-01-01

    The overexpression, crystallization and preliminary diffraction analysis of E. coli YegS are reported. yegS is a gene encoding a 32 kDa cytosolic protein with unknown function but with strong sequence homology to a family of structurally uncharacterized eukaryotic non-protein kinases: diacylglycerol kinases, sphingosine kinases and ceramide kinases. Here, the overexpression, crystallization and preliminary diffraction analysis of Escherichia coli YegS are reported. The crystals belong to space group P2 1 , with unit-cell parameters a = 42.4, b = 166.1, c = 48.5 Å, β = 96.97°. The presence of a dimer in the asymmetric unit was estimated to give a Matthews coefficient (V M ) of 2.5 Å 3 Da −1 and a solvent content of 50.8%(v/v). Single-wavelength diffraction data were collected to a resolution of 1.9 Å using synchrotron radiation

  15. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1, Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana P. Sankova

    2017-11-01

    Full Text Available There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST and the N-terminal domain (ectodomain of human high affinity copper transporter CTR1 (hNdCTR1, which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell’s copper metabolism and its chelating properties are discussed.

  16. Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

    Science.gov (United States)

    Tagliavia, Marcello; Cuttitta, Angela

    2016-01-01

    High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

  17. Tropomyosin Receptor Kinase A Expression on Merkel Cell Carcinoma Cells.

    Science.gov (United States)

    Wehkamp, Ulrike; Stern, Sophie; Krüger, Sandra; Hauschild, Axel; Röcken, Christoph; Egberts, Friederike

    2017-11-01

    Merkel cell carcinoma (MCC) is a malignant neuroendocrine skin tumor frequently associated with the Merkel cell polyomavirus. Immune checkpoint therapy showed remarkable results, although not all patients are responsive to this therapy. Anti-tropomyosin receptor kinase A (TrkA)-targeted treatment has shown promising results in several tumor entities. To determine TrkA expression in MCC as a rationale for potential targeted therapy. This case series study investigated the MCC specimens of 55 patients treated at the Department of Dermatology, University Hospital of Schleswig-Holstein, Kiel, Germany, from January 1, 2005, through December 31, 2015. Thirty-nine of the 55 samples were suitable for further histopathologic examination. Expression of TrkA was explored by immunohistochemical analysis. Diagnosis of MCC was confirmed by staining positive for cytokeratin 20 (CK20) and synaptophysin. Expression of TrkA on the tumor cells. Specimens of 39 patients (21 women and 18 men; mean [SD] age, 75.0 [7.8] years) underwent immunohistochemical investigation. Thirty-eight of 38 specimens expressed CK20 and synaptophysin on the MCC tumor cells (100% expression). Merkel cell polyomavirus was detected in 32 of 38 specimens (84%). Tropomyosin receptor kinase A was found in all 36 evaluable specimens on the tumor cells; 34 (94%) showed a weak and 2 (6%) showed a strong cytoplasmic expression. In addition, strongly positive perinuclear dots were observed in 30 of 36 specimens (83%). Tropomyosin receptor kinase A was expressed on MCC tumor cells in 100% of evaluable specimens. This result may lead to the exploration of new targeted treatment options in MCC, especially for patients who do not respond to anti-programmed cell death protein 1 treatment.

  18. Cloning and expression of the Escherichia coli K-12 sad gene.

    OpenAIRE

    Marek, L E; Henson, J M

    1988-01-01

    The Escherichia coli K-12 sad gene, which encodes an NAD-dependent succinic semialdehyde dehydrogenase, was cloned into a high-copy-number vector. Minicells carrying a sad+ plasmid produced a 55,000-dalton peptide, the probable sad gene product.

  19. Acquisition of carbapenem resistance by plasmid-encoded-AmpC-expressing Escherichia coli

    NARCIS (Netherlands)

    Tommassen - van Boxtel, Ria; Wattel, Agnes A.; Arenas Busto, Jesus; Goessens, Wil H.F.; Tommassen, J

    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

  20. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Kausik [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Ramagopal, Udupi A. [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Nathenson, Stanley G., E-mail: nathenso@aecom.yu.edu [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Almo, Steven C., E-mail: nathenso@aecom.yu.edu [Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States)

    2009-05-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.

  1. 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

    International Nuclear Information System (INIS)

    Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.

    2009-01-01

    1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL

  2. 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.

  3. DNA synthesis and uv resistance in Escherichia coli K12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Slezarikova, V [Slovenska Akademia Vied, Bratislava (Czechoslovakia). Vyskumny Ustav Onkologicky

    1976-01-01

    The influence was studied of preirradiation inhibition of proteosynthesis by amino acids starvation on survival and DNA synthesis in E. coli K 12 cells, which differ by their genetic features with regard to a certain type of repair. The surviving fraction was studied by appropriate dilution of cell suspension and spreading on agar plates. DNA synthesis was investigated by the incorporation of thymine-2-/sup 14/C. In our conditions a correlation was found between cell survival and the resistance of DNA replication to UV radiation in cells proficient in excision and post-replication repair. This correlation was not found in the excision deficient strain. It is concluded that enhanced resistance of DNA replication is not a sufficient condition for enhanced cell resistance.

  4. Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

    Directory of Open Access Journals (Sweden)

    Schaudinn Christoph

    2005-01-01

    Full Text Available Abstract Background Serotyping of O-(lipopolysaccharide and H-(flagellar antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies. Results The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4 and P12b (O15:H17 was determined and both were found 99.3% (1043 of 1050 nucleotides identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella. Conclusion The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of

  5. Oncomirs Expression Profiling in Uterine Leiomyosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Bruna Cristine de Almeida

    2017-12-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that act as regulators of gene expression at the post-transcriptional level. They play a key role in several biological processes. Their abnormal expression may lead to malignant cell transformation. This study aimed to evaluate the expression profile of 84 miRNAs involved in tumorigenesis in immortalized cells of myometrium (MM, uterine leiomyoma (ULM, and uterine leiomyosarcoma (ULMS. Specific cell lines were cultured and qRT-PCR was performed. Thirteen miRNAs presented different expression profiles in ULM and the same thirteen in ULMS compared to MM. Eight miRNAs were overexpressed, and five were underexpressed in ULM. In ULMS cells, five miRNAs exhibited an overexpression and eight were down-regulated. Six miRNAs (miR-1-3p, miR-130b-3p, miR-140-5p, miR-202-3p, miR-205-5p, and miR-7-5p presented a similar expression pattern in cell lines compared to patient samples. Of these, only three miRNAs showed significant expression in ULM (miR-1-3p, miR-140-5p, and miR-7-5p and ULMS (miR-1-3p, miR-202-3p, and miR-7-5p. Our preliminary approach identified 24 oncomirs with an altered expression profile in ULM and ULMS cells. We identified four differentially expressed miRNAs with the same profile when compared with patients’ samples, which strongly interacted with relevant genes, including apoptosis regulator (BCL2, epidermal growth factor receptor (EGFR, vascular endothelial growth factor A (VEGFA, insulin like growth factor 1 receptor (IGF1R,serine/threonine kinase (RAF1, receptor tyrosine kinase (MET, and bHLH transcription factor (MYCN. This led to alterations in their mRNA-target.

  6. Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

    Directory of Open Access Journals (Sweden)

    Markiv Anatoliy

    2011-11-01

    trafficking studies. Assembling the single chain antibody with monomeric fluorescent protein linker facilitates optimal variable domain pairing and alters the isoelectric point of the recombinant 4D5-8 protein conferring solubility at physiological pH 7.4. The efficient intracellular expression of these functional molecules opens up the possibility of developing an alternative approach for tagging intracellular targets with fluorescent proteins for a range of molecular cell biology imaging studies.

  7. Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

    Directory of Open Access Journals (Sweden)

    Zach Hensel

    Full Text Available DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R and O(L (separated by 2.3 kb, mediated by the λ repressor CI (accession number P03034, play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.

  8. Dissecting stem cell differentiation using single cell expression profiling

    OpenAIRE

    Moignard, Victoria Rachel; Göttgens, Berthold

    2016-01-01

    Many assumptions about the way cells behave are based on analyses of populations. However, it is now widely recognized that even apparently pure populations can display a remarkable level of heterogeneity. This is particularly true in stem cell biology where it hinders our understanding of normal development and the development of strategies for regenerative medicine. Over the past decade technologies facilitating gene expression analysis at the single cell level have become widespread, provi...

  9. Dental enamel cells express functional SOCE channels.

    Science.gov (United States)

    Nurbaeva, Meerim K; Eckstein, Miriam; Concepcion, Axel R; Smith, Charles E; Srikanth, Sonal; Paine, Michael L; Gwack, Yousang; Hubbard, Michael J; Feske, Stefan; Lacruz, Rodrigo S

    2015-10-30

    Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.

  10. A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli

    Directory of Open Access Journals (Sweden)

    Apte-Deshpande Anjali

    2010-05-01

    Full Text Available Abstract Background The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc. Results We describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP as the reporter gene and is constructed in such a way that the E. coli cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts. Conclusions This is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.

  11. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  12. Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study

    Directory of Open Access Journals (Sweden)

    Silvia Y Bando

    2010-09-01

    Full Text Available Enteroinvasive Escherichia coli (EIEC and Shigellaspp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigellashare many genetic and biochemical similarities, the illness caused by Shigellais more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneriwith cultured J774 macrophage-like cells. We evaluated several phenotypes: (i bacterial escape from macrophages after phagocytosis, (ii macrophage death induced by EIEC and S. flexneri, (iii macrophage cytokine expression in response to infection and (iv expression of plasmidial (pINV virulence genes. The results showed thatS. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

  13. Effect of rat ovary irradiation or OVX on the expression of COLI and TGF-β1 mRNA in the rat bone

    International Nuclear Information System (INIS)

    Gao Yanhong; Gao Jianjun; Jin Weifang; Wang Hongfu

    2003-01-01

    To observe the effects of exposure of rat ovary to radiation or OVX on the expression of TGF-β 1 and COLI in the rat bone. The mRNA levels of TGF-β 1 and COLI in rat tibiae were measured with RT-PCR after the rat ovaries were irradiated by 50 Gy of 137 Cs γ-rays or OVX. For both the radiation group and the OVX group, the COLI mRNA level in the rat bone increased, whereas the TGF-β 1 decreased. Irradiation of ovary and OVX affect the expression of COLI and TGF-β 1 mRNA in bone probably in a similar way which is related to estrogen decrease

  14. On the efficient bio-incorporation of 5-hydroxy-tryptophan in recombinant proteins expressed in Escherichia coli with T7 RNA polymerase-based vectors.

    Science.gov (United States)

    Oliveira-Souza, Wellington P; Bronze, Fellipe; Broos, Jaap; Marcondes, Marcelo F M; Oliveira, Vitor

    2017-10-21

    Biosynthetic incorporation of non-canonic amino acids is an attractive strategy to introduce new properties in recombinant proteins. Trp analogs can be incorporated in recombinant proteins replacing regular Trp during protein translation into a Trp-auxotrophic cell host. This straightforward method however, is limited to few analogs recognized and accepted by the cellular protein production machinery. 5-hydroxy-tryptophan (5OH-Trp) can be bio-incorporated using E. coli as expression host however; we have experienced very low incorporation yields - amount of protein containing regular Trp/amount of protein containing the Trp analog - during expressions of 5OH-Trp labeled proteins. Furthermore, this low incorporation yield were verified especially when the widely-used vectors based on the T7 RNA polymerase were used. Testing different 5OH-Trp incorporation protocols we verified that in these T7-based systems, the production of the T7 RNA polymerase is driven by the same elements - lac promoter/IPTG - as the target protein. Consequently, the bio-incorporation of the 5OH-Trp residues also occurs in this crucial enzyme, but, the produced T7 RNA polymerase labeled with 5OH-Trp is inactive or much less active. In the present work, we describe an efficient method to overcome this mentioned problem and bio-incorporate 5OH-Trp in proteins expressed in E. coli., using vectors based on the T7 RNA polymerase-T7 promoter. The two-step induction protocol here described showed incorporation efficiencies of 5OH-Trp higher than 90%. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli

    Directory of Open Access Journals (Sweden)

    Mohammadreza Nassiri

    2015-10-01

    Full Text Available Background: Degradation of phytic acid to inorganic phosphate in domestic animals’ diets requires thermostable phytase. Although Basillus subtilis phytase shows a potential to be degraded phytate complex in high temperature, the enzyme activities and yields need to be increased to make them possible for industrial application. Methods: The phytase gene from Bacillus subtilis DR8886 was isolated from Dig Rostam hot mineral spring in Iran and cloned into pET21(+ and pET32(+. Expression was induced with 1.5 mM IPTG and the proteins were purified. Results: The recombinant protein affected by thioredoxin (Trx from pET32a-PhyC was estimated to constitute about 31% of the total soluble protein in the cells; its concentration was 3.5 μg/ml, and its maximal phytase activity was 15.9 U/ml, whereas the recombinant phytase from pET21a-PhyC was estimated to comprise about 19% of the total soluble protein; its concentration was 2.2 μg/ml, and its maximal phytase activity was 69 U/ml. The molecular masses of recombinant phytase with and without Trx were about 60 kDa and 42 kDa, respectively. Zymography confirmed that the recombinant enzymes were active. Although the concentration of the alkaline phytase expressed by pET32a was approximately 59% greater than that expressed by pET21, its phytase activity was approximately 77% less. Conclusion: This study showed that the peripheral gene (Trx encoded by the pET32a (+ vector are the principal reason for the decrease in recombinant phytase enzyme activity.

  16. Atypical Enteropathogenic Escherichia coli Strains form Biofilm on Abiotic Surfaces Regardless of Their Adherence Pattern on Cultured Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Hebert F. Culler

    2014-01-01

    Full Text Available The aim of this study was to determine the capacity of biofilm formation of atypical enteropathogenic Escherichia coli (aEPEC strains on abiotic and biotic surfaces. Ninety-one aEPEC strains, isolated from feces of children with diarrhea, were analyzed by the crystal violet (CV assay on an abiotic surface after 24 h of incubation. aEPEC strains representing each HEp-2 cell type of adherence were analyzed after 24 h and 6, 12, and 18 days of incubation at 37°C on abiotic and cell surfaces by CFU/cm2 counting and confocal laser scanning microscopy (CLSM. Biofilm formation on abiotic surfaces occurred in 55 (60.4% of the aEPEC strains. There was no significant difference in biofilm biomass formation on an abiotic versus prefixed cell surface. The biofilms could be visualized by CLSM at various developmental stages. aEPEC strains are able to form biofilm on an abiotic surface with no association with their adherence pattern on HEp-2 cells with the exception of the strains expressing UND (undetermined adherence. This study revealed the capacity of adhesion and biofilm formation by aEPEC strains on abiotic and biotic surfaces, possibly playing a role in pathogenesis, mainly in cases of persistent diarrhea.

  17. 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.

  18. [Nitrogen oxide is involved in the regulation of the Fe-S cluster assembly in proteins and the formation of biofilms by Escherichia coli cells].

    Science.gov (United States)

    Vasil'eva, S V; Streltsova, D A; Starostina, I A; Sanina, N A

    2013-01-01

    The functions of nitrogen oxide (NO) in the regulation of the reversible processes of Fe-S cluster assembly in proteins and the formation of Escherichia coli biofilms have been investigated. S-nitrosoglutathione (GSNO) and crystalline nitrosyl complexes of iron with sulfur-containing aliphatic ligands cisaconite (CisA) and penaconite have been used as NO donors for the first time. Wild-type E. coli cells of the strain MC4100, mutants deltaiscA and deltasufA, and the double paralog mutant deltaiscA/sufA with deletions in the alternative pathways of Fe2+ supply for cluster assembly (all derived from the above-named strain) were used in this study. Plankton growth of bacterial cultures, the mass of mature biofilms, and the expression of the SoxRS[2Fe-2S] regulon have been investigated and shown to depend on strain genotype, the process of Fe-S cluster assembly in iron-sulfur proteins, NO donor structure, and the presence of Fe2+ chelator ferene in the incubation medium. The antibiotic ciprofloxacine (CF) was used as an inhibitor of E. coli biofilm formation in the positive control. NO donors regulating Fe-S cluster assembly in E. coli have been shown to control plankton growth of the cultures and the process of mature biofilm formation; toxic doses of NO caused a dramatic (3- to 4-fold) stimulation of cell entry into biofilms as a response to nitrosative stress; NO donors CisA and GSNO in physiological concentrations suppressed the formation of mature biofilms, and the activity of these compounds was comparable to that of CE Regulation of both Fe-S cluster assembly in iron-sulfur proteins and biofilm formation by NO is indicative of the connection between these processes in E. coli.

  19. Cloning, expression, purification and initial crystallographic studies of UbiG: a methyltransferase involved in ubiquinone biosynthesis in Escherichia coli

    International Nuclear Information System (INIS)

    Costa, M.A.F.; Magalhaes, R.D.; Nagem, R.A.P.; Ferreira-Junior, J.R.; Barros, M.H.

    2012-01-01

    Full text: Ubiquinone is a molecule that functions as an electron carrier in the respiratory chain in living organisms. Some clinical phenotypes, including, encephalomyopathy, has been associated with ubiquinone deficiency, raising the interest in the biosynthetic pathway of this molecule. This pathway was proposed mainly from the results of the genetic analysis of mutants of E. coli. UbiG is a methyltransferase involved in ubiquinone biosynthesis in E. coli. In this work we have cloned, expressed, purified and made initial crystallographic assessments of UbiG for later determination of its three-dimensional structure. The gene encoding UbiG was amplified from E. coli genomic DNA by polymerase chain reaction. The 753 bases pairs amplicon was inserted into the expression plasmid pMCSG7 by ligation independent cloning system and transformed into BL21(DE3) E. coli strain. The expression of UbiG, verified by SDS polyacrylamide gel, showed a protein of approximately 29kDa after IPTG induction. The recombinant UbiG, in the soluble fraction of the cellular lysate, was purified by affinity chromatography and the molecular weight of recombinant UbiG of approximately 29 kDa was confirmed by mass spectrometry. After removal of His-tag by TEV protease, another affinity chromatography was performed and UbiG, without His-tag, was observed in flow-through fraction. In Size-Exclusion Chromatography (SEC), the recombinant UbiG showed a unique peak with correct molecular weight of a monomer. Analysis of CD indicated that recombinant UbiG has 31,80% of alpha helix at 20 deg C and DLS showed that 70.9% of the sample is still monomeric in solution even five days after purification. Initial crystallization studies were performed with Crystal Screen 1 and Crystal Screen 2 from Hampton Research. Needle-shaped microcrystals of UbiG were obtained using a precipitant solution consisting of 0,1M lithium sulfate, 0,1M Tris pH 7,5 and 30% w/v polyethylene glycol 4,000. (author)

  20. Lactobacillus zeae protects Caenorhabditis elegans from enterotoxigenic Escherichia coli-caused death by inhibiting enterotoxin gene expression of the pathogen.

    Directory of Open Access Journals (Sweden)

    Mengzhou Zhou

    Full Text Available BACKGROUND: The nematode Caenorhabditis elegans has become increasingly used for screening antimicrobials and probiotics for pathogen control. It also provides a useful tool for studying microbe-host interactions. This study has established a C. elegans life-span assay to preselect probiotic bacteria for controlling K88(+ enterotoxigenic Escherichia coli (ETEC, a pathogen causing pig diarrhea, and has determined a potential mechanism underlying the protection provided by Lactobacillus. METHODOLOGY/PRINCIPAL FINDINGS: Life-span of C. elegans was used to measure the response of worms to ETEC infection and protection provided by lactic acid-producing bacteria (LAB. Among 13 LAB isolates that varied in their ability to protect C. elegans from death induced by ETEC strain JG280, Lactobacillus zeae LB1 offered the highest level of protection (86%. The treatment with Lactobacillus did not reduce ETEC JG280 colonization in the nematode intestine. Feeding E. coli strain JFF4 (K88(+ but lacking enterotoxin genes of estA, estB, and elt did not cause death of worms. There was a significant increase in gene expression of estA, estB, and elt during ETEC JG280 infection, which was remarkably inhibited by isolate LB1. The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode. However, the elt clone killed only approximately 40% of worms. The killing by the clones could also be prevented by isolate LB1. The same isolate only partially inhibited the gene expression of enterotoxins in both ETEC JG280 and E. coli DH5α in-vitro. CONCLUSIONS/SIGNIFICANCE: The established life-span assay can be used for studies of probiotics to control ETEC (for effective selection and mechanistic studies. Heat-stable enterotoxins appeared to be the main factors responsible for the death of C. elegans. Inhibition of ETEC enterotoxin production, rather than interference of its intestinal colonization, appears to be the

  1. Cloning, expression, purification and initial crystallographic studies of UbiG: a methyltransferase involved in ubiquinone biosynthesis in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Costa, M.A.F.; Magalhaes, R.D.; Nagem, R.A.P. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil); Ferreira-Junior, J.R.; Barros, M.H. [Universidade de Sao Paulo (USP), SP (Brazil)

    2012-07-01

    Full text: Ubiquinone is a molecule that functions as an electron carrier in the respiratory chain in living organisms. Some clinical phenotypes, including, encephalomyopathy, has been associated with ubiquinone deficiency, raising the interest in the biosynthetic pathway of this molecule. This pathway was proposed mainly from the results of the genetic analysis of mutants of E. coli. UbiG is a methyltransferase involved in ubiquinone biosynthesis in E. coli. In this work we have cloned, expressed, purified and made initial crystallographic assessments of UbiG for later determination of its three-dimensional structure. The gene encoding UbiG was amplified from E. coli genomic DNA by polymerase chain reaction. The 753 bases pairs amplicon was inserted into the expression plasmid pMCSG7 by ligation independent cloning system and transformed into BL21(DE3) E. coli strain. The expression of UbiG, verified by SDS polyacrylamide gel, showed a protein of approximately 29kDa after IPTG induction. The recombinant UbiG, in the soluble fraction of the cellular lysate, was purified by affinity chromatography and the molecular weight of recombinant UbiG of approximately 29 kDa was confirmed by mass spectrometry. After removal of His-tag by TEV protease, another affinity chromatography was performed and UbiG, without His-tag, was observed in flow-through fraction. In Size-Exclusion Chromatography (SEC), the recombinant UbiG showed a unique peak with correct molecular weight of a monomer. Analysis of CD indicated that recombinant UbiG has 31,80% of alpha helix at 20 deg C and DLS showed that 70.9% of the sample is still monomeric in solution even five days after purification. Initial crystallization studies were performed with Crystal Screen 1 and Crystal Screen 2 from Hampton Research. Needle-shaped microcrystals of UbiG were obtained using a precipitant solution consisting of 0,1M lithium sulfate, 0,1M Tris pH 7,5 and 30% w/v polyethylene glycol 4,000. (author)

  2. Sensibilization of escherichia coli cells by cholesterol incorporated into their membrane

    International Nuclear Information System (INIS)

    Breslev, S.E.; Rozenberg, O.A.; Noskin, L.A.; Stepanova, I.M.; Beketova, A.G.; Loshakova, L.V.; Kovaleva, I.G.

    1984-01-01

    It has been established earlier that a level of cell radiosensitivity is defined by membrane viscosity changing in a wide temperature range. Therefore in epsilon coli cells of a natural type lethal doses of gamma rays are increased approximately a 3.5 times at 45 deg C, as compared to 4 deg C. Cholesterol changing a phase state of membrane lipids was used as a modifying factor. Liposomes were used with the goal of effective bacteria transfer to a membrane. It is established that liposomes without cholesterol do not affect their radioresistance and an increase of its content leads to resistance decrease. The effect is attained only at a sufficient long time of incubation of cells with liposomes (10-16 h). At 4 deg C lipids of E. coli membrane are in a solid-crystalline state independently on pholesterol presence, because of this, radiosensitivity does not change. Temperature increase up to 45 deg C transfer a part of lipids to a liquid-crystalline state, thus decreasing membrane viscosity. In this case cholesterol manifests itself. The authors explain viscosity increase with a violation in functioning of those enzyme systems, which activity is connected with membrane structural state, including enzymes of DNA repair. The authors assume that the radiosensibilization effect of cholesterol introduction into a bacterial membrane in high-temperature cell irradiation is explained by this phenomenon

  3. Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.

    OpenAIRE

    Frothingham, R; Meeker-O'Connell, W A; Talbot, E A; George, J W; Kreuzer, K N

    1996-01-01

    Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which al...

  4. CTX-M-1 β-lactamase expression in Escherichia coli is dependent on cefotaxime concentration, growth phase and gene location

    DEFF Research Database (Denmark)

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

    2015-01-01

    blaCTX-M-1 mRNA expression and CTX-M-1 protein levels were dependent on cefotaxime concentration, growth phase and gene location. These results provide insight into the expression of cephalosporin resistance in CTX-M-1-producing E. coli, improving our understanding of the relationship between ant...

  5. MEMBRANE LEc EXPRESSION IN BREAST CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Ya. A. Udalova

    2009-01-01

    Full Text Available Affine chromatography was used to isolate Lec antibodies from the sera of a healthy female donor with the high titers of these anti- bodies, which were labeled with biotin. The study enrolled 51 patients with primary breast cancer (BC. Antigen expression was found by immunohistochemistry and flow cytometry. With these two techniques being used, the detection rate of Lec expression in BC cells was 65% (33/51; the antigen was most frequently found by flow cytometry as compared with immunohistochemistry: 72 and 58% of cases, respectively.

  6. Metallothionein gene expression in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Deeksha Pal

    2014-01-01

    Full Text Available Introduction: Metallothioneins (MTs are a group of low-molecular weight, cysteine-rich proteins. In general, MT is known to modulate three fundamental processes: (1 the release of gaseous mediators such as hydroxyl radical or nitric oxide, (2 apoptosis and (3 the binding and exchange of heavy metals such as zinc, cadmium or copper. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers. Most of the previous studies primarily used immunohistochemistry to analyze localization of MT in renal cell carcinoma (RCC. No information is available on the gene expression of MT2A isoform in different types and grades of RCC. Materials and Methods: In the present study, total RNA was isolated from 38 histopathologically confirmed cases of RCC of different types and grades. Corresponding adjacent normal renal parenchym