Serum from Nipah Virus Patients Recognises Recombinant Viral Proteins Produced in Escherichia coli.

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Tiong, Vunjia; Lam, Chui-Wan; Phoon, Wai-Hong; AbuBakar, Sazaly; Chang, Li-Yen

2017-01-24

The genes for Nipah virus (NiV) proteins were amplified from viral RNA, cloned into the plasmid pTriEx-3 Hygro, expressed, and purified using immobilized metal affinity chromatography. The recombinant N, F, and G NiV proteins (rNiV-N, rNiV-F, and rNiV-G), were successfully expressed in Escherichia coli and purified with a yield of 4, 16, and 4 mg/L, respectively. All 3 recombinant viral proteins reacted with all 19 samples of NiV-positive human sera. The rNiV-N and rNiV-G proteins were the most immunogenic. The recombinant viral proteins did not react with any of the 12 NiV-negative sera. However, serum from a patient with a late-onset relapsing NiV infection complication was found to be primarily reactive to rNiV-G only. Additionally, there is a distinctive variation in the profile of antigen-reactive bands between the sample from a case of relapsing NiV encephalitis and that of acute NiV infection. The overall findings of this study suggest that the recombinant viral proteins have the potential to be developed further for use in the detection of NiV infection, and continuous biosurveillance of NiV infection in resource-limited settings.

Escherichia coli ArgR mutants defective in cer/Xer recombination, but not in DNA binding.

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Sénéchal, Hélène; Delesques, Jérémy; Szatmari, George

2010-04-01

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls the expression of the arginine biosynthetic genes and is required as an accessory factor for Xer site-specific recombination at cer and related recombination sites in plasmids. We used the technique of pentapeptide scanning mutagenesis to isolate a series of ArgR mutants that were considerably reduced in cer recombination, but were still able to repress an argA::lacZ fusion. DNA sequence analysis showed that all of the mutants mapped to the same nucleotide, resulting in a five amino acid insertion between residues 149 and 150 of ArgR, corresponding to the end of the alpha6 helix. A truncated ArgR containing a stop codon at residue 150 displayed the same phenotype as the protein with the five amino acid insertion, and both mutants displayed sequence-specific DNA-binding activity that was L-arginine dependent. These results show that the C-terminus of ArgR is more important in cer/Xer site-specific recombination than in DNA binding.

Succinic acid production from xylose mother liquor by recombinant Escherichia coli strain.

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Wang, Honghui; Pan, Jiachuan; Wang, Jing; Wang, Nan; Zhang, Jie; Li, Qiang; Wang, Dan; Zhou, Xiaohua

2014-11-02

Succinic acid (1,4-butanedioic acid) is identified as one of important building-block chemicals. Xylose mother liquor is an abundant industrial residue in xylitol biorefining industry. In this study, xylose mother liquor was utilized to produce succinic acid by recombinant Escherichia coli strain SD121, and the response surface methodology was used to optimize the fermentation media. The optimal conditions of succinic acid fermentation were as follows: 82.62 g L -1 total initial sugars, 42.27 g L -1 MgCO 3 and 17.84 g L -1 yeast extract. The maximum production of succinic acid was 52.09 ± 0.21 g L -1 after 84 h with a yield of 0.63 ± 0.03 g g -1 total sugar, approaching the predicted value (53.18 g L -1 ). It was 1.78-fold of the production of that obtained with the basic medium. This was the first report on succinic acid production from xylose mother liquor by recombinant E. coli strains with media optimization using response surface methodology. This work suggested that the xylose mother liquor could be an alternative substrate for the economical production of succinic acid by recombinant E. coli strains.

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

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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)

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli

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Lynn C. Thomason

2016-09-01

Full Text Available Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion.

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.

Prediction of recombinant protein overexpression in Escherichia coli using a machine learning based model (RPOLP).

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Habibi, Narjeskhatoon; Norouzi, Alireza; Mohd Hashim, Siti Z; Shamsir, Mohd Shahir; Samian, Razip

2015-11-01

Recombinant protein overexpression, an important biotechnological process, is ruled by complex biological rules which are mostly unknown, is in need of an intelligent algorithm so as to avoid resource-intensive lab-based trial and error experiments in order to determine the expression level of the recombinant protein. The purpose of this study is to propose a predictive model to estimate the level of recombinant protein overexpression for the first time in the literature using a machine learning approach based on the sequence, expression vector, and expression host. The expression host was confined to Escherichia coli which is the most popular bacterial host to overexpress recombinant proteins. To provide a handle to the problem, the overexpression level was categorized as low, medium and high. A set of features which were likely to affect the overexpression level was generated based on the known facts (e.g. gene length) and knowledge gathered from related literature. Then, a representative sub-set of features generated in the previous objective was determined using feature selection techniques. Finally a predictive model was developed using random forest classifier which was able to adequately classify the multi-class imbalanced small dataset constructed. The result showed that the predictive model provided a promising accuracy of 80% on average, in estimating the overexpression level of a recombinant protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

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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.

Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli.

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Gao, Dongfang; Wang, Shengjun; Li, Haoran; Yu, Huili; Qi, Qingsheng

2015-04-10

The Gram-negative bacterium Escherichia coli has been widely used as a cell factory for the production of proteins and specialty chemicals because it is the best characterized host with many available expression and regulation systems. However, recombinant proteins produced in Escherichia coli are generally intracellular and often found in the form of inclusion bodies. Extracellular production of proteins is advantageous compared with intracellular production because extracellular proteins can be purified more easily and can avoid protease attack, which results in higher product quality. In this study, we found a catalytic domain of a cellulase (Cel-CD) and its N-terminus can be employed as carriers for extracellular production of recombinant proteins. In this report, we identified the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. that can be secreted into the medium from recombinant E. coli BL21 (DE3) in large quantities without its native signal peptide. By subcellular location analysis, we proved that the secretion was a two-step process and the N-terminal sequence of the full length Cel-CD played a crucial function in secretion. Both the Cel-CD and its N-terminal sequence can serve as carriers for efficient extracellular production of select target proteins. Fusion of heterologous proteins with N20 from Cel-CD can carry the target proteins out of the cells with a concentration from 101 to 691 mg/L in flask cultivation. The extracellular recombinant proteins with a relative high purity. The results suggested that this system has a potential application in plant biomass conversion and industrial production of enzymes and therapeutic proteins.

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

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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.

Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

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2012-01-01

Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed

Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

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Jordà Joel

2012-05-01

Full Text Available Abstract Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic

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

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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.

Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL

International Nuclear Information System (INIS)

Douette, Pierre; Navet, Rachel; Gerkens, Pascal; Galleni, Moreno; Levy, Daniel; Sluse, Francis E.

2005-01-01

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein

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

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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.

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

Interaction of Escherichia coli with growing salad spinach plants.

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Warriner, Keith; Ibrahim, Faozia; Dickinson, Matthew; Wright, Charles; Waites, William M

2003-10-01

In this study, the interaction of a bioluminescence-labeled Escherichia coli strain with growing spinach plants was assessed. Through bioluminescence profiles, the direct visualization of E. coli growing around the roots of developing seedlings was accomplished. Subsequent in situ glucuronidase (GUS) staining of seedlings confirmed that E. coli had become internalized within root tissue and, to a limited extent, within hypocotyls. When inoculated seeds were sown in soil microcosms and cultivated for 42 days, E. coli was recovered from the external surfaces of spinach roots and leaves as well as from surface-sterilized roots. When 20-day-old spinach seedlings (from uninoculated seeds) were transferred to soil inoculated with E. coli, the bacterium became established on the plant surface, but internalization into the inner root tissue was restricted. However, for seedlings transferred to a hydroponic system containing 10(2) or 10(3) CFU of E. coli per ml of the circulating nutrient solution, the bacterium was recovered from surface-sterilized roots, indicating that it had been internalized. Differences between E. coli interactions in the soil and those in the hydroponic system may be attributed to greater accessibility of the roots in the latter model. Alternatively, the presence of a competitive microflora in soil may have restricted root colonization by E. coli. The implications of this study's findings with regard to the microbiological safety of minimally processed vegetables are discussed.

Effect of mutagens, chemotherapeutic agents and defects in DNA repair genes on recombination in F' partial diploid Escherichia coli

International Nuclear Information System (INIS)

Norin, A.J.; Goldschmidt, E.P.

1979-01-01

The ability of mutagenic agents, nonmutagenic substances and defects in DNA repair to alter the genotype of F' partial diploid (F30) Escherichia coli was determined. The frequency of auxotrophic mutants and histidine requiring (His - ) haploid colonies was increased by mutagen treatment but Hfr colonies were not detected in F30 E. coli even with specific selection techniques. Genotype changes due to nonreciprocal recombination were determined by measuring the frequency of His - homogenotes, eg. F' hisC780, hisI + /hisC780, hisI + , arising from a His + heterogenote, F' hisC780 hisI + /hisC + , his1903. At least 75% of the recombinants were homozygous for histidine alleles which were present on the F' plasmid (exogenote) of the parental hetergenote rather than for histidine alleles on the chromosome. Mutagens, chemotherapeutic agents which block DNA synthesis and a defective DNA polymerase I gene, polA1, were found to increase the frequency of nonreciprocal recombination. A defect in the ability to excise thymine dimers, uvrC34, did not increase spontaneous nonreciprocal recombination. However, UV irradiation but not methyl methanesulfonate (MMS) induced greater recombination in this excision-repair defective mutant than in DNA-repair-proficient strains. (Auth.)

Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917.

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Fiege, Kerstin; Querebillo, Christine Joy; Hildebrandt, Peter; Frankenberg-Dinkel, Nicole

2018-05-15

Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.

The unconventional xer recombination machinery of Streptococci/Lactococci

NARCIS (Netherlands)

Le Bourgeois, Pascal; Bugarel, Marie; Campo, Nathalie; Daveran-Mingot, Marie-Line; Labonte, Jessica; Lanfranchi, Daniel; Lautier, Thomas; Pages, Carine; Ritzenthaler, Paul

Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving

Induction of genetic recombination in the lambda bacteriophage by ultraviolet irradiation of the Escherichia Coli cells. III. Role of the ruvA and recN genes

International Nuclear Information System (INIS)

Alcantara D, D.

1987-05-01

The objective of this work is to determine the paper of the genes ruvA and recN in the stimulation of the recombination of Lambda for UV irradiation of Escherichia Coli, taking into account that both genes are inducible, they belong to the group of genes that participate in the SOS response and that a deficiency in its expression reduces the capacity to repair and recombiner the DNA. (Author)

Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose production in recombinant Escherichia coli.

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Lee, Won-Heong; Shin, So-Yeon; Kim, Myoung-Dong; Han, Nam Soo; Seo, Jin-Ho

2012-03-01

Guanosine 5'-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5'-diphosphate (GDP)-L-fucose. In this study, improvement of GDP-L-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-L-fucose. The effects of overexpression of inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine-inosine kinase (Gsk) on GDP-L-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-L-fucose production. Maximum GDP-L-fucose concentration of 305.5 ± 5.3 mg l(-1) was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes. Such an enhancement of GDP-L-fucose production could be due to the increase in the intracellular level of GMP.

Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

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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.

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

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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. 

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

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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.

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.

Anti-aggregatory effect of cyclodextrins in the refolding process of recombinant growth hormones from Escherichia coli inclusion bodies

DEFF Research Database (Denmark)

Bajorunaite, Egle; Cirkovas, Andrejus; Radzevicius, Kostas

2009-01-01

Cyclodextrins with different ring size and ring substituents were tested for recombinant mink and porcine growth hormones aggregation suppression in the refolding process from Escherichia coli inclusion bodies. Methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin show a positive effect...... on the aggregation suppression of both proteins. The influence of different methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin concentrations on the renaturation yield of both growth hormones was investigated. Moreover, methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin suppress not only folding...

Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

OpenAIRE

Eggers, Jessica; Steinbüchel, Alexander

2014-01-01

The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this p...

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

Science.gov (United States)

Thomason, Lynn C; Costantino, Nina; Court, Donald L

2016-09-13

Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single

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)

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)

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.

Vanillin production by recombinant strains of Escherichia coli Produção de vanilina por linhagens recombinantes de Escherichia coli

Directory of Open Access Journals (Sweden)

Attilio Converti

2003-11-01

Full Text Available Vanillin production from ferulate was studied using different recombinant strains of Escherichia coli. To prevent the occurrence of aerobic conditions and then possible product oxidation, tests were performed in Erlenmeyer flasks under mild mixing (150 rpm. Among other transformants, E. coli JM109(pBB1 appeared to be the best vanillin producer, being able to convert no less than 95% of starting ferulate to the product within 1h. This yield decreased down to 72% after 72h, likely because of a non-specific oxidase activity responsible for vanillin oxidation to vanillate.A produção de vanilina a partir de ácido ferúlico foi estudada utilizando-se diferentes linhagens recombinantes de Escherichia coli. Para prevenir a ocorrência de condições de aerobiose e a possível oxidação do produto, os ensaios foram realizados em frascos Erlenmeyer sob agitação moderada (150 rpm. E. coli JM109 (pBBI mostrou-se o melhor produtor de vanilina entre os demais agentes transformantes, sendo capaz de converter 95% do ácido ferúlico inicial em produto após 1h, rendimento este que decresceu para 72% após 72h, provavelmente devido à atividade de uma oxidase não-específica responsável pela oxidação de vanilina a ácido vanílico.

Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag.

Science.gov (United States)

Raran-Kurussi, Sreejith; Waugh, David S

2017-01-01

Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His 6 - or a dual His 6 -MBP tagged fusion protein by Gateway ® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His 6 tag or a His 6 -MBP tag can be made on the basis of this solubility test.

Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.

Science.gov (United States)

Lee, Won-Heong; Chin, Young-Wook; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

2011-08-01

Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

Analysis of the efficiency of recombinant Escherichia coli strain cultivation in a gas-vortex bioreactor.

Science.gov (United States)

Savelyeva, Anna V; Nemudraya, Anna A; Podgornyi, Vladimir F; Laburkina, Nadezhda V; Ramazanov, Yuriy A; Repkov, Andrey P; Kuligina, Elena V; Richter, Vladimir A

2017-09-01

The levels of aeration and mass transfer are critical parameters required for an efficient aerobic bioprocess, and directly depend on the design features of exploited bioreactors. A novel apparatus, using gas vortex for aeration and mass transfer processes, was constructed in the Center of Vortex Technologies (Novosibirsk, Russia). In this paper, we compared the efficiency of recombinant Escherichia coli strain cultivation using novel gas-vortex technology with conventional bioprocess technologies such as shake flasks and bioreactors with mechanical stirrers. We demonstrated that the system of aeration and agitation used in gas-vortex bioreactors provides 3.6 times higher volumetric oxygen transfer coefficient in comparison with mechanical bioreactor. The use of gas-vortex bioreactor for recombinant E. coli strain cultivation allows to increase the efficiency of target protein expression at 2.2 times for BL21(DE3)/pFK2 strain and at 3.5 times for auxotrophic C600/pRT strain (in comparison with stirred bioreactor). © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli

International Nuclear Information System (INIS)

Moreau, P.L.

1988-01-01

Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA

Anti-proliferative activity of recombinant melittin expressed in ...

African Journals Online (AJOL)

Recombinant melittin was then successfully expressed in Escherichia coli. The activity of affinity-purified recombinant melittin was determined in human leukemic U937 cells. Results show that the recombinant melittin had the same anti-proliferative activity in human leukemic U937 cells in vitro as natural one. This shows the ...

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant Escherichia coli strain FBR5

Energy Technology Data Exchange (ETDEWEB)

Saha, Badal C.; Nichols, Nancy N.; Qureshi, Nasib; Cotta, Michael A. [U.S. Department of Agriculture, Agricultural Research Services Peoria, IL (United States). Bioenergy Reserach Unit

2011-11-15

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H2SO4) pretreated (160 C, 10 min) and enzymatically saccharified (pH 5.0, 45 C, 72 h) WS (86 g/l) was 50.0 {+-} 1.4 g/l. The hydrolyzate contained 1,184 {+-} 19 mg furfural and 161 {+-} 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 C. However, it produced 21.9 {+-} 0.3 g ethanol from non-abated WSH (total sugars, 44.1 {+-} 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 C for 15 h. The bacterium produced 21.6 {+-} 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 {+-} 0.4 g) at pH 6.0. It produced 24.9 {+-} 0.3 g ethanol in 96 h and 26.7 {+-} 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield. (orig.)

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.

Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid.

Science.gov (United States)

Sun, Jingchen; Yao, Lunguang; Yao, Ning; Xu, Hua; Jin, Pengfei; Kan, Yunchao

2010-12-01

The present study elaborates a cost-effective and transfectant-free method for generating recombinant Bombyx mori (silkworm) nucleopolyhedrovirus in silkworm larvae and pupae by injecting invasive Escherichia coli carrying BmBacmid [BmNPV (B. mori nucleopolyhedrovirus)-Bacmid] into larval haemocoel. Up to 109 PFU (plaque-forming units)/ml of infective recombinant baculovirus was generated in the silkworm by intrahaemocoelic injection with 106 DAP (diaminopimelic acid) auxotrophic and BmBacmid containing E. coli cells expressing both invasin and listeriolysin. Thus 1 ml of overnight culture of E. coli is sufficient to inject more than 2000 larvae, while DAP costing up to $1 is enough to inject about 4000 larvae. Recombinant proteins can be controlled to be expressed mainly in pupae by adjusting the injection dose, too. In this new method, many original manipulations have been eliminated, including BmBacmid preparation and the subsequent complex transfection procedures. Hence it is a time- and cost-saving means for large-scale injection of B. mori for recombinant baculovirus production in comparison with the traditional transfection methods, which may play an important role in the industrial development of the BmNPV-silkworm bioreactor.

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.

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

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.)

Successful recombinant production of Allochromatium vinosum cytochrome c' requires coexpression of cmm genes in heme-rich Escherichia coli JCB712

International Nuclear Information System (INIS)

Evers, Toon H.; Merkx, Maarten

2005-01-01

Cytochrome c' from the purple photosynthetic bacterium Allochromatium vinosum (CCP) displays a unique, reversible dimer-to-monomer transition upon binding of NO, CO, and CN - . This small, four helix bundle protein represents an attractive model for the study of other heme protein biosensors, provided a recombinant expression system is available. Here we report the development of an efficient expression system for CCP that makes use of a maltose binding protein fusion strategy to enhance periplasmic expression and allow easy purification by affinity chromatography. Coexpression of cytochrome c maturase genes and the use of a heme-rich Escherichia coli strain were found to be necessary to obtain reasonable yields of cytochrome c'. Characterization using circular dichroism, UV-vis spectroscopy, and size-exclusion chromatography confirms the native-like properties of the recombinant protein, including its ligand-induced monomerization

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.

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

Science.gov (United States)

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

2017-07-01

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

Fermentation of sweet whey by recombinant Escherichia coli K011 Fermentação de soro de leite por Escherichia coli KO11 recombinante

Directory of Open Access Journals (Sweden)

Amarildo Ricardo Leite

2000-09-01

Full Text Available The production of ethanol from sweet whey using the recombinant Escherichia coli KO11, in batch fermentation, was tested. The maximum ethanol yield was reached after 96h, representing only 38% of the theoretical yield. The supplementation of whey with components of LB broth increased the maximum yield to 96% in 72h. The addition of 0.5% yeast extract to whey resulted in maximum yield of 74% at 36h and it increased to over 100% when yeast extract and trace metals solution (Fe++, Mn++ and Zn++ were added.A produção de etanol a partir de soro de leite empregando a cepa Escherichia coli KO11 recombinante, em fermentação de batelada, foi testada. O rendimento máximo de etanol foi obtido em 96h, representando apenas 38% do rendimento teórico. A suplementação do soro com os componentes do caldo LB aumentou o rendimento para 96% em 72h. A adição de 0,5% de extrato de levedura ao soro resultou em um rendimento máximo de 74% em 36h que aumentou para acima de 100% quando se adicionou extrato de levedura e uma solução de metais traço (Fe++, Mn++ e Zn++.

A review of machine learning methods to predict the solubility of overexpressed recombinant proteins in Escherichia coli.

Science.gov (United States)

Habibi, Narjeskhatoon; Mohd Hashim, Siti Z; Norouzi, Alireza; Samian, Mohammed Razip

2014-05-08

Over the last 20 years in biotechnology, the production of recombinant proteins has been a crucial bioprocess in both biopharmaceutical and research arena in terms of human health, scientific impact and economic volume. Although logical strategies of genetic engineering have been established, protein overexpression is still an art. In particular, heterologous expression is often hindered by low level of production and frequent fail due to opaque reasons. The problem is accentuated because there is no generic solution available to enhance heterologous overexpression. For a given protein, the extent of its solubility can indicate the quality of its function. Over 30% of synthesized proteins are not soluble. In certain experimental circumstances, including temperature, expression host, etc., protein solubility is a feature eventually defined by its sequence. Until now, numerous methods based on machine learning are proposed to predict the solubility of protein merely from its amino acid sequence. In spite of the 20 years of research on the matter, no comprehensive review is available on the published methods. This paper presents an extensive review of the existing models to predict protein solubility in Escherichia coli recombinant protein overexpression system. The models are investigated and compared regarding the datasets used, features, feature selection methods, machine learning techniques and accuracy of prediction. A discussion on the models is provided at the end. This study aims to investigate extensively the machine learning based methods to predict recombinant protein solubility, so as to offer a general as well as a detailed understanding for researches in the field. Some of the models present acceptable prediction performances and convenient user interfaces. These models can be considered as valuable tools to predict recombinant protein overexpression results before performing real laboratory experiments, thus saving labour, time and cost.

Multiplex Genome Editing in Escherichia coli

DEFF Research Database (Denmark)

Ingemann Jensen, Sheila; Nielsen, Alex Toftgaard

2018-01-01

Lambda Red recombineering is an easy and efficient method for generating genetic modifications in Escherichia coli. For gene deletions, lambda Red recombineering is combined with the use of selectable markers, which are removed through the action of, e.g., flippase (Flp) recombinase. This PCR......-based engineering method has also been applied to a number of other bacteria. In this chapter, we describe a recently developed one plasmid-based method as well as the use of a strain with genomically integrated recombineering genes, which significantly speeds up the engineering of strains with multiple genomic...

Co-solute assistance in refolding of recombinant proteins | Gerami ...

African Journals Online (AJOL)

Prokaryotic expression system is the most widely used host for the production of recombinant proteins but inclusion body formation is a major bottleneck in the production of recombinant proteins in prokaryotic cells, especially in Escherichia coli. In vitro refolding of inclusion body into the the proteins with native ...

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

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

African Journals Online (AJOL)

Expression and purification of recombinant Shiga toxin 2B from Escherichia coli O157:H7. ... (SDS-PAGE) and StxB2 yield was 450 μg ml-1 confirmed by Bradford assay. Recombinant Stx2B protein was produced in highly pure yield using ...

Decolorization of dyes by recombinase CotA from Escherichia coli ...

African Journals Online (AJOL)

The CotA laccase could efficiently decolorize anthraquinone and azo dyes in 24 h. The decolourization capacity of this recombinant laccase suggested that it could be a useful biocatalyst for the treatment of dye-containing effluents. Key words: Recombinant CotA laccase, Escherichia coli, purification, dye decolorization.

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering.

Science.gov (United States)

Zhai, Yafei; Han, Donglei; Pan, Ying; Wang, Shuaishuai; Fang, Junqiang; Wang, Peng; Liu, Xian-wei

2015-02-01

Guanosine 5'-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5'-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue. Copyright © 2014 Elsevier Inc. All rights reserved.

Induction of genetic recombination in the lambda bacteriophage by ultraviolet irradiation of the Escherichia Coli cells. III. Role of the ruvA and recN genes; Induccion de recombinacion genetica en el bacteriofago lambda por irradiacion ultravioleta de las celulas de Escherichia Coli. III. Papel de los genes ruvA and recN

Energy Technology Data Exchange (ETDEWEB)

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

1987-05-15

The objective of this work is to determine the paper of the genes ruvA and recN in the stimulation of the recombination of Lambda for UV irradiation of Escherichia Coli, taking into account that both genes are inducible, they belong to the group of genes that participate in the SOS response and that a deficiency in its expression reduces the capacity to repair and recombiner the DNA. (Author)

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

Science.gov (United States)

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2017-04-01

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

Vanillin production using metabolically engineered Escherichia coli under non-growing conditions

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Fava Fabio

2007-04-01

Full Text Available Abstract Background Vanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details. Results Effect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting cells of E. coli strain JM109 harbouring a low-copy number vector and a promoter exhibiting a low activity to drive the expression of the catabolic genes. Optimization of the bioconversion of ferulic acid to vanillin was accomplished by a response surface methodology. The experimental conditions that allowed us to obtain high values for response functions were 3.3 mM ferulic acid and 4.5 g/L of biomass, with a yield of 70.6% and specific productivity of 5.9 μmoles/g × min after 3 hours of incubation. The final concentration of vanillin in the medium was increased up to 3.5 mM after a 6-hour incubation by sequential spiking of 1.1 mM ferulic acid. The resting cells could be reused up to four times maintaining the production yield levels over 50%, thus increasing three times the vanillin obtained per gram of biomass. Conclusion Ferulic acid can be efficiently converted to vanillin, without accumulation of undesirable vanillin reduction/oxidation products

Vanillin production using metabolically engineered Escherichia coli under non-growing conditions.

Science.gov (United States)

Barghini, Paolo; Di Gioia, Diana; Fava, Fabio; Ruzzi, Maurizio

2007-04-16

Vanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details. Effect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting cells of E. coli strain JM109 harbouring a low-copy number vector and a promoter exhibiting a low activity to drive the expression of the catabolic genes. Optimization of the bioconversion of ferulic acid to vanillin was accomplished by a response surface methodology. The experimental conditions that allowed us to obtain high values for response functions were 3.3 mM ferulic acid and 4.5 g/L of biomass, with a yield of 70.6% and specific productivity of 5.9 micromoles/g x min after 3 hours of incubation. The final concentration of vanillin in the medium was increased up to 3.5 mM after a 6-hour incubation by sequential spiking of 1.1 mM ferulic acid. The resting cells could be reused up to four times maintaining the production yield levels over 50%, thus increasing three times the vanillin obtained per gram of biomass. Ferulic acid can be efficiently converted to vanillin, without accumulation of undesirable vanillin reduction/oxidation products, using E. coli JM109 cells expressing genes from the ferulic

Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

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Nida Toufiq

Full Text Available Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L. cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.

Genetic dependence of recombination in recD mutants of Escherichia coli

International Nuclear Information System (INIS)

Lovett, S.T.; Luisi-DeLuca, C.; Kolodner, R.D.

1988-01-01

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations

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.

Limiting factors in Escherichia colifed-batch production of recombinant proteins

DEFF Research Database (Denmark)

Sanden, A.M.; Prytz, I.; Tubelekas, I.

2003-01-01

recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation......recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation...

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

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Willson Richard C

2010-12-01

Full Text Available Abstract Background Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Results Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. Conclusions The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use

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.

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.

Recombination Blurs Phylogenetic Groups Routine Assignment in Escherichia coli: Setting the Record Straight

Science.gov (United States)

Turrientes, María-Carmen; González-Alba, José-María; del Campo, Rosa; Baquero, María-Rosario; Cantón, Rafael; Baquero, Fernando; Galán, Juan Carlos

2014-01-01

The characterization of population structures plays a main role for understanding outbreaks and the dynamics of bacterial spreading. In Escherichia coli, the widely used combination of multiplex-PCR scheme together with goeBURST has some limitations. The purpose of this study is to show that the combination of different phylogenetic approaches based on concatenated sequences of MLST genes results in a more precise assignment of E. coli phylogenetic groups, complete understanding of population structure and reconstruction of ancestral clones. A collection of 80 Escherichia coli strains of different origins was analyzed following the Clermont and Doumith's multiplex-PCR schemes. Doumith's multiplex-PCR showed only 1.7% of misassignment, whereas Clermont's-2000 protocol reached 14.0%, although the discrepancies reached 30% and 38.7% respectively when recombinant C, F and E phylogroups were considered. Therefore, correct phylogroup attribution is highly variable and depends on the clonal composition of the sample. As far as population structure of these E. coli strains, including 48 E. coli genomes from GenBank, goeBURST provides a quite dispersed population structure; whereas NeighborNet approach reveals a complex population structure. MLST-based eBURST can infer different founder genotypes, for instance ST23/ST88 could be detected as the founder genotypes for STC23; however, phylogenetic reconstructions might suggest ST410 as the ancestor clone and several evolutionary trajectories with different founders. To improve our routine understanding of E. coli molecular epidemiology, we propose a strategy based on three successive steps; first, to discriminate three main groups A/B1/C, D/F/E and B2 following Doumith's protocol; second, visualization of population structure based on MLST genes according to goeBURST, using NeighborNet to establish more complex relationships among STs; and third, to perform, a cost-free characterization of evolutionary trajectories in variants

Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

International Nuclear Information System (INIS)

1984-01-01

This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro

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.

Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli.

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Karen Einsfeldt

Full Text Available L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.

Hydrogen production by recombinant Escherichia coli strains

Science.gov (United States)

Maeda, Toshinari; Sanchez‐Torres, Viviana; Wood, Thomas K.

2012-01-01

Summary The production of hydrogen via microbial biotechnology is an active field of research. Given its ease of manipulation, the best‐studied bacterium Escherichia coli has become a workhorse for enhanced hydrogen production through metabolic engineering, heterologous gene expression, adaptive evolution, and protein engineering. Herein, the utility of E. coli strains to produce hydrogen, via native hydrogenases or heterologous ones, is reviewed. In addition, potential strategies for increasing hydrogen production are outlined and whole‐cell systems and cell‐free systems are compared. PMID:21895995

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.

Parallel steady state studies on a milliliter scale accelerate fed-batch bioprocess design for recombinant protein production with Escherichia coli.

Science.gov (United States)

Schmideder, Andreas; Cremer, Johannes H; Weuster-Botz, Dirk

2016-11-01

In general, fed-batch processes are applied for recombinant protein production with Escherichia coli (E. coli). However, state of the art methods for identifying suitable reaction conditions suffer from severe drawbacks, i.e. direct transfer of process information from parallel batch studies is often defective and sequential fed-batch studies are time-consuming and cost-intensive. In this study, continuously operated stirred-tank reactors on a milliliter scale were applied to identify suitable reaction conditions for fed-batch processes. Isopropyl β-d-1-thiogalactopyranoside (IPTG) induction strategies were varied in parallel-operated stirred-tank bioreactors to study the effects on the continuous production of the recombinant protein photoactivatable mCherry (PAmCherry) with E. coli. Best-performing induction strategies were transferred from the continuous processes on a milliliter scale to liter scale fed-batch processes. Inducing recombinant protein expression by dynamically increasing the IPTG concentration to 100 µM led to an increase in the product concentration of 21% (8.4 g L -1 ) compared to an implemented high-performance production process with the most frequently applied induction strategy by a single addition of 1000 µM IPGT. Thus, identifying feasible reaction conditions for fed-batch processes in parallel continuous studies on a milliliter scale was shown to be a powerful, novel method to accelerate bioprocess design in a cost-reducing manner. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1426-1435, 2016. © 2016 American Institute of Chemical Engineers.

Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli

Science.gov (United States)

Finkenwirth, Friedrich; Kirsch, Franziska

2013-01-01

Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [3H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane. PMID:23836870

Engineering the productivity of recombinant Escherichia coli for limonene formation from glycerol in minimal media.

Science.gov (United States)

Willrodt, Christian; David, Christian; Cornelissen, Sjef; Bühler, Bruno; Julsing, Mattijs K; Schmid, Andreas

2014-08-01

The efficiency and productivity of cellular biocatalysts play a key role in the industrial synthesis of fine and bulk chemicals. This study focuses on optimizing the synthesis of (S)-limonene from glycerol and glucose as carbon sources using recombinant Escherichia coli. The cyclic monoterpene limonene is extensively used in the fragrance, food, and cosmetic industries. Recently, limonene also gained interest as alternative jet fuel of biological origin. Key parameters that limit the (S)-limonene yield, related to genetics, physiology, and reaction engineering, were identified. The growth-dependent production of (S)-limonene was shown for the first time in minimal media. E. coli BL21 (DE3) was chosen as the preferred host strain, as it showed low acetate formation, fast growth, and high productivity. A two-liquid phase fed-batch fermentation with glucose as the sole carbon and energy source resulted in the formation of 700 mg L(org) (-1) (S)-limonene. Specific activities of 75 mU g(cdw) (-1) were reached, but decreased relatively quickly. The use of glycerol as a carbon source resulted in a prolonged growth and production phase (specific activities of ≥50 mU g(cdw) (-1) ) leading to a final (S)-limonene concentration of 2,700 mg L(org) (-1) . Although geranyl diphosphate (GPP) synthase had a low solubility, its availability appeared not to limit (S)-limonene formation in vivo under the conditions investigated. GPP rerouting towards endogenous farnesyl diphosphate (FPP) formation also did not limit (S)-limonene production. The two-liquid phase fed-batch setup led to the highest monoterpene concentration obtained with a recombinant microbial biocatalyst to date. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Effects of UV radiation on genetic recombination

International Nuclear Information System (INIS)

Vlahovic, K.; Zahradka, D.; Petranovic, M.; Petranovic, D.

1996-01-01

We have used the model consisting of Escherichia coli cells and l phage to study the effects of UV radiation on genetic recombination. We found two radiation induced processes that reduce or inhibit genetic recombination. One such process leads to the inability of prophage to excise itself from the irradiated bacterial chromosome by the site-specific recombination. The other process was shown to inhibit a type of general recombination by which the prophage transfers one of its genetic markers to the infecting homologous phage. Loss of the prophage ability to take part in both site-specific and general recombination was shown to develop in recB + but not in recB cells. From this we infer that the loss of prophage recombinogenicity in irradiated cells is a consequence of one process in which RecBCD enzyme (the product of recB, recC and recD genes) plays an essential role. (author)

Genetic characterization of somatic recombination in Trichoderma pseudokoningii

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Barcellos Fernando Gomes

2003-01-01

Full Text Available Crossing experiments via hyphal anastomosis between two strains contrasting for auxotrophic markers of Trichoderma pseudokoningii were conducted to characterize the somatic recombination process in this specie. Four crossings were made and a total of 1052 colonies obtained from conidial suspensions of the heterokaryotic colonies were analyzed. Sixty-eight recombinant colonies, from four growing generations, were analyzed for the auxotrophic markers. Of the 68 colonies analyzed, 58 were stable after four generations and the remainders were unstable, reverting to one of the parentals. Most of the recombinant colonies were unstable through subculture and after four growing generations they showed the leu ino met markers (auxotrophic for leucin, inositol and metionin respectively. The unstable recombinant colonies showed irregular growing borders, sparse sporulation and frequent sector formation. The results suggest the occurrence of recombination mechanisms in the heterokaryon (somatic recombination, different from those described for the parasexual cycle or parameiosis. Therefore, we proposed the ocurrence of nuclei degradation from one parental (non prevalent parental in the heterokaryon and that the resulting chromosomal fragments may be incorporated into whole nuclei of the another parental (prevalent parental. However the parameiosis as originally described cannot be excluded.

Maximized Autotransporter-Mediated Expression (MATE for Surface Display and Secretion of Recombinant Proteins in Escherichia coli

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Shanna Sichwart

2015-01-01

Full Text Available A new optimized system for the surface display and secretion of recombinant proteins is described, termed MATE (maximized autotransporter-mediated expression. It is based on an artificial gene consisting of the coding region for the signal peptide of CtxB, a multiple cloning site for passenger gene insertion, flanked by coding sequences for linear epitopes for monoclonal antibodies and OmpT, and factor Xa protease cleavage sites followed by a codon-optimized DNA sequence of the linker and the β-barrel of the type V autotransporter EhaA from Escherichia coli under control of an IPTG-inducible T5 promoter. The MATE system enabled the continuous secretion of recombinant passenger mCherry via OmpT-mediated cleavage, using native OmpT protease activity in E. coli when grown at 37 °C. It is the first example to show that native OmpT activity is sufficient to facilitate the secretion of a correctly folded target protein in preparative amounts obtaining 240 μg of purified mCherry from 800 mL of crude culture supernatant. Because the release of mCherry was achieved by a simple transfer of the encoding plasmid from an OmpT-negative to an OmpT-positive strain, it bears the option to use surface display for screening purposes and secretion for production of the selected variant. A single plasmid could therefore be used for continuous secretion in OmpT-positive strains or surface display in OmpT-negative strains. In conclusion, the MATE system appears to be a versatile tool for the surface display and for the secretion of target proteins in E. coli.

New strategies for genetic engineering Pseudomonas syringae using recombination

Science.gov (United States)

Here we report that DNA oligonucleotides (oligos) introduced directly into bacteria by electroporation can recombine with the bacterial chromosome. This phenomenon was identified in Pseudomonas syringae and we subsequently found that Escherichia coli, Salmonella typhimurium and Shigella flexneri are...

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.

Production of a recombinant swollenin from Trichoderma harzianum in Escherichia coli and its potential synergistic role in biomass degradation.

Science.gov (United States)

Santos, Clelton A; Ferreira-Filho, Jaire A; O'Donovan, Anthonia; Gupta, Vijai K; Tuohy, Maria G; Souza, Anete P

2017-05-16

Fungal swollenins (SWOs) constitute a class of accessory proteins that are homologous to canonical plant expansins. Expansins and expansin-related proteins are well known for acting in the deagglomeration of cellulose structure by loosening macrofibrils. Consequently, SWOs can increase the accessibility and efficiency of the other enzymes involved in the saccharification of cellulosic substrates. Thus, SWOs are promising targets for improving the hydrolysis of plant biomass and for use as an additive to enhance the efficiency of an enzyme cocktail designed for the production of biofuels. Here, we report the initial characterization of an SWO from Trichoderma harzianum (ThSwo) that was successfully produced using Escherichia coli as a host. Initially, transcriptome and secretome data were used to compare swo gene expression and the amount of secreted ThSwo. The results from structural modeling and phylogenetic analysis of the ThSwo protein showed that ThSwo does preserve some structural features of the plant expansins and family-45 glycosyl hydrolase enzymes, but it evolutionarily diverges from both of these protein classes. Recombinant ThSwo was purified at a high yield and with high purity and showed secondary folding similar to that of a native fungal SWO. Bioactivity assays revealed that the purified recombinant ThSwo created a rough and amorphous surface on Avicel and displayed a high synergistic effect with a commercial xylanase from T. viride, enhancing its hydrolytic performance up to 147 ± 7%. Many aspects of the structure and mechanism of action of fungal SWOs remain unknown. In the present study, we produced a recombinant, active SWO from T. harzianum using a prokaryotic host and confirmed its potential synergistic role in biomass degradation. Our work paves the way for further studies evaluating the structure and function of this protein, especially regarding its use in biotechnology.

Recombinant Cyclophilins Lack Nuclease Activity

OpenAIRE

Manteca, Angel; Sanchez, Jesus

2004-01-01

Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.

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...

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

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Lisa Gerner

2016-09-01

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

Cloning of the nptII gene of Escherichia coli and construction of a recombinant strain harboring functional recA and nptII antibiotic resistance.

Science.gov (United States)

Ghanem, S

2011-01-01

In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.

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

Effects of substitutions at position 180 in the Escherichia coli RNA ...

Indian Academy of Sciences (India)

Escherichia coli RNA polymerase, two mutant variants of the protein with substitutions for either alanine or glutamic .... promoter signals utilized for in vitro transcription assays and ..... free recombinant protein using a self-cleavable affinity tag.

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

Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks.

Science.gov (United States)

Valdez-Cruz, Norma A; Reynoso-Cereceda, Greta I; Pérez-Rodriguez, Saumel; Restrepo-Pineda, Sara; González-Santana, Jesus; Olvera, Alejandro; Zavala, Guadalupe; Alagón, Alejandro; Trujillo-Roldán, Mauricio A

2017-07-25

Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom. Comparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (k L a ≈ 80 h -1 ) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and β-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response. In moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in

Functional Requirements for DjlA- and RraA-Mediated Enhancement of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR.

Science.gov (United States)

Gialama, Dimitra; Delivoria, Dafni Chrysanthi; Michou, Myrsini; Giannakopoulou, Artemis; Skretas, Georgios

2017-06-16

In previous work, we have generated the engineered Escherichia coli strains SuptoxD and SuptoxR, which upon co-expression of the effector genes djlA or rraA, respectively, are capable of suppressing the cytotoxicity caused by membrane protein (MP) overexpression and of producing dramatically enhanced yields for a variety of recombinant MPs of both prokaryotic and eukaryotic origin. Here, we investigated the functional requirements for DnaJ-like protein A (DjlA)- and regulator of ribonuclease activity A (RraA)-mediated enhancement of recombinant MP production in these strains and show that: (i) DjlA and RraA act independently, that is, the beneficial effects of each protein on recombinant MP production occur through a mechanism that does not involve the other, and in a non-additive manner; (ii) full-length and membrane-bound DjlA is required for exerting its beneficial effects on recombinant MP production in E. coli SuptoxD; (iii) the MP production-promoting properties of DjlA in SuptoxD involve the action of the molecular chaperone DnaK but do not rely on the activation of the regulation of capsular synthesis response, a well-established consequence of djlA overexpression; (iv) the observed RraA-mediated effects in E. coli SuptoxR involve the ribonucleolytic activity of RNase E, but not that of its paralogous ribonuclease RNase G; and (v) DjlA and RraA are unique among similar E. coli proteins in their ability to promote bacterial recombinant MP production. These observations provide important clues about the molecular requirements for suppressed toxicity and enhanced MP accumulation in SuptoxD/SuptoxR and will guide future studies aiming to decipher the exact mechanism of DjlA- and RraA-mediated enhancement of recombinant MP production in these strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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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

Comparative analysis of immune effects in mice model: Clonorchis sinensis cysteine protease generated from recombinant Escherichia coli and Bacillus subtilis spores.

Science.gov (United States)

Wu, Zhanshuai; Tang, Zeli; Shang, Mei; Zhao, Lu; Zhou, Lina; Kong, Xiangzhan; Lin, Zhipeng; Sun, Hengchang; Chen, Tingjin; Xu, Jin; Li, Xuerong; Huang, Yan; Yu, Xinbing

2017-07-01

Clonorchiasis remains a nonnegligible public health problem in endemic areas. Cysteine protease of Clonorchis sinensis (CsCP) plays indispensable roles in the parasitic physiology and pathology, and has been exploited as a promising drug and vaccine candidate. In recent years, development of spore-based vaccines against multiple pathogens has attracted many investigators' interest. In previous studies, the recombinant Escherichia coli (BL21) and Bacillus subtilis spores expressing CsCP have been successfully constructed, respectively. In this study, the immune effects of CsCP protein purified from recombinant BL21 (rCsCP) and B. subtilis spores presenting CsCP (B.s-CsCP) in Balb/c mice model were conducted with comparative analysis. Levels of specific IgG, IgG1 and IgG2a were significantly increased in sera from both rCsCP and B.s-CsCP intraperitoneally immunized mice. Additionally, recombinant spores expressing abundant fusion CsCP (0.03125 pg/spore) could strongly enhance the immunogenicity of CsCP with significantly higher levels of IgG and isotypes. Compared with rCsCP alone, intraperitoneal administration of mice with spores expressing CsCP achieved a better effect of fighting against C. sinensis infection by slowing down the process of fibrosis. Our results demonstrated that a combination of Th1/Th2 immune responses could be elicited by rCsCP, while spores displaying CsCP prominently induced Th1-biased specific immune responses, and the complex cytokine network maybe mediates protective immune responses against C. sinensis. This work further confirmed that the usage of B. subtilis spores displaying CsCP is an effective way to against C. sinensis.

Development of bisphenol A-removing recombinant Escherichia coli by monomeric and dimeric surface display of bisphenol A-binding peptide.

Science.gov (United States)

Maruthamuthu, Murali Kannan; Hong, Jiyeon; Arulsamy, Kulandaisamy; Somasundaram, Sivachandiran; Hong, SoonHo; Choe, Woo-Seok; Yoo, Ik-Keun

2018-04-01

Peptide-displaying Escherichia coli cells were investigated for use in adsorptive removal of bisphenol A (BPA) both in Luria-Bertani medium including BPA or ATM thermal paper eluted wastewater. Two recombinant strains were constructed with monomeric and dimeric repeats of the 7-mer BPA-binding peptide (KSLENSY), respectively. Greater than threefold increased adsorption of BPA [230.4 µmol BPA per g dry cell weight (DCW)] was found in dimeric peptide-displaying cells compared to monomeric strains (63.4 µmol per g DCW) in 15 ppm BPA solution. The selective removal of BPA from a mixture of BPA analogs (bisphenol F and bisphenol S) was verified in both monomeric and dimeric peptide-displaying cells. The binding chemistry of BPA with the peptide was assumed, based on molecular docking analysis, to be the interaction of BPA with serine and asparagine residues within the 7-mer peptide sequence. The peptide-displaying cells also functioned efficiently in thermal paper eluted wastewater containing 14.5 ppm BPA.

Immunogenicity in chickens with orally administered recombinant chicken-borne Lactobacillus saerimneri expressing FimA and OmpC antigen of O78 avian pathogenic Escherichia coli.

Science.gov (United States)

Ma, Sun-Ting; Ding, Guo-Jie; Huang, Xue-Wei; Wang, Zi-Wei; Wang, Li; Yu, Mei-Ling; Shi, Wen; Jiang, Yan-Ping; Tang, Li-Jie; Xu, Yi-Gang; Li, Yi-Jing

2018-03-01

Avian colibacillosis is responsible for economic losses to poultry producers worldwide. To combat this, we aimed to develop an effective oral vaccine for chicken against O78 avian pathogenic Escherichia coli (APEC) infection through a Lactobacillus delivery system. Eight Lactobacillus strains isolated from the intestines of broiler chickens were evaluated based on their in vitro adherence ability to assess their potential as a delivery vector. Fimbrial subunit A (FimA) and outer-membrane protein C (OmpC) of APEC with and without fusion to dendritic cell-targeting peptide (DCpep) and microfold cell-targeting peptide (Co1) were displayed on the surface of Lactobacillus saerimneri M-11 and yielded vaccine groups (pPG-ompC-fimA/M-11 and pPG-ompC-fimA-Co1-DCpep/M-11, respectively). The colonization of the recombinant strains in vivo was assessed and the immunogenicity and protective efficacy of orally administered recombinant strains in chickens were evaluated. The colonization of the recombinant strains in vivo revealed no significant differences between the recombinant and wild-type strains. Chickens orally administered with vaccine groups showed significantly higher levels of OmpC/FimA-specific IgG in serum and mucosal IgA in cecum lavage, nasal lavage and stool compared to the pPG/M-11 group. After challenge with APEC CVCC1553, better protective efficacy was observed in chickens orally immunized with pPG-ompC-fimA/M-11 and pPG-ompC-fimA-Co1-DCpep/M-11, but no significant differences were observed between the two groups. Recombinant chicken-borne L. saerimneri M-11 showed good immunogenicity in chickens, suggesting that it may be a promising vaccine candidate against APEC infections. However, the activity of mammalian DCpep and Co1 was not significant in chickens.

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.

Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli

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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

Effects of recombinant human collagen VI from Escherichia coli on ...

African Journals Online (AJOL)

Jane

2011-07-20

Jul 20, 2011 ... In this study, we reported the cloning and over expression of a gene coding for human collagen peptide. (CP6) in Escherichia coli and investigated the protective effects of CP6 on UVA-irradiated human skin fibroblasts cells. The collagen peptide (CP6) was highly soluble and the expression level was.

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.

Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli

Science.gov (United States)

Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

2016-01-01

S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

Science.gov (United States)

Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

2015-09-01

A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

Engineering Escherichia coli to grow constitutively on D-xylose using the carbon-efficient Weimberg pathway

Science.gov (United States)

Rossoni, Luca; Carr, Reuben; Baxter, Scott; Cortis, Roxann; Thorpe, Thomas; Eastham, Graham; Stephens, Gill

2018-01-01

Bio-production of fuels and chemicals from lignocellulosic C5 sugars usually requires the use of the pentose phosphate pathway (PPP) to produce pyruvate. Unfortunately, the oxidation of pyruvate to acetyl-coenzyme A results in the loss of 33 % of the carbon as CO2, to the detriment of sustainability and process economics. To improve atom efficiency, we engineered Escherichia coli to utilize d-xylose constitutively using the Weimberg pathway, to allow direct production of 2-oxoglutarate without CO2 loss. After confirming enzyme expression in vitro, the pathway expression was optimized in vivo using a combinatorial approach, by screening a range of constitutive promoters whilst systematically varying the gene order. A PPP-deficient (ΔxylAB), 2-oxoglutarate auxotroph (Δicd) was used as the host strain, so that growth on d-xylose depended on the expression of the Weimberg pathway, and variants expressing Caulobacter crescentus xylXAB could be selected on minimal agar plates. The strains were isolated and high-throughput measurement of the growth rates on d-xylose was used to identify the fastest growing variant. This strain contained the pL promoter, with C. crescentus xylA at the first position in the synthetic operon, and grew at 42 % of the rate on d-xylose compared to wild-type E. coli using the PPP. Remarkably, the biomass yield was improved by 53.5 % compared with the wild-type upon restoration of icd activity. Therefore, the strain grows efficiently and constitutively on d-xylose, and offers great potential for use as a new host strain to engineer carbon-efficient production of fuels and chemicals via the Weimberg pathway. PMID:29458683

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.

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

Impact of Ralstonia eutropha's poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB storage in recombinant Escherichia coli.

Science.gov (United States)

Eggers, Jessica; Steinbüchel, Alexander

2014-12-01

The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay

Microsatellite primers for fungus-growing ants

DEFF Research Database (Denmark)

Villesen, Palle; Gertsch, P J; Boomsma, JJ

2002-01-01

We isolated five polymorphic microsatellite loci from a library of two thousand recombinant clones of two fungus-growing ant species, Cyphomyrmex longiscapus and Trachymyrmex cf. zeteki. Amplification and heterozygosity were tested in five species of higher attine ants using both the newly...... developed primers and earlier published primers that were developed for fungus-growing ants. A total of 20 variable microsatellite loci, developed for six different species of fungus-growing ants, are now available for studying the population genetics and colony kin-structure of these ants....

Microsatellite Primers for Fungus-Growing Ants

DEFF Research Database (Denmark)

Villesen Fredsted, Palle; Gertsch, Pia J.; Boomsma, Jacobus Jan (Koos)

2002-01-01

We isolated five polymorphic microsatellite loci from a library of two thousand recombinant clones of two fungus-growing ant species, Cyphomyrmex longiscapus and Trachymyrmex cf. zeteki. Amplification and heterozygosity were tested in five species of higher attine ants using both the newly...... developed primers and earlier published primers that were developed for fungus-growing ants. A total of 20 variable microsatellite loci, developed for six different species of fungus-growing ants, are now available for studying the population genetics and colony kin-structure of these ants....

Cloning and over-expression of Penicillin G acylase in Escherichia ...

African Journals Online (AJOL)

The aim of this study is to screen for PGA producing Escherichia coli isolates as well as the cloning and recombinant expression of PGA for high level enzyme production. Bacteria isolated from environmental and clinical samples were identified by standard microbiological tests and then E. coli isolates were subjected to

Expression and evaluation of IgE-binding capacity of recombinant Pacific mackerel parvalbumin

OpenAIRE

Hamada, Yuki; Tanaka, Hiroyuki; Sato, Ayako; Ishizaki, Shoichiro; Nagashima, Yuji; Shiomi, Kazuo

2004-01-01

Background: Parvalbumin is the major and cross-reactive allergen in fish. Sufficient amounts of IgE-reactive recombinant fish parvalbumin are needed for diagnosis and immunotherapy of fish allergy. Methods: A DNA fragment corresponding to parvalbumin of the Pacific mackerel Scomber japonicus was synthesized and cloned into the expression vector pGEX-6p-3 to produce glutathione S-transferase (GST)-fusion parvalbumin in Escherichia coli. The GST-free recombinant parvalbumin was purified usin...

New vaccine strategies against enterotoxigenic Escherichia coli: II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine

Directory of Open Access Journals (Sweden)

M.O. Lásaro

1999-02-01

Full Text Available The induction of systemic (IgG and mucosal (IgA antibody responses against the colonization factor I antigen (CFA/I of enterotoxigenic Escherichia coli (ETEC was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

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 ...

Suppression of colorectal tumorigenesis by recombinant Bacteroides fragilis enterotoxin-2 in vivo

OpenAIRE

Lv, You; Ye, Tao; Wang, Hui-Peng; Zhao, Jia-Ying; Chen, Wen-Jie; Wang, Xin; Shen, Chen-Xia; Wu, Yi-Bin; Cai, Yuan-Kun

2017-01-01

AIM To evaluate the impact of recombinant Bacteroides fragilis enterotoxin-2 (BFT-2, or Fragilysin) on colorectal tumorigenesis in mice induced by azoxymethane/dextran sulfate sodium (AOM/DSS). METHODS Recombinant proBFT-2 was expressed in Escherichia coli strain Rosetta (DE3) and BFT-2 was obtained and tested for its biological activity via colorectal adenocarcinoma cell strains SW-480. Seventy C57BL/6J mice were randomly divided into a blank (BC; n = 10), model (AD; n = 20), model + low-dos...

Relationship among the repair and genetic recombination mechanisms. II. Effect of gamma radiation on the lambda recombination in E. coli AB1157 and AB1886

International Nuclear Information System (INIS)

Alcantara D, D.

1988-08-01

The objective of the present work is to determine if the radiation gamma that is a good inductor of the answer SOS of Escherichia Coli but that it produces alterations in the DNA very different to those taken place by the light UV, it is able to stimulate the viral recombination. (Author)

Expression and Characterisation of Recombinant Rhodocyclus tenuis High Potential Iron-Sulphur Protein

DEFF Research Database (Denmark)

Caspersen, Michael Bjerg; Bennet, K.; Christensen, Hans Erik Mølager

2000-01-01

The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein...

CRMAGE: CRISPR Optimized MAGE Recombineering

DEFF Research Database (Denmark)

Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten Otto Alexander

2016-01-01

A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli...... that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red...

Chromosomal Fragmentation in "Escherichia Coli": Its Absence in "mutT" Mutants and Its Mechanisms in "seqA" Mutants

Science.gov (United States)

Rotman, Ella Rose

2009-01-01

Chromosomal fragmentation in "Escherichia coli" is a lethal event for the cell unless mended by the recombinational repair proteins RecA, RecBCD, and RuvABC. Certain mutations exacerbate problems that cause the cell to be dependent on the recombinational repair proteins for viability. We tested whether the absence of the MutT protein caused…

Recombinant protein expression in microbial systems

OpenAIRE

Rosano, Germán L.; Ceccarelli, Eduardo A.

2014-01-01

The emergence of recombinant DNA technology during the early 70's set a revolution in molecular biology. This set of techniques was strengthened even further later on with the introduction of the polymerase chain reaction and allowed scientists to explore and understand essential life processes in an easy and straightforward way. It also marked the birth of the modern biotech industry. At that time, it was shown that eukaryotic DNA could be propagated in Escherichia coli (Morrow et al., 1974)...

Production and purification of avian antibodies (IgYs from inclusion bodies of a recombinant protein central in NAD+ metabolism

Directory of Open Access Journals (Sweden)

Paula A. Moreno-González

2013-08-01

Full Text Available The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models.  The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems frequently prompts the protein to precipitate, forming insoluble aggregates of limited utility (inclusion bodies. A methodology for the production of avian polyclonal antibodies, using recombinant protein from inclusion bodies is presented in this article.In order to produce the antigen, a recombinant Nicotinamide mononucleotide adenylyltransferase from Giardia intestinalis (His-GiNMNAT was expressed in Escherichia coli.  The protein was purified through solubilization from inclusion bodies prior to its renaturalization.  Antibodies were purified from egg yolk of immunized hens by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography.The purified antibodies were tested against His-GiNMNAT protein in Western blot essays. From one egg yolk, 14.4 mg of highly pure IgY were obtained; this antibody was able to detect 15ng of His-GiNMNAT.  IgY specificity was improved by means of antigen affinity purification, allowing its use for parasite protein recognition.

Comparison of hydrogen-production capability of four different Enterobacteriaceae strains under growing and non-growing conditions

Energy Technology Data Exchange (ETDEWEB)

Seol, Eunhee; Kim, Seohyoung; Raj, S. Mohan; Park, Sunghoon [Department of Chemical and Biochemical Engineering, Pusan National University, Busan 609-735 (Korea)

2008-10-15

Non-growing cells can function as whole-cell biocatalysts for hydrogen (H{sub 2}) production, a process that has recently drawn much attention. In order to evaluate their potential as whole-cell biocatalysts, we compared the H{sub 2}-production capability of four Enterobacteriaceae strains (Citrobacter amalonaticus Y19, Escherichia coli K-12 MG1655, Escherichia coli DJT135, and Enterobacter aerogenes) under growing and non-growing conditions. We evaluated their H{sub 2}-production activity at varying temperatures (25-45 C) and pH conditions (6.0-8.0) using glucose or formate as the carbon source. Under growing conditions with 10 mM glucose as a substrate, E. aerogenes exhibited the highest H{sub 2}-production activity (17.0 {+-} 0.2 {mu}mol H{sub 2} mg cell{sup -1} h{sup -1}) among the four strains, but the final H{sub 2} yield was similar (1.7-1.8 mol H{sub 2} mol{sup -1} glucose) in all four strains. H{sub 2} production in the four strains proceeded through a formate-dependent pathway that involved the formate hydrogen lyase (FHL) complex. Under non-growing conditions with 20 mM formate as a substrate, we obtained high H{sub 2}-production activities, in the range of 95.5-195.2 {mu}mol H{sub 2} mg cell{sup -1} h{sup -1}, with E. coli DJT135 exhibiting the highest activity (195.2 {mu}mol H{sub 2} mg{sup -1} h{sup -1}) at pH 6.0 and 45 C. In contrast, using glucose as the carbon substrate in non-growing cell experiments greatly reduced the H{sub 2}-production activity to 6.1-7.7 {mu}mol H{sub 2} mg cell{sup -1} h{sup -1}. This study indicated that formate is a better substrate than glucose for H{sub 2} production by non-growing cells, and that the H{sub 2}-production performance among the strains did not vary significantly, with the exception of E. coli K-12 MG1655. (author)

Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli.

Science.gov (United States)

Liu, Siqing; Bischoff, Kenneth M; Anderson, Amber M; Rich, Joseph O

2016-09-01

A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-β-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid, plus the generally

Polyclonal Antibodies to a Recombinant Coat Protein of Potato Virus A

Czech Academy of Sciences Publication Activity Database

Čeřovská, Noemi; Moravec, Tomáš; Velemínský, Jiří

2002-01-01

Roč. 46, - (2002), s. 147-151 ISSN 0001-723X R&D Projects: GA ČR GA310/00/0381 Institutional research plan: CEZ:AV0Z5038910 Keywords : Potato virus A * recombinant coat protein * Escherichia coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.660, year: 2002

Crystallization and preliminary crystallographic analysis of recombinant immunoglobulin G-binding protein from Streptococcus suis

Energy Technology Data Exchange (ETDEWEB)

Khan, Abdul Hamid; Chu, Fuliang; Feng, Youjun; Zhang, Qinagmin [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Qi, Jianxun [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Gao, George Fu, E-mail: gaof@im.ac.cn [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China)

2008-08-01

Crystallization of recombinant IgG-binding protein expressed in Escherichia coli using the hanging-drop vapour-diffusion method is described. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å. Streptococcus suis, an important zoonotic pathogen, expresses immunoglobulin G-binding protein, which is thought to be helpful to the organism in eluding the host defence system. Recombinant IgG-binding protein expressed in Escherichia coli has been crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å and one molecule in the asymmetric unit. Diffraction data were collected to 2.60 Å resolution.

IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins

NARCIS (Netherlands)

Vornhagen, R; Hinderer, W; Sonneborn, HH; Bein, G; Matter, L; The, T. Hauw; Enders, G; Jahn, G; Plachter, B

Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and

Recombinant Lipoproteins as Novel Vaccines with Intrinsic Adjuvant.

Science.gov (United States)

Chong, Pele; Huang, Jui-Hsin; Leng, Chih-Hsiang; Liu, Shih-Jen; Chen, Hsin-Wei

2015-01-01

A core platform technology for high production of recombinant lipoproteins with built-in immunostimulator for novel subunit vaccine development has been established. This platform technology has the following advantages: (1) easily convert antigen into lipidated recombinant protein using a fusion sequence containing lipobox and express high level (50-150mg/L) in Escherichia coli; (2) a robust high-yield up- and downstream bioprocess for lipoprotein production is successfully developed to devoid endotoxin contamination; (3) the lipid moiety of recombinant lipoproteins, which is identical to that of bacterial lipoproteins is recognized as danger signals by the immune system (Toll-like receptor 2 agonist), so both innate and adaptive immune responses can be induced by lipoproteins; and (4) successfully demonstrate the feasibility and safety of this core platform technology in meningococcal group B subunit vaccine, dengue subunit vaccine, novel subunit vaccine against Clostridium difficile-associated diseases, and HPV-based immunotherapeutic vaccines in animal model studies. © 2015 Elsevier Inc. All rights reserved.

Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication

DEFF Research Database (Denmark)

Nielsen, Henrik Jørck; Youngren, Brenda; Hansen, Flemming G.

2007-01-01

Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chro......Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division......, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple......-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive...

Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.

Directory of Open Access Journals (Sweden)

Asma Rehman

Full Text Available Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4 vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1-2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.

Recombination of a fast expanding plasma

International Nuclear Information System (INIS)

Salvat, M.

1979-05-01

The goal of the following calculations is to determine numerically the recombination of dense plasmas (for instance of laser-produced plasmas). The recombination is computed for plasmas with initial densities of 10 24 27 [m -3 ] and with initial temperatures >= 50 eV. The ionization of the plasma remains essentially constant during the early phase of expansion. The time for which the ionization is 'frozen-in' grows with decreasing initial density and with increasing initial temperature. (orig.) [de

Refolding in high hydrostatic pressure of recombinant proteins from inclusion bodies in Escherichia Coli

International Nuclear Information System (INIS)

Balduino, Keli Nunes

2009-01-01

The expression of proteins as inclusion bodies in bacteria is a widely used alternative for production of recombinant protein. However, the aggregation is a problem often encountered during refolding of these proteins. High hydrostatic pressure are able to solubilise the inclusion bodies in the presence of low concentrations of denaturant reagents, encouraging refolding protein with high efficiency and reduce costs. This work aims to refolding of recombinant proteins expressed in Escherichia coli from inclusion bodies using high hydrostatic pressure. Three toxins, all featuring five or more disulfide bonds were studied: NXH8, Natterin 2 and Bothropstoxin 1. Suspensions of inclusion bodies of the three proteins were pressurized to 2000 bars for 16 hours. The buffers were optimized for refolding of the three proteins. The buffer used in the refolding of NXH8 was 50 mM Tris HCl, pH 9.0 with proportion of 1GSH: 4GSSG at a concentration of 6 mM and 2 M GdnHCl. Inclusion bodies were used in O.D. (A600nm) of 0.5. After refolding process, dialysis was performed at pH 7.0. The final yield of obtaining soluble NXH8 was 40% (28,6 mg of soluble NXH8/L of culture medium). The refolding of Bothropstoxin 1 was obtained in refolding buffer of Tris HCl 50 mM, pH 7,5 with proportion of 2 GSH: GSSG 3 and concentration of 3 mM and 1 M GdnHCl. Use with a suspension of O.D. (A600nm) of 0.5. The final yield of recovery of Bothropstoxin 1 refolded was 32% (9,2 mg of refolded Bothropstoxin 1/L of culture medium). The refolding of Natterin 2 was performed in the refolding buffer: 20 mM Tris HCl pH 9.0 at a ratio of 2 GSH: 3GSSG and concentration of 10 mM and 1 M GdnHCl and inclusion bodies O.D. (A600nm) of 6.0. The yield of Natterin 2 refolded was 20% (3,7 mg/L of culture medium). Physico-chemical and biological analysis were performed by SDS-PAGE, western blot, scanning electron microscopy, biological tests in vivo and in vitro and structural. The analysis conducted in NXH8 did not show

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.

Role of the RecF pathway of recombination in the metabolism of uv-irradiated DNA in Escherichia coli K-12

International Nuclear Information System (INIS)

Rothman, R.H.

1976-01-01

The RecF pathway of genetic recombination in Escherichia coli is potentially capable of supporting wild type levels of recombination, but in wild type cells it plays a relatively minor role in this process. RecF and recL single mutants were found to be ultraviolet-sensitive but recombination proficient. These observations led to the hypothesis that the main function of the RecF pathway lies in the metabolism of uv-damaged DNA. The role of reF and recL in pathways of recovery from uv-irradiation has been examined. Both recF - and recL - inhibited post-replication joining of DNA fragments synthesized on uv-damaged DNA templates (post-replication repair). The addition of a uvrB5 mutation to the single mutants did not affect the cell's ability to complete post-replication repair in the case of recL, but did completely prevent completion of joining in the case of recF. It was hypothesized that recF is an endonuclease weakly indirectly suppressible by the presence of functional correndo II. It is suggested that recF is necessary to cleave the crossed strand intermediate at the end of repair. RecL, in addition to its involvement in post-replication repair, was also found to be involved in excision repair. A uvrB recB recC recF multiple mutant was as sensitive as a uvrB recA strain, suggesting that it is devoid of any repair abilities. RecB - was shown to have an inhibitory effect of post-replication repair. The uvrB recF mutant, however, was totally devoid of post-replication repair even though recB + contributed to the recovery of the strain. Thus the role of recB in post-replication repair is unclear. Lastly, the effects of recF and recL on uv-inducible repair was studied. W-reactivation of uv-irradiated lambda was used as an assay for inducible repair. The conclusions from these experiments were unclear. They seemed to imply that W-reactivation is effected by the combined action of excision repair and post-replication repair

Escherichia coli as a production host for novel enzymes from basidiomycota.

Science.gov (United States)

Zelena, Katerina; Eisele, Nadine; Berger, Ralf G

2014-12-01

Many enzymes from basidiomycota have been identified and more recently characterized on the molecular level. This report summarizes the potential biotechnological applications of these enzymes and evaluates recent advances in their heterologous expression in Escherichia coli. Being one of the most widely used hosts for the production of recombinant proteins, there are, however, recurrent problems of recovering substantial yields of correctly folded and active enzymes. Various strategies for the efficient production of recombinant proteins from basidiomycetous fungi are reviewed including the current knowledge on vectors and expression strains, as well as methods for enhancing the solubility of target expression products and their purification. Research efforts towards the refolding of recombinant oxidoreductases and hydrolases are presented to illustrate successful production strategies. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2016-02-01

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

Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling

DEFF Research Database (Denmark)

Kristensen, Janni; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

2005-01-01

We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23...... therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only...... proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome....

The recombinant 120-kilodalton protein of Ehrlichia chaffeensis, a potential diagnostic tool.

OpenAIRE

Yu, X J; Crocquet-Valdes, P; Cullman, L C; Walker, D H

1996-01-01

DNA encoding two repeat units of 120-kDa protein of Ehrlichia chaffeensis was cloned into the expression vector pGEX and expressed in Escherichia coli. The sensitivity and specificity of a dot blot assay for detection of human antibodies with the recombinant protein were 86 and 100%, respectively, compared with an indirect immunofluorescence assay.

Differences in mutagenic and recombinational DNA repair in enterobacteria

International Nuclear Information System (INIS)

Sedgwick, S.G.; Goodwin, P.A.

1985-01-01

The incidence of recombinational DNA repair and inducible mutagenic DNA repair has been examined in Escherichia coli and 11 related species of enterobacteria. Recombinational repair was found to be a common feature of the DNA repair repertoire of at least 6 genera of enterobacteria. This conclusion is based on observations of (i) damage-induced synthesis of RecA-like proteins, (ii) nucleotide hybridization between E. coli recA sequences and some chromosomal DNAs, and (iii) recA-negative complementation by plasmids showing SOS-inducible expression of truncated E. coli recA genes. The mechanism of DNA damage-induced gene expression is therefore sufficiently conserved to allow non-E. coli regulatory elements to govern expression of these cloned truncated E. coli recA genes. In contrast, the process of mutagenic repair, which uses umuC+ umuD+ gene products in E. coli, appeared less widespread. Little ultraviolet light-induced mutagenesis to rifampicin resistance was detected outside the genus Escherichia, and even within the genus induced mutagenesis was detected in only 3 out of 6 species. Nucleotide hybridization showed that sequences like the E. coli umuCD+ gene are not found in these poorly mutable organisms. Evolutionary questions raised by the sporadic incidence of inducible mutagenic repair are discussed

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin

Directory of Open Access Journals (Sweden)

Carlos Eduardo Pouey da Cunha

Full Text Available BACKGROUND The B subunit of Escherichia coli heat-labile enterotoxin (LTB is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA were assessed by enzyme-linked immunosorbent assay (ELISA. The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

Induction of intrachromosomal homologous recombination in whole plants

International Nuclear Information System (INIS)

Puchta, H.; Swoboda, P.; Hohn, B.

1995-01-01

The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced several fold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed. (author)

Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli.

Science.gov (United States)

Pardavé-Alejandre, H D; Alvarado-Yaah, J E; Pompa-Mera, E N; Muñoz-Medina, J E; Sárquiz-Martínez, B; Santacruz-Tinoco, C E; Manning-Cela, R G; Ortíz-Navarrete, V; López-Macías, C; González-Bonilla, C R

2018-03-01

To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and β domains, whereas the second consisted of a 314 amino acid from α and truncated β domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli.

Science.gov (United States)

Kawaguchi, Hideo; Katsuyama, Yohei; Danyao, Du; Kahar, Prihardi; Nakamura-Tsuruta, Sachiko; Teramura, Hiroshi; Wakai, Keiko; Yoshihara, Kumiko; Minami, Hiromichi; Ogino, Chiaki; Ohnishi, Yasuo; Kondo, Ahikiko

2017-07-01

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

Participation of different genes in the ruptures repair of double chain in Escherichia coli stumps exposed to gamma radiation; Participacion de diferentes genes en la reparacion de rupturas de doble cadena en cepas de Escherichia coli expuestas a radiacion gamma

Energy Technology Data Exchange (ETDEWEB)

Serment G, J. H.; Martinez M, E.; Alcantara D, D., E-mail: jorge.serment@inin.gob.mx [ININ, Departamento de Biologia, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

2013-05-01

All living organisms are naturally exposed to radiation from different sources. Ionizing radiation produces a plethora of lesions upon DNA that can be categorized as single and double strand breaks and base damage. Among them, unrepaired double strand breaks (Dbs) have the greatest biological significance, since they are responsible of cell death. In Escherichia coli this kind of lesions are repaired mostly by homologous recombination. In this work the participation of some recombination genes in the repair of Dbs is evaluated. Escherichia coli defective strains were exposed to gamma radiation and incubated for different periods in ideal conditions. Both micro electrophoresis and pulse field gel electrophoresis techniques were used to evaluate the kinetics of repair of such lesions, reflecting the importance of each defective gene in the process. (Author)

Evaluation of software sensors for on-line estimation of culture conditions in an Escherichia coli cultivation expressing a recombinant protein.

Science.gov (United States)

Warth, Benedikt; Rajkai, György; Mandenius, Carl-Fredrik

2010-05-03

Software sensors for monitoring and on-line estimation of critical bioprocess variables have mainly been used with standard bioreactor sensors, such as electrodes and gas analyzers, where algorithms in the software model have generated the desired state variables. In this article we propose that other on-line instruments, such as NIR probes and on-line HPLC, should be used to make more reliable and flexible software sensors. Five software sensor architectures were compared and evaluated: (1) biomass concentration from an on-line NIR probe, (2) biomass concentration from titrant addition, (3) specific growth rate from titrant addition, (4) specific growth rate from the NIR probe, and (5) specific substrate uptake rate and by-product rate from on-line HPLC and NIR probe signals. The software sensors were demonstrated on an Escherichia coli cultivation expressing a recombinant protein, green fluorescent protein (GFP), but the results could be extrapolated to other production organisms and product proteins. We conclude that well-maintained on-line instrumentation (hardware sensors) can increase the potential of software sensors. This would also strongly support the intentions with process analytical technology and quality-by-design concepts. 2010 Elsevier B.V. All rights reserved.

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.

Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling.

Directory of Open Access Journals (Sweden)

Ying Hou

Full Text Available Phenylpyruvic acid (PPA is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L-1, temperature 35°C and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L-1. The total maximal production (mass conversion rate reached 29.8 ± 2.1 g·L-1 (99.3% and 75.1 ± 2.5 g·L-1 (93.9% in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation.

Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling.

Science.gov (United States)

Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng; Chen, Jian

2016-01-01

Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L-1, temperature 35°C) and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation) was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L-1. The total maximal production (mass conversion rate) reached 29.8 ± 2.1 g·L-1 (99.3%) and 75.1 ± 2.5 g·L-1 (93.9%) in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation.

Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling

Science.gov (United States)

Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Liu, Long; Du, Guocheng; Chen, Jian

2016-01-01

Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L−1, temperature 35°C) and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation) was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L−1. The total maximal production (mass conversion rate) reached 29.8 ± 2.1 g·L−1 (99.3%) and 75.1 ± 2.5 g·L−1 (93.9%) in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation. PMID:27851793

Phosphoribosylpyrophosphate (PRPP)-less mutants of Escherichia coli

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne

1989-01-01

A DNA fragment encoding kanamycin resistance was inserted in vitro into a plasmid-borne prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli. The resulting plasmids were subsequently transferred to the chromosome by homologous recombination and the haploid strains prs-3::Kan......R and prs-4::KanR were obtained. These strains were fully viable, but required guanosine, uridine, histidine, tryptophan and nicotinamide mononucleotide. There was no phosphoribosylpyrophosphate synthetase activity or phosphoribosylpyrophosphate pool in the mutant strains. These results show...

Participation of different genes in the ruptures repair of double chain in Escherichia coli stumps exposed to gamma radiation

International Nuclear Information System (INIS)

Serment G, J. H.; Martinez M, E.; Alcantara D, D.

2013-01-01

All living organisms are naturally exposed to radiation from different sources. Ionizing radiation produces a plethora of lesions upon DNA that can be categorized as single and double strand breaks and base damage. Among them, unrepaired double strand breaks (Dbs) have the greatest biological significance, since they are responsible of cell death. In Escherichia coli this kind of lesions are repaired mostly by homologous recombination. In this work the participation of some recombination genes in the repair of Dbs is evaluated. Escherichia coli defective strains were exposed to gamma radiation and incubated for different periods in ideal conditions. Both micro electrophoresis and pulse field gel electrophoresis techniques were used to evaluate the kinetics of repair of such lesions, reflecting the importance of each defective gene in the process. (Author)

Production of Two Novel Methoxy-Isoflavones from Biotransformation of 8-Hydroxydaidzein by Recombinant Escherichia coli Expressing O-Methyltransferase SpOMT2884 from Streptomyces peucetius

Science.gov (United States)

Chiang, Chien-Min; Ding, Hsiou-Yu; Tsai, Ya-Ting; Chang, Te-Sheng

2015-01-01

Biotransformation of 8-hydroxydaidzein by recombinant Escherichia coli expressing O-methyltransferase (OMT) SpOMT2884 from Streptomyces peucetius was investigated. Two metabolites were isolated and identified as 7,4′-dihydroxy-8-methoxy-isoflavone (1) and 8,4′-dihydroxy-7-methoxy-isoflavone (2), based on mass, 1H-nuclear magnetic resonance (NMR) and 13C-NMR spectrophotometric analysis. The maximum production yields of compound (1) and (2) in a 5-L fermenter were 9.3 mg/L and 6.0 mg/L, respectively. The two methoxy-isoflavones showed dose-dependent inhibitory effects on melanogenesis in cultured B16 melanoma cells under non-toxic conditions. Among the effects, compound (1) decreased melanogenesis to 63.5% of the control at 25 μM. This is the first report on the 8-O-methylation activity of OMT toward isoflavones. In addition, the present study also first identified compound (1) with potent melanogenesis inhibitory activity. PMID:26610478

Genome Editing in Escherichia coli with Cas9 and synthetic CRISPRs

Energy Technology Data Exchange (ETDEWEB)

Peng, Ze; Richardson, Sarah; Robinson, David; Deutsch, Samuel; Cheng, Jan-Fang

2014-03-14

Recently, the Cas9-CRISPR system has proven to be a useful tool for genome editing in eukaryotes, which repair the double stranded breaks made by Cas9 with non-homologous end joining or homologous recombination. Escherichia coli lacks non-homologous end joining and has a very low homologous recombination rate, effectively rendering targeted Cas9 activity lethal. We have developed a heat curable, serializable, plasmid based system for selectionless Cas9 editing in arbitrary E. coli strains that uses synthetic CRISPRs for targeting and -red to effect repairs of double stranded breaks. We have demonstrated insertions, substitutions, and multi-target deletions with our system, which we have tested in several strains.

Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

Czech Academy of Sciences Publication Activity Database

Čeřovská, Noemi; Moravec, Tomáš; Rosecká, Pavla; Dědič, P.; Filigarová, Marie

2003-01-01

Roč. 151, č. 4 (2003), s. 195-200 ISSN 0931-1785 R&D Projects: GA ČR GA522/01/1121 Institutional research plan: CEZ:AV0Z5038910 Keywords : potato mop-top virus * recombinant coat protein * Escherichia Coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.557, year: 2003

Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli.

Science.gov (United States)

Weickert, M J; Pagratis, M; Curry, S R; Blackmore, R

1997-01-01

Accumulation of soluble recombinant hemoglobin (rHb1.1) in Escherichia coli requires proper protein folding, prosthetic group (heme) addition, and subunit assembly. This served as a new model system for the study of the effects of temperature, protein synthesis rates, and protein accumulation rates on protein solubility in E. coli. Fermentation expression of rHb1.1 at 30 degrees C from cultures containing a medium or high globin gene dosage (pBR-based or pUC-based plasmids with rHb1.1 genes under the control of the tac promoter) was compared. A medium gene dosage resulted in rHb1.1 accumulating to approximately 7% of the soluble cell protein, of which 78% was soluble. A high globin gene dosage resulted in a > or = 3-fold increase in total globin to 23 to 24% of the soluble cell protein, but 70% was insoluble. Accumulation of insoluble rHb1.1 began immediately upon induction. The proportion of rHb1.1 from the high globin gene dosage that accumulated as insoluble globin was affected by reducing (i) the inducer concentration and (ii) the temperature. Reducing the inducer concentration reduced globin synthesis up to eightfold but increased the proportion of soluble rHb1.1 to 93%. In contrast, total globin protein synthesis was barely affected by reducing the temperature from 30 to 26 degrees C, while soluble globin accumulation increased > 2-fold to approximately 15% of the soluble cell protein. The contrast between the effects of reducing rates of protein synthesis and accumulation and those of reducing temperature suggests that lower temperature stabilizes one or more folding intermediates. We propose a simplified physical model which integrates protein synthesis, folding, and heme association. This model shows that temperature-dependent apoglobin stability is the most critical factor in soluble rHb1.1 accumulation. PMID:9361418

Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

International Nuclear Information System (INIS)

Wilhelm, O.G.; Jaskunas, S.R.; Vlahos, C.J.; Bang, N.U.

1990-01-01

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis

A recombinant multi-antigen vaccine with broad protection potential against avian pathogenic Escherichia coli.

Directory of Open Access Journals (Sweden)

Angelica Van Goor

Full Text Available Chickens are a major source of protein worldwide, yet infectious diseases continue to threaten the poultry industry. Avian pathogenic Escherichia coli (APEC, a subgroup of extraintestinal pathogenic E. coli (ExPEC, causes colibacillosis in chickens resulting in economic loss because of treatment, condemnation of products, and death. In this study, we evaluated a recombinant antigens (rAg vaccine combining common ExPEC surface proteins EtsC, OmpA, OmpT, and TraT for broad protective potential against APEC infections in chickens. The specific objectives were to evaluate antibody (serum and cytokines (lymphoid organs responses to vaccination; in vitro bactericidal ability of serum and splenocytes against multiple APEC serotypes; and in vivo protection against APEC challenge in chickens. Groups of four-day old chickens (N = 10 were vaccinated twice (two-week interval subcutaneously with rAgs alone or in combination and CpG adjuvant or PBS (control. IgY antibody in the serum and mRNA expression of IL-1β, IL-6, IL-18, IFN-γ, IL-4, IFN-β, and IL-8 in bursa, spleen, and thymus were measured using ELISA and RT-qPCR, respectively. Serum and splenocytes were tested for their bactericidal ability in vitro against multiple APEC isolates. Vaccinated and non-vaccinated chickens were challenged with 108 CFU of APEC-O2 via air sac at 31 days post first vaccination. Vaccine protection was determined by the decrease of bacterial loads in blood and organs (lung, heart, spleen, and liver, as well as gross colibacillosis lesion scores in air sac, heart, and liver. Vaccination significantly (P < 0.05 elicited IgY against specific antigens, induced immune related mRNA expression in the spleen and bursa, reduced in vitro growth of multiple APEC serotypes, and decreased bacterial loads in the heart and spleen, and gross lesion scores of the air sac, heart and liver in chickens. The vaccine reported may be used to provide broad protection against APEC strains

Microtiter miniature shaken bioreactor system as a scale-down model for process development of production of therapeutic alpha-interferon2b by recombinant Escherichia coli.

Science.gov (United States)

Tan, Joo Shun; Abbasiliasi, Sahar; Kadkhodaei, Saeid; Tam, Yew Joon; Tang, Teck-Kim; Lee, Yee-Ying; Ariff, Arbakariya B

2018-01-04

Demand for high-throughput bioprocessing has dramatically increased especially in the biopharmaceutical industry because the technologies are of vital importance to process optimization and media development. This can be efficiently boosted by using microtiter plate (MTP) cultivation setup embedded into an automated liquid-handling system. The objective of this study was to establish an automated microscale method for upstream and downstream bioprocessing of α-IFN2b production by recombinant Escherichia coli. The extraction performance of α-IFN2b by osmotic shock using two different systems, automated microscale platform and manual extraction in MTP was compared. The amount of α-IFN2b extracted using automated microscale platform (49.2 μg/L) was comparable to manual osmotic shock method (48.8 μg/L), but the standard deviation was 2 times lower as compared to manual osmotic shock method. Fermentation parameters in MTP involving inoculum size, agitation speed, working volume and induction profiling revealed that the fermentation conditions for the highest production of α-IFN2b (85.5 μg/L) was attained at inoculum size of 8%, working volume of 40% and agitation speed of 1000 rpm with induction at 4 h after the inoculation. Although the findings at MTP scale did not show perfect scalable results as compared to shake flask culture, but microscale technique development would serve as a convenient and low-cost solution in process optimization for recombinant protein.

High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii

DEFF Research Database (Denmark)

Ramos Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

2017-01-01

Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post......-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas...... in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins....

Analysis of the lipidated recombinant outer surface protein A from Borrelia burgdorferi by mass spectrometry

NARCIS (Netherlands)

Bouchon, B.; Klein, Michele; Bischoff, Rainer; Van Dorsselaer, A.; Roitsch, C.

1997-01-01

The outer surface protein A, OspA, from the spirochete Borrelia burgdorferi is a lipoprotein of 25 kDa. The recombinant OspA (rOspA) expressed in Escherichia coli has been purified and analyzed by electrospray mass spectrometry (ESMS). A heterogenous spectrum gave a measured mass of 28,462 +/- 9 Da

Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein

DEFF Research Database (Denmark)

Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld

1992-01-01

A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two...... areas of repeated amino acid sequences. Antibodies against recombinant GLURP489-1271, as well as against a synthetic peptide corresponding to GLURP899-916, and against a synthetic peptide representing the major glutamate rich repeat sequence from the P. falciparum ring erythrocyte surface antigen (Pf155...... between the anti-GLURP489-1271 and anti-(EENV)6 antibody responses. The data provide indirect evidence for a protective role of antibodies reacting with recombinant GLURP489-1271 as well as with the synthetic peptide (EENV)6 from the Pf155/RESA....

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...

Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

DEFF Research Database (Denmark)

Rolf, B; Oudenampsen-Krüger, E; Börchers, T

1995-01-01

The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

Expression of recombinant Arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification.

Science.gov (United States)

Chahardooli, Mahmood; Niazi, Ali; Aram, Farzaneh; Sohrabi, Seyyed Mohsen

2016-01-30

Lactoferricin (LFcin) is a strong cationic peptide released from the N-terminus of lactoferrin by gastric pepsin digestion. LFcin has some important properties, including high antimicrobial activity. To date, lactoferricins have been isolated and characterised from various animal species, but not from camel. The aim of this study was to characterise and express recombinant camel lactoferricin (LFcinC) in Pichia pastoris and investigate its antimicrobial activity. After methanol induction, LFcinC was expressed and secreted into a culture broth medium and the results determined by concentrated supernatant culture medium showed high antimicrobial activity against the following microorganisms: Escherichia coli PTCC 1330 (ATCC 8739), Staphylococcus aureus PTCC 1112 (ATCC 6538), Pseudomonas aeruginosa PTCC 1074 (ATCC 9027), Bacillus subtilis PTCC 1023 (ATCC 6633), and Candida albicans PTCC 5027 (ATCC 10231). Thermal stability was clarified with antibacterial activity against Escherichia coli PTCC 1330 (ATCC 8739). Results confirmed that camel lactoferricin had suitable antimicrobial activity and its production by Pichia pastoris can be used for recombinant production. © 2015 Society of Chemical Industry.

Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae

Directory of Open Access Journals (Sweden)

Eivind B. Drejer

2018-05-01

Full Text Available Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to properly synthesize, fold, and excrete these in high yields, and thermophilic Bacillaceae represent one potentially interesting group of microorganisms for such purposes. A number of thermophilic Bacillaceae including B. methanolicus, B. coagulans, B. smithii, B. licheniformis, Geobacillus thermoglucosidasius, G. kaustophilus, and G. stearothermophilus are investigated concerning physiology, genomics, genetic tools, and technologies, altogether paving the way for their utilization as hosts for recombinant production of thermostable and other difficult-to-express proteins. Moreover, recent successful deployments of CRISPR/Cas9 in several of these species have accelerated the progress in their metabolic engineering, which should increase their attractiveness for future industrial-scale production of proteins. This review describes the biology of thermophilic Bacillaceae and in particular focuses on genetic tools and methods enabling use of these organisms as hosts for recombinant protein production.

Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

OpenAIRE

Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

2011-01-01

Objective(s) Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics ...

Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization

NARCIS (Netherlands)

Slos, P.; Speck, D.; Accart, N.; Kolbe, H.V.; Schubnel, D.; Bouchon, B.; Bischoff, Rainer; Kieny, M.P.

1994-01-01

The gene coding for cholera toxin subunit B (CT-B) was fused to a modified ompA signal sequence and subsequently cloned into a high expression vector based on the regulatory signals of the arabinose operon of Salmonella typhimurium. Upon induction of gene expression in Escherichia coli, a product of

High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

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Blanca Iglesias-Figueroa

2016-06-01

Full Text Available In this study, bovine lactoferrin (bLf, an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin demonstrated antibacterial activity against Escherichia coli (E. coli BL21DE3, Staphylococcus aureus (S. aureus FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly.

Effect of phytoplankton on Escherichia coli survival in laboratory microcosms

Science.gov (United States)

Fecal contamination of water sources is an important water quality issue for agricultural irrigation ponds. Escherichia coli is a common microbial indicator used to evaluate recreational and irrigation water quality. Nuisance algae commonly grow in low- or no-flow irrigation water source The objecti...

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.

Effect of uv-irradiation on genetic recombination of Simian virus 40 mutants

International Nuclear Information System (INIS)

Gentil, A.; Margot, A.; Sarasin, A.

1983-01-01

Genetic recombination in monkey kidney cells has been studied using Simian virus 40 (SV40) as a molecular probe. Control or uv-irradiated cells have been co-infected with two thermosensitive mutants of SV40, tsA58 and tsA30. Recombination between the two viral genomes gives rise to a wild type virus phenotype, able to grow at the restrictive temperature of 41 0 C, which was taken as a measure of the recombination activity of the host cells. Results show that recombination takes place at a low frequency when viruses are not uv-irradiated. Irradiation of one or both viruses increases drastically recombination frequency. Pretreatment of the host cells with uv-light or mitomycin C 24 hours before being infected does not increase recombination frequency measured in our experimental conditions. 23 references, 5 tables

Combinations of SPR and MS for Characterizations of Native and Recombinant Proteins in Cell Lysates

DEFF Research Database (Denmark)

Borch, Jonas; Roepstorff, Peter

2006-01-01

Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni2+ for SPR analysis of the specific Ni2+ binding response...... and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands...... of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein...

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

Science.gov (United States)

Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1994-01-01

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

Adaptation and heterogeneity of Escherichia coli MC1000 growing in complex environments

DEFF Research Database (Denmark)

Puentes-Téllez, Pilar; Hansen, Martin Asser; Sørensen, Søren

2013-01-01

In a study aiming to assess bacterial evolution in complex growth media, we evaluated the long-term adaptive response of Escherichia coli MC1000 in Luria-Bertani (LB) medium. Seven parallel populations were founded and followed over 150 days in sequential batch cultures under three different oxygen...... conditions (defined environments), and 19 evolved forms were isolated. The emergence of forms with enhanced fitness was evident in competition experiments of all evolved forms versus the ancestral strain. The evolved forms were then subjected to phenotypic and genomic analyses relative to the ancestor...... in galR, a repressor of the galactose operon. Concomitantly, the new forms revealed enhanced growth on galactose as well as galactose-containing disaccharides. This response was likely driven by the LB medium....

Detection of Antibodies to U.S. Isolates of Avian Pneumovirus by a Recombinant Nucleocapsid Protein-Based Sandwich Enzyme-Linked Immunosorbent Assay

OpenAIRE

Gulati, Baldev R.; Munir, Shirin; Patnayak, Devi P.; Goyal, Sagar M.; Kapur, Vivek

2001-01-01

The nucleocapsid (N) protein of subgroup C (United States-specific) avian pneumovirus (APV/US) was expressed in Escherichia coli, and antibodies to the recombinant N protein were shown to specifically recognize the ≈47-kDa N protein of APV/US by Western immunoblot analysis. The recombinant APV/US N protein was used in a sandwich-capture enzyme-linked immunosorbent assay (ELISA), and the resulting assay was found to be more sensitive and specific than the routine indirect ELISA for the detecti...

Escherichia coli F4 fimbriae specific lama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhea

NARCIS (Netherlands)

Harmsen, M.M.; Solt, van C.B.; Hoogendoorn, A.; Zijderveld, van F.G.; Niewold, T.A.; Meulen, van der J.

2005-01-01

Oral administration of polyclonal antibodies directed against enterotoxigenic Escherichia coli (ETEC) F4 fimbriae is used to protect against piglet post-weaning diarrhoea. For cost reasons, we aim to replace these polyclonal antibodies by recombinant llama single-domain antibody fragments (VHHs)

The Contribution of Genetic Recombination to CRISPR Array Evolution.

Science.gov (United States)

Kupczok, Anne; Landan, Giddy; Dagan, Tal

2015-06-16

CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and

Assignment of two new host range types to the P2 family of temperate coliphages.

Science.gov (United States)

Poon, A P; Dhillon, T S

1986-04-01

Six non-inducible coliphages which grow on Escherichia coli C but not on K12 (C-specific) were shown to be antigenically related to P2. All six were shown to be P4 helpers and some of them could also recombine with P2.

Construction of heterologous gene expression cassettes for the development of recombinant Clostridium beijerinckii.

Science.gov (United States)

Oh, Young Hoon; Eom, Gyeong Tae; Kang, Kyoung Hee; Joo, Jeong Chan; Jang, Young-Ah; Choi, Jae Woo; Song, Bong Keun; Lee, Seung Hwan; Park, Si Jae

2016-04-01

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

Science.gov (United States)

Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

2016-10-01

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

Construction and characterisation of a genetically engineered Escherichia coli strain for the epoxide hydrolase-catalysed kinetic resolution of epoxides

NARCIS (Netherlands)

Visser, H.; Oliveira Vil Filho, de M.; Liese, A.; Weijers, C.A.G.M.; Verdoes, J.C.

2003-01-01

The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell

An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding.

Science.gov (United States)

Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad

2017-09-01

The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols

Energy Technology Data Exchange (ETDEWEB)

Yu, Chao; Cao, Yujin; Zou, Huibin; Xian, Mo [Chinese Academy of Sciences, Qingdao (China). Key Lab. of Biofuels

2011-02-15

Confronted with the gradual and inescapable exhaustion of the earth's fossil energy resources, the bio-based process to produce platform chemicals from renewable carbohydrates is attracting growing interest. Escherichia coli has been chosen as a workhouse for the production of many valuable chemicals due to its clear genetic background, convenient to be genetically modified and good growth properties with low nutrient requirements. Rational strain development of E. coli achieved by metabolic engineering strategies has provided new processes for efficiently biotechnological production of various high-value chemical building blocks. Compared to previous reviews, this review focuses on recent advances in metabolic engineering of the industrial model bacteria E. coli that lead to efficient recombinant biocatalysts for the production of high-value organic acids like succinic acid, lactic acid, 3-hydroxypropanoic acid and glucaric acid as well as alcohols like 1,3-propanediol, xylitol, mannitol, and glycerol with the discussion of the future research in this area. Besides, this review also discusses several platform chemicals, including fumaric acid, aspartic acid, glutamic acid, sorbitol, itaconic acid, and 2,5-furan dicarboxylic acid, which have not been produced by E. coli until now. (orig.)

Development of recombinant vaccine candidate molecule against Shigella infection.

Science.gov (United States)

Chitradevi, S T S; Kaur, G; Sivaramakrishna, U; Singh, D; Bansal, A

2016-10-17

Shigellosis is an acute bacillary diarrheal disease caused by the gram negative bacillus Shigella. The existence of multiple Shigella serotypes and their growing resistance to antibiotics stress the urgent need for the development of vaccine that is protective across all serotypes. Shigella's IpaB antigen is involved in translocon pore formation, promotes bacterial invasion and induces apoptosis in macrophages. S. Typhi GroEL (Hsp 60) is the immunodominant antigen inducing both arms of immunity and has been explored as adjuvant in this study. The present study evaluates the immunogenicity and protective efficacy of recombinant IpaB domain-GroEL fusion protein in mice against lethal Shigella infection. The IpaB domain and GroEL genes were fused using overlap extension PCR and cloned in pRSETA expression vector. Fused gene was expressed in Escherichia coli BL-21 cells and the resulting 90 KDa fusion protein was purified by affinity chromatography. Intranasal (i.n.) immunization of mice with fusion protein increased the IgG and IgA antibody titers as compared to the group immunized with IpaB and GroEL and control PBS immunized group. Also IgG1 and IgG2a antibodies induced in fusion protein immunized mice were higher than co-immunized group. Significant increase in lymphocyte proliferation and cytokine levels (IFN-γ, IL-4 and IL-10), indicates induction of both Th1 and Th2 immune responses in both immunized groups. Immunization with fusion protein protected 90-95% of mice whereas 80-85% survivability was observed in co-immunized group against lethal challenge with S. flexneri, S. boydii and S. sonnei. Passive immunization conferred 60-70% protection in mice against all these Shigella species. Organ burden and histopathology studies also revealed significant decrease in lung infection as compared to the co-immunized group. Since IpaB is the conserved dominant molecule in all Shigella species, this study will lead to an ideal platform for the development of safe

The Use of Recombinant Hemagglutinine Protein of Rinderpest Virus in Enzyme Immunoassay

OpenAIRE

BULUT, Hakan; BOLAT, Yusuf

2003-01-01

In this study, Rinderpest virus (RPV) recombinant hemagglutinine protein (rH) fused with protein A region of Staphylococcus aureus was expressed in Escherichia coli and purified by IgG affinity chromatography. rH protein was also used to establish enzyme immunoassay. Therefore, to prevent IgG binding to the protein A the wells coated with the rH proteins were blocked by human serum. Afterwards, RPV antigens were added to the wells to evaluate this assay. To this end, serum from mice immunized...

Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis

Directory of Open Access Journals (Sweden)

Pacífico Lucila G

2007-01-01

Full Text Available Abstract Background Recombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-α and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 μg/mL in the presence of Polymyxin B (10 μg/mL. Results The levels of cytokines were measured using ELISA. There was greater than 90 % reduction (p S. mansoni recombinant proteins in the presence of Polymyxin B, a reduction in the levels of TNF-α and IL-10 was also observed. However, the percentage of reduction was lower when compared to the cultures stimulated with LPS, probably because these proteins are able to induce the production of these cytokines by themselves. Conclusion This study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-α and IL-10 production.

Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

Science.gov (United States)

Martínez-Alonso, Mónica; Villaverde, Antonio

2010-01-01

Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

Mutational Analysis of the RecJ Exonuclease of Escherichia coli: Identification of Phosphoesterase Motifs

OpenAIRE

Sutera, Vincent A.; Han, Eugene S.; Rajman, Luis A.; Lovett, Susan T.

1999-01-01

The recJ gene, identified in Escherichia coli, encodes a Mg+2-dependent 5′-to-3′ exonuclease with high specificity for single-strand DNA. Genetic and biochemical experiments implicate RecJ exonuclease in homologous recombination, base excision, and methyl-directed mismatch repair. Genes encoding proteins with strong similarities to RecJ have been found in every eubacterial genome sequenced to date, with the exception of Mycoplasma and Mycobacterium tuberculosis. Multiple genes encoding protei...

Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells

International Nuclear Information System (INIS)

Moonen, P.; Mermod, J.J.; Ernst, J.F.; Hirschi, M.; DeLamarter, J.F.

1987-01-01

Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coli. The E. coli produced-protein-lacking any carbohydrate- had by far the highest specific activity observed for the recombinant hGM-CSFs

Growing Escherichia coli mutants deficient in riboflavin biosynthesis with non-limiting riboflavin results in sensitization to inactivation by broad-spectrum near-ultraviolet light (320-400 nm)

International Nuclear Information System (INIS)

Lloyd, R.E.; Rinkenberger, J.L.; Hug, B.A.; Tuveson, R.W.

1990-01-01

Two mutants of Escherichia coli unable to synthesize riboflavin were grown with limiting (2 μg ml -1 ) and non-limiting (10 μg ml -1 ) concentrations of riboflavin. These riboflavin auxotrophs when grown to exponential phase with non-limiting riboflavin are more sensitive to broad spectrum near-ultraviolet light (NUV, 320-400 nm) inactivation than when they are grown with limiting riboflavin. Exponential phase cells of the riboflavin auxotrophs grown with limiting riboflavin are sensitized when irradiated in saline supplemented with riboflavin. This suggests that extracellular riboflavin is important as a NUV sensitizer when intracellular levels of riboflavin are reduced. The concentration of riboflavin in crude extracts from exponentially growing cells correlates well with the sensitivity of these mutants to NUV inactivation. The level of riboflavin supplementation has little effect on the NUV sensitivity of the parental strain. (author)

Unusual Structure of the attB Site of the Site-Specific Recombination System of Lactobacillus delbrueckii Bacteriophage mv4

Science.gov (United States)

Auvray, Frédéric; Coddeville, Michèle; Ordonez, Romy Catoira; Ritzenthaler, Paul

1999-01-01

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3′ end of a tRNASer gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNASer gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model. PMID:10572145

Characterization of recombinant terrelysin, a hemolysin of Aspergillus terreus.

Science.gov (United States)

Nayak, Ajay P; Blachere, Françoise M; Hettick, Justin M; Lukomski, Slawomir; Schmechel, Detlef; Beezhold, Donald H

2011-01-01

Fungal hemolysins are potential virulence factors. Some fungal hemolysins belong to the aegerolysin protein family that includes cytolysins capable of lysing erythrocytes and other cells. Here, we describe a hemolysin from Aspergillus terreus called terrelysin. We used the genome sequence database to identify the terrelysin sequence based on homology with other known aegerolysins. Aspergillus terreus mRNA was isolated, transcribed to cDNA and the open reading frame for terrelysin amplified by PCR using specific primers. Using the pASK-IBA6 cloning vector, we produced recombinant terrelysin (rTerrelysin) as a fusion product in Escherichia coli. The recombinant protein was purified and using MALDI-TOF MS determined to have a mass of 16,428 Da. Circular dichroism analysis suggests the secondary structure of the protein to be predominantly β-sheet. Results from thermal denaturation of rTerrelysin show that the protein maintained the β-sheet confirmation up to 65°C. Polyclonal antibody to rTerrelysin recognized a protein of approximately 16.5 kDa in mycelial extracts from A. terreus.

The Escherichia coli transcriptome linked to growth fitness

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Bei-Wen Ying

2016-03-01

Full Text Available A series of Escherichia coli strains with varied genomic sequences were subjected to high-density microarray analyses to elucidate the fitness-correlated transcriptomes. Fitness, which is commonly evaluated by the growth rate during the exponential phase, is not only determined by the genome but is also linked to growth conditions, e.g., temperature. We previously reported genetic and environmental contributions to E. coli transcriptomes and evolutionary transcriptome changes in thermal adaptation. Here, we describe experimental details on how to prepare microarray samples that truly represent the growth fitness of the E. coli cells. A step-by-step record of sample preparation procedures that correspond to growing cells and transcriptome data sets that are deposited at the GEO database (GSE33212, GSE52770, GSE61739 are also provided for reference. Keywords: Transcriptome, Growth fitness, Escherichia coli, Microarray

Expression of recombinant Antibodies

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André eFrenzel

2013-07-01

Full Text Available Recombinant antibodies are highly specific detection probes in research, diagnostics and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with human-like post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

Immune responses to recombinants of the South African vaccine strain of lumpy skin disease virus generated by using thymidine kinase gene insertion.

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Wallace, David B; Viljoen, Gerrit J

2005-04-27

The South African vaccine strain of lumpy skin disease virus (type SA-Neethling) is currently being developed as a vector for recombinant vaccines of economically important livestock diseases throughout Africa. In this study, the feasibility of using the viral thymidine kinase gene as the site of insertion was investigated and recombinant viruses were evaluated in animal trials. Two separate recombinants were generated and selected for homogeneity expressing either the structural glycoprotein gene of bovine ephemeral fever virus (BEFV) or the two structural glycoprotein genes of Rift Valley fever virus (RVFV). Both recombinants incorporate the enhanced green fluorescent protein (EGFP) as a visual marker and the Escherichia coli guanine phosphoribosyl transferase (gpt) gene for dominant positive selection. The LSDV-RVFV recombinant construct (rLSDV-RVFV) protected mice against virulent RVFV challenge. In a small-scale BEFV-challenge cattle trial the rLSDV-BEFV construct failed to fully protect the cattle against virulent challenge, although both a humoral and cellular BEFV-specific immune response was elicited.

Pharmacokinetics, pharmacodynamics, efficacy, and safety of a new recombinant asparaginase preparation in children with previously untreated acute lymphoblastic leukemia: A randomized phase 2 clinical trial

NARCIS (Netherlands)

R. Pieters (Rob); I.M. Appel (Inge); H.J. Kuehnel; I. Tetzlaff-Fohr (Iris); U. Pichlmeier (Uwe); I. van der Vaart (Inekee); E. Visser (Eline); R.L. Stigter

2008-01-01

textabstractThe pharmacokinetics, pharmacodynam-ics, efficacy, and safety of a new recom-binant Escherichia coli - asparaginase preparation was compared withAsparagi-nase medac. Thirty-two children with acute lymphoblastic leukemia were randomized to receive one of both agents at a dose of 5000

Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

Science.gov (United States)

Targovnik, Alexandra M; Arregui, Mariana B; Bracco, Lautaro F; Urtasun, Nicolas; Baieli, Maria F; Segura, Maria M; Simonella, Maria A; Fogar, Mariela; Wolman, Federico J; Cascone, Osvaldo; Miranda, Maria V

2016-01-01

In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

Science.gov (United States)

Yamaguchi, Hiroshi; Miyazaki, Masaya

2014-02-20

Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

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Hiroshi Yamaguchi

2014-02-01

Full Text Available Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

The expression and serological reactivity of recombinant canine herpesvirus 1 glycoprotein D

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MarkéŽta Vaňkov‡á

2016-01-01

Full Text Available The aim of this work was to express recombinant glycoprotein D of canine herpesvirus 1 in bacterial cells and to evaluate its diagnostic sensitivity and specificity when compared to traditional serological methods. The gene fragment coding glycoprotein D of canine herpesvirus 1 was amplified by polymerase chain reaction, cloned into plasmid vector and expressed in Escherichia coli cells. Recombinant protein was then purified and used as an antigen in immunoblot for a detection of canine herpesvirus 1 specific antibodies. Antibody testing was performed on the panel of 100 canine sera by immunoblot with recombinant glycoprotein D as antigen and compared with indirect immunofluorescence assay. Serum samples were collected from 83 dogs with no history of canine herpesvirus 1 or reproductive disorders, and from 17 dogs from breeding kennels with a history of canine herpesvirus 1 related reproductive disorders. Sensitivity of glycoprotein D based immunoblot was 89.2% and specificity was 93%. Kappa value was calculated to be 0.8 between immunoblot and indirect immunofluorescence assay. Antibodies against canine herpesvirus 1 infection were detected in 33% of samples by immunoblot assay. Our study confirms that recombinant glycoprotein D expressed in bacterial cells could be used as a suitable and sensitive antigen for immunological tests and that herpesvirus infection seems to be common among the canine population in the Czech Republic.

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.

Optimization of the recombinant production and purification of a self-assembling peptide in Escherichia coli

NARCIS (Netherlands)

Rad-Malekshahi, Mazda; Flement, Matthias; Hennink, Wim E.; Mastrobattista, Enrico

2014-01-01

Background: Amphiphilic peptides are important building blocks to generate nanostructured biomaterials for drug delivery and tissue engineering applications. We have shown that the self-assembling peptide SA2 (Ac-AAVVLLLWEE) can be recombinantly produced in E. coli when fused to the small

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

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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.

A novel chimeric protein composed of recombinant Mycoplasma hyopneumoniae antigens as a vaccine candidate evaluated in mice.

Science.gov (United States)

de Oliveira, Natasha Rodrigues; Jorge, Sérgio; Gomes, Charles Klazer; Rizzi, Caroline; Pacce, Violetta Dias; Collares, Thais Farias; Monte, Leonardo Garcia; Dellagostin, Odir Antônio

2017-03-01

Enzootic Pneumonia (EP) is caused by the Mycoplasma hyopneumoniae pathogenic bacteria, and it represents a significant respiratory disease that is responsible for major economic losses within the pig industry throughout the world. The bacterins that are currently commercially available have been proven to offer only partial protection against M. hyopneumoniae, and the development of more efficient vaccines is required. Several recombinant antigens have been evaluated via different immunization strategies and have been found to be highly immunogenic. This work describes the construction and immunological characterization of a multi-antigen chimera composed of four M. hyopneumoniae antigens: P97R1, P46, P95, and P42. Immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in Escherichia coli, and the chimeric protein was recognized by specific antibodies against each subunit, as well as by convalescent pig sera. The immunogenic properties of the chimera were then evaluated in a mice model through two recombinant vaccines that were formulated as follows: (1) purified chimeric protein plus adjuvant or (2) recombinant Escherichia coli bacterin. The immune response induced in BALB/c mice immunized with each formulation was characterized in terms of total IgG levels, IgG1, and IgG2a isotypes against each antigen present in the chimera. The results of the study indicated that novel chimeric protein is a potential candidate for the future development of a more effective vaccine against EP. Copyright © 2017 Elsevier B.V. All rights reserved.

Export of Cytochrome P450 105D1 to the Periplasmic Space of Escherichia coli

OpenAIRE

Kaderbhai, Mustak A.; Ugochukwu, Cynthia C.; Kelly, Steven L.; Lamb, David C.

2001-01-01

CYP105D1, a cytochrome P450 from Streptomyces griseus, was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase and placed under the transcriptional control of the native phoA promoter. Heterologous expression in E. coli phosphate-limited medium resulted in abundant synthesis of recombinant CYP105D1 that was translocated across the bacterial inner membrane and processed to yield authentic, heme-incorporated P450 within the periplasmic space. Cell ext...

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

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Ashok Pandey

2011-01-01

Full Text Available Microbial phytases, especially from fungal and bacterial sources, have received much attention as food additives in human nutrition and as feed supplements for monogastric animals. An effective expression screening method for recombinant production of this enzyme on a small scale is industrially desirable. An effort has been made in this work to clone and express phytase genes from Aspergillus sp. and Escherichia coli with the selected host, vector and inducer combination. Albeit the formation of insoluble inclusion bodies by fungal phytase, recombinant E. coli appA was effectively expressed in a cost-effective manner in the periplasm of BL21plysS using an inducer concentration of 0.01 mM in 4 h of growth. Enzyme was purified in three consecutive steps and functional studies were carried out.

RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.

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Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael; Korolev, Sergey

2014-10-17

Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Recombinant production of plant lectins in microbial systems for biomedical application – the frutalin case study

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Carla eOliveira

2014-08-01

Full Text Available Frutalin is a homotetrameric partly-glycosylated alpha-D-galactose-binding lectin of biomedical interest from Artocarpus incisa (breadfruit seeds, belonging to the jacalin-related lectins family. As other plant lectins, frutalin is a heterogeneous mixture of several isoforms possibly with distinct biological activities. The main problem of using such lectins as biomedical tools is that batch-to-batch variation in isoforms content may lead to inconstant results. The production of lectins by recombinant means has the advantage of obtaining high amounts of proteins with defined amino-acid sequences and more precise properties. In this mini review, we provide the strategies followed to produce two different forms of frutalin in two different microbial systems: Escherichia coli and Pichia pastoris. The processing and functional properties of the recombinant frutalin obtained from these hosts are compared to those of frutalin extracted from breadfruit. Emphasis is given particularly to recombinant frutalin produced in P. pastoris, which showed a remarkable capacity as biomarker of human prostate cancer and as apoptosis-inducer of cancer cells. Recombinant frutalin production opens perspectives for its development as a new tool in human medicine.

Recombinant production of plant lectins in microbial systems for biomedical application – the frutalin case study

Science.gov (United States)

Oliveira, Carla; Teixeira, José A.; Domingues, Lucília

2014-01-01

Frutalin is a homotetrameric partly glycosylated α-D-galactose-binding lectin of biomedical interest from Artocarpus incisa (breadfruit) seeds, belonging to the jacalin-related lectins family. As other plant lectins, frutalin is a heterogeneous mixture of several isoforms possibly with distinct biological activities. The main problem of using such lectins as biomedical tools is that “batch-to-batch” variation in isoforms content may lead to inconstant results. The production of lectins by recombinant means has the advantage of obtaining high amounts of proteins with defined amino-acid sequences and more precise properties. In this mini review, we provide the strategies followed to produce two different forms of frutalin in two different microbial systems: Escherichia coli and Pichia pastoris. The processing and functional properties of the recombinant frutalin obtained from these hosts are compared to those of frutalin extracted from breadfruit. Emphasis is given particularly to recombinant frutalin produced in P. pastoris, which showed a remarkable capacity as biomarker of human prostate cancer and as apoptosis-inducer of cancer cells. Recombinant frutalin production opens perspectives for its development as a new tool in human medicine. PMID:25152749

Quantification and persistence of recombinant DNA of Roundup Ready corn and soybean in rotation.

Science.gov (United States)

Lerat, Sylvain; Gulden, Robert H; Hart, Miranda M; Powell, Jeff R; England, Laura S; Pauls, K Peter; Swanton, Clarence J; Klironomos, John N; Trevors, Jack T

2007-12-12

The presence of the recombinant cp4 epsps gene from Roundup Ready (RR) corn and RR soybean was quantified using real-time PCR in soil samples from a field experiment growing RR and conventional corn and soybean in rotation. RR corn and RR soybean cp4 epsps persisted in soil for up to 1 year after seeding. The concentration of recombinant DNA in soil peaked in July and August in RR corn and RR soybean plots, respectively. A small fraction of soil samples from plots seeded with conventional crops contained recombinant DNA, suggesting transgene dispersal by means of natural process or agricultural practices. This research will aid in the understanding of the persistence of recombinant DNA in agricultural cropping systems.

Biochemical and immunological characterization of recombinant allergen Lol p 1.

Science.gov (United States)

Tamborini, E; Faccini, S; Lidholm, J; Svensson, M; Brandazza, A; Longhi, R; Groenlund, H; Sidoli, A; Arosio, P

1997-11-01

Pollen from perennial rye grass (Lolium perenne), a major cause of type-I allergy worldwide, contains a complex mixture of allergenic proteins among which Lol p 1 is one of the most important. We describe the expression, purification and characterization of a recombinant Lol p 1 overproduced in Escherichia coli. The recombinant allergen, expressed in high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specifically recombinant Lol p 1 and natural Lol p 1 of pollen extract. Recombinant Lol p 1 was used to develop ImmunoCAP assays for analysis of 150 sera that were Radioallergosorbent test positive to L. perenne pollen. In 130 of them (87%) the assay detected a significant level of IgE antibodies to Lol p 1, reaching on average 37% of the level obtained with a test for IgE to the whole grass pollen extract. To map epitopes on Lol p 1, we produced three deletion mutants [des-(116-240)-Lol p 1, des-(1-88)-Lol p 1 and des-(133-189)-Lol p 1], which were efficiently expressed in bacteria. These all showed a strong reactivity with the specific rabbit IgG antibodies, but lacked most or all the allergenic properties of recombinant Lol p 1. A study of the antigenic structure of Lol p 1 was performed using the three deletion mutants and a set of 17-18-residue overlapping synthetic peptides covering the whole allergen sequence. The results indicate that human IgE and rabbit IgG antibodies bind to distinct regions of Lol p 1, and that at least some important IgE epitopes are mainly conformational. The findings suggest that recombinant allergens constitute useful reagents for further development of serological diagnosis of allergy, and that it should be possible to produce immunogenic fragments of allergenic proteins without allergenic properties.

Co-production of hydrogen and ethanol by Escherichia coli SS1 and its recombinant

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Chiu-Shyan Soo

2017-11-01

Conclusions: HybC could improve glycerol consumption rate and ethanol productivity of E. coli despite lower hydrogen and ethanol yields. Higher glycerol consumption rate of recombinant hybC could be an advantage for bioconversion of glycerol into biofuels. This study could serve as a useful guidance for dissecting the role of hydrogenase in glycerol metabolism and future development of effective strain for biofuels production.

Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli

Science.gov (United States)

Overhage, Jörg; Steinbüchel, Alexander; Priefert, Horst

2003-01-01

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter−1. This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter−1 after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter−1, besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter−1. The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue. PMID:14602615

Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

Science.gov (United States)

Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

2014-03-01

Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate. Copyright © 2014 Elsevier Inc. All rights reserved.

Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD

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Moon Yuseok

2009-01-01

Full Text Available Abstract Background ATP binding cassette (ABC transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA, lipase ABC transporter domains (LARDs were designed for the secretion of fusion proteins. Results The LARDs included four glycine-rich repeats comprising a β-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD to three types of proteins; green fluorescent protein (GFP, epidermal growth factor (EGF and cytoplasmic transduction peptide (CTP. These fusion proteins were expressed in Escherichia coli together with ABC transporter of either P. fluorescens or Erwinia chrysanthemi. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant. Conclusion The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in E. coli supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in Pseudomonas using LARDs or TliA as a C-terminal signal sequence.

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

Yeast synthetic biology for the production of recombinant therapeutic proteins.

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Kim, Hyunah; Yoo, Su Jin; Kang, Hyun Ah

2015-02-01

The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Purification and Fibrillation of Full-Length Recombinant PrP.

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Makarava, Natallia; Savtchenko, Regina; Baskakov, Ilia V

2017-01-01

Misfolding and aggregation of prion protein are related to several neurodegenerative diseases in humans such as Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. A growing number of applications in the prion field including assays for detection of PrP Sc and methods for production of PrP Sc de novo require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, oxidative modifications, or truncation, which is typically generated as a result of spontaneous oxidation or degradation. We also describe methods for preparation of amyloid fibrils from recombinant PrP in vitro. Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of PrP Sc for development of prion diagnostics including generation of PrP Sc -specific antibody.

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

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Ha, Jang-Ho; Kim, Ha-Neul; Moon, Ki-Beom; Jeon, Jae-Heung; Jung, Dai-Hyun; Kim, Su-Jung; Mason, Hugh S; Shin, Seo-Yeon; Kim, Hyun-Soon; Park, Kyung-Mok

2017-07-01

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana . Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana . The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana . The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana -derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli -derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana- derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana -derived recombinant human acidic fibroblast growth factor

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

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Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

2015-07-01

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

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

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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.

Recombinant Programming

OpenAIRE

Pawlak , Renaud; Cuesta , Carlos; Younessi , Houman

2004-01-01

This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and allows the programmer to recombine these sequences through the definition of cohesive code units called extensions. The output of such recombination is a mesh that can be used by the interpretation la...

Evaluating Recombinant Antigen ROP1 Efficacy in Diagnosis of Toxoplasma Gondii Infection

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F Keshavarzi

2015-07-01

Full Text Available Introduction:Toxoplasma gondii is a ubiquitous obligate intracellular parasite with a relatively broad host range infecting both mammals and birds. Toxoplasma proteins are strong antigens that can begin strong immune reactions, among which Rhoptry protein 1 (ROP1 can be named discharging from rhoptry cell-organ. ROP1 is regarded as a competitor for recombinant vaccines against toxoplasmosis. Therefore, the main objective of the current study was to evaluate the cloning and expression of ROP1 Toxoplasma gondii in a cloning vector as well as to create this recombinant antigen in order to be applied for later uses. Methods:Genomic DNA of Toxoplasma gondii was removed and reproduced by PCR, then the PCR product was cloned into the EcoR1 and BamH1 sites of cloning vector, pUET1, and transformed into Escherichia coli BL21 plysS strain. Moreover, pcROP1 was sub-cloned into the HindIII and EcoRI sites of the pcDNA3 in order to produce recombining eukaryotic declaration vector. The cloned ROP1 was verified by PCR, limitation enzymes (HindIII and BglΙ digestion and nucleotide sequencing. Then, this recombinant antigen was covered applying IgM and ELISAIgG. Results:The study results demonstrated that a fragment of 757 bp was separated. In addition, nucleotide sequence analysis of the ROP1 cloned in pUET1vector revealed high homology (96% with RH strain Gene Bank Accession (No. M71274. Conclusion:The recombinant ROP1 antigen in an IgM Rec-ELISA test can be replaced with the tachyzoite antigen in IgG and IgM serologic tests.

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

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Raran-Kurussi, Sreejith; Waugh, David S

2016-07-01

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

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis.

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Lennon, V A; Lambert, E H; Leiby, K R; Okarma, T B; Talib, S

1991-04-01

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis.

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Godfrey, Charlotte L; Mead, Emma J; Daramola, Olalekan; Dunn, Sarah; Hatton, Diane; Field, Ray; Pettman, Gary; Smales, C Mark

2017-08-01

mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5' untranslated regions that might be utilised to enhance recombinant transcript translation. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Refolding in high hydrostatic pressure of recombinant proteins from inclusion bodies in Escherichia Coli; Renaturacao em altas pressoes hidrostaticas de proteinas recombinantes agregadas em corpos de inclusao produzidos em Escherichia Coli

Energy Technology Data Exchange (ETDEWEB)

Balduino, Keli Nunes

2009-07-01

The expression of proteins as inclusion bodies in bacteria is a widely used alternative for production of recombinant protein. However, the aggregation is a problem often encountered during refolding of these proteins. High hydrostatic pressure are able to solubilise the inclusion bodies in the presence of low concentrations of denaturant reagents, encouraging refolding protein with high efficiency and reduce costs. This work aims to refolding of recombinant proteins expressed in Escherichia coli from inclusion bodies using high hydrostatic pressure. Three toxins, all featuring five or more disulfide bonds were studied: NXH8, Natterin 2 and Bothropstoxin 1. Suspensions of inclusion bodies of the three proteins were pressurized to 2000 bars for 16 hours. The buffers were optimized for refolding of the three proteins. The buffer used in the refolding of NXH8 was 50 mM Tris HCl, pH 9.0 with proportion of 1GSH: 4GSSG at a concentration of 6 mM and 2 M GdnHCl. Inclusion bodies were used in O.D. (A600nm) of 0.5. After refolding process, dialysis was performed at pH 7.0. The final yield of obtaining soluble NXH8 was 40% (28,6 mg of soluble NXH8/L of culture medium). The refolding of Bothropstoxin 1 was obtained in refolding buffer of Tris HCl 50 mM, pH 7,5 with proportion of 2 GSH: GSSG 3 and concentration of 3 mM and 1 M GdnHCl. Use with a suspension of O.D. (A600nm) of 0.5. The final yield of recovery of Bothropstoxin 1 refolded was 32% (9,2 mg of refolded Bothropstoxin 1/L of culture medium). The refolding of Natterin 2 was performed in the refolding buffer: 20 mM Tris HCl pH 9.0 at a ratio of 2 GSH: 3GSSG and concentration of 10 mM and 1 M GdnHCl and inclusion bodies O.D. (A600nm) of 6.0. The yield of Natterin 2 refolded was 20% (3,7 mg/L of culture medium). Physico-chemical and biological analysis were performed by SDS-PAGE, western blot, scanning electron microscopy, biological tests in vivo and in vitro and structural. The analysis conducted in NXH8 did not show

Recombiner

International Nuclear Information System (INIS)

Kikuchi, Nobuo.

1983-01-01

Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)

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

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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.

Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

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Matej Zábrady

Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

A recombinant Toscana virus nucleoprotein in a diagnostic immunoblot test system.

Science.gov (United States)

Schwarz, T F; Gilch, S; Schätzl, H M

1998-01-01

Sandfly fever, a vector-borne disease endemic in the Mediterranean region, is caused by Toscana virus (TOS). The disease is increasingly important as a travel-related infection. Serological diagnosis is currently dependent on viral antigens derived from TOS-infected cell cultures. In this study, we report the cloning and expression of the TOS nucleoprotein (N) in Escherichia coli and evaluation of the recombinant (r) TOS N protein as an antigen for immunoblot assays. The TOS N gene was amplified by reverse-transcriptase polymerase chain reaction and cloned into the bacterial expression vector pTrcHis-A. Sera with known TOS antibody status were used to evaluate the immunoblot assay. The expressed rTOS N protein was purified and used as antigen for immunoblots. By recombinant immunoblot, the TOS antibody status (IgM and/or IgG) of the test panel was correctly identified. No cross-reactivity was detected. The rTOS N protein is useful as an antigen for immunoblot assays, and will enable more laboratories to perform TOS antibody diagnosis.

Effect of the mutation of recB21 of Escherichia coli on indirect recombinogenesis of bacteriophage lambda

International Nuclear Information System (INIS)

Castillo-Villanueva, A.

1993-01-01

In this work two undamaged amber lambda mutants were crossed in UV-irradiated Escherichia coli host cells and the total and recombinant λ + progenies scored after one lytic cycle. In a wild - type strain, such treatment produces an stimulation of 5-7 times in the production of recombinant λ + particles, accompanied by a variable but consistent increase in the total phage pro genie too. The effect has been designed as indirect recombinogenesis of bacteriophage lambda because it is elicited among undamaged λ genomes by the UV irradiation of host cells. Through the use of recB21 mutants we tested the role of the RecBCD enzyme as a whole in the effect just described and observed that in those hosts the effect is absent. The RecBCD enzyme of Escherichia coli has different activities, important for both the genetic recombination and recovery from DNA damage. Among those activities is that of ATP-dependent double-stranded DNA exonuclease, by means of which the enzyme digests the lineal molecules of viral DNA, produced during the late phase of lambda lytic cycle. To face such a destructive activity, λ encodes the Gam protein which inhibits all the activities of RecBCD; so the ability of RecBCD to act in the phage response, may be due either to a residual activity of the enzyme in lambda infected host cells or to an unknown Gam non-inhibited activity of the RecBCD enzyme. because the synthesis of the RecBCD is constitutive, the apparent inducibility of the λ response should be due to another reason such as an increase in the molecular substrates on which the enzyme acts. (Author)

The population and evolutionary dynamics of homologous gene recombination in bacterial populations.

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Bruce R Levin

2009-08-01

Full Text Available In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination -- broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT -- plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1 the contribution of HGR to the rate of adaptive evolution in these populations and (2 the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1 HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2 once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent

Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins.

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David J Leibly

Full Text Available Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2, proline, xylitol, NDSB 201, CTAB and K(2PO(4 solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40% were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA) vaccine to prevent anthrax in adults.

Science.gov (United States)

Brown, Bruce K; Cox, Josephine; Gillis, Anita; VanCott, Thomas C; Marovich, Mary; Milazzo, Mark; Antonille, Tanya Santelli; Wieczorek, Lindsay; McKee, Kelly T; Metcalfe, Karen; Mallory, Raburn M; Birx, Deborah; Polonis, Victoria R; Robb, Merlin L

2010-11-05

The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA). A total of 73 healthy adults ages 18-40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg) of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29) was observed in the group receiving 25 µg Alhydrogel®-formulated rPA. The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses. ClinicalTrials.gov NCT00057525.

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

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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.

Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein.

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Litai Zhang

Full Text Available Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4 emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats.

Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

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N.S. Sato

2004-07-01

Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

Relationship among the repair and genetic recombination mechanisms. II. Effect of gamma radiation on the lambda recombination in E. coli AB1157 and AB1886; Relacion entre los mecanismos de reparacion y la recombinacion genetica. II. efecto de la radiacion gamma sobre la recombinacion de lambda en E. Coli AB1157 y AB1886

Energy Technology Data Exchange (ETDEWEB)

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

1988-08-15

The objective of the present work is to determine if the radiation gamma that is a good inductor of the answer SOS of Escherichia Coli but that it produces alterations in the DNA very different to those taken place by the light UV, it is able to stimulate the viral recombination. (Author)

Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase.

Science.gov (United States)

Jameson, Guy N L; Cosper, Michele Mader; Hernández, Heather L; Johnson, Michael K; Huynh, Boi Hanh

2004-02-24

Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe-2S]-containing BioB with Fe(2+) and S(2)(-) produces a form of BioB that contains approximately one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per monomer ([2Fe-2S]/[4Fe-4S] BioB). In the absence of added Fe, the [2Fe-2S]/[4Fe-4S] BioB is active and can produce up to approximately 0.7 equiv of biotin per monomer. To better define the roles of the Fe-S clusters in the BioB reaction, Mössbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the states of the Fe-S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe-4S](2+) cluster is stable during the reaction and present in the SAM-bound form, supporting the current consensus that the functional role of the [4Fe-4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5'-deoxyadenosyl radical. The results also demonstrate that approximately (2)/(3) of the [2Fe-2S] clusters are degraded by the end of the turnover experiment (24 h at 25 degrees C). A transient species with spectroscopic properties consistent with a [2Fe-2S](+) cluster is observed during turnover, suggesting that the degradation of the [2Fe-2S](2+) cluster is initiated by reduction of the cluster. This observed degradation of the [2Fe-2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe-2S] cluster put forth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry 40, 8352-8358). Interestingly, degradation of the [2Fe-2S](2+) cluster was found not to parallel

Frequency and organization of papA homologous DNA sequences among uropathogenic digalactoside-binding Escherichia coli strains.

OpenAIRE

Denich, K; Craiu, A; Rugo, H; Muralidhar, G; O'Hanley, P

1991-01-01

The frequency of selected papA DNA sequences among 89 digalactoside-binding, uropathogenic Escherichia coli strains was evaluated with 12 different synthetic 15-base probes corresponding to papA genes from four digalactoside-binding piliated recombinant strains (HU849, 201B, and 200A). The papA probes encode amino acids which are common at the carboxy terminus of all strains, adjacent to the proximal portion of the intramolecular disulfide loop of strain 210B, or predicted to constitute the t...

Expression of Recombinant Streptokinase from Streptococcus Pyogenes and its Reaction with Infected Human and Murine Sera

Science.gov (United States)

Molaee, Neda; Abtahi, Hamid; Mosayebi, Ghasem

2013-01-01

Objective(s): Streptokinase (SKa) is an antigenic protein which is secreted by Streptococcus pyogenes. Streptokinase induces inflammation by complement activation, which may play a role in post infectious diseases. In the present study, recombinant streptokinase from S. pyogenes was produced and showed that recombinant SKa protein was recognized by infected human sera using Western blot analysis. Materials and Methods: In this study, the ska gene from S. pyogenes was amplified and cloned into pET32a which is a prokaryotic expression vector. pET32a-ska was transformed to Escherichia coli BL21 (DE3) pLysS and gene expression was induced by IPTG. Protein production was improved by modification of composition of the bacterial culture media and altering the induction time by IPTG. The expressed protein was purified by affinity chromatography using the Ni-NTA resin. The integrity of the product was confirmed by Westernblot analysis using infected mice. Serum reactivity of five infected individuals was further analyzed against the recombinant SKa protein. Results: Data indicated that recombinant SKa protein from S. pyogenes can be recognized by patient and mice sera. The concentration of the purified recombinant protein was 3.2 mg/L of initial culture. The highest amount of the expressed protein after addition of IPTG was obtained in a bacterial culture without glucose with the culture optical density of 0.8 (OD600 = 0.8). Conclusion : Present data shows, recombinant SKa protein has same epitopes with natural form of this antigen. Recombinant SKa also seemed to be a promising antigen for the serologic diagnosis of S. pyogenes infections. PMID:24171077

Expression of Recombinant Streptokinase from Streptococcus Pyogenes and Its Reaction with Infected Human and Murine Sera

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Neda Molaee

2013-09-01

Full Text Available   Objective(s: Streptokinase (SKa is an antigenic protein which is secreted by Streptococcus pyogenes. Streptokinase induces inflammation by complement activation, which may play a role in post infectious diseases. In the present study, recombinant streptokinase from S. pyogenes was produced and showed that recombinant SKa protein was recognized by infected human sera using Western blot analysis.   Materials and Methods: In this study, the ska gene from S. pyogenes was amplified and cloned into pET32a which is a prokaryotic expression vector. pET32a-ska was transformed to Escherichia coli BL21 (DE3 pLysS and gene expression was induced by IPTG. Protein production was improved by modification of composition of the bacterial culture media and altering the induction time by IPTG. The expressed protein was purified by affinity chromatography using the Ni-NTA resin. The integrity of the product was confirmed by Westernblot analysis using infected mice. Serum reactivity of five infected individuals was further analyzed against the recombinant SKa protein. Results: Data indicated that recombinant SKa protein from S. pyogenes can be recognized by patient and mice sera. The concentration of the purified recombinant protein was 3.2 mg/L of initial culture. The highest amount of the expressed protein after addition of IPTG was obtained in a bacterial culture without glucose with the culture optical density of 0.8 (OD600 = 0.8. Conclusion : Present data shows, recombinant SKa protein has same epitopes with natural form of this antigen. Recombinant SKa also seemed to be a promising antigen for the serologic diagnosis of S. pyogenes infections.

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

Genomic diversity of Escherichia isolates from diverse habitats.

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Seungdae Oh

Full Text Available Our understanding of the Escherichia genus is heavily biased toward pathogenic or commensal isolates from human or animal hosts. Recent studies have recovered Escherichia isolates that persist, and even grow, outside these hosts. Although the environmental isolates are typically phylogenetically distinct, they are highly related to and phenotypically indistinguishable from their human counterparts, including for the coliform test. To gain insights into the genomic diversity of Escherichia isolates from diverse habitats, including freshwater, soil, animal, and human sources, we carried out comparative DNA-DNA hybridizations using a multi-genome E. coli DNA microarray. The microarray was validated based on hybridizations with selected strains whose genome sequences were available and used to assess the frequency of microarray false positive and negative signals. Our results showed that human fecal isolates share two sets of genes (n>90 that are rarely found among environmental isolates, including genes presumably important for evading host immune mechanisms (e.g., a multi-drug transporter for acids and antimicrobials and adhering to epithelial cells (e.g., hemolysin E and fimbrial-like adhesin protein. These results imply that environmental isolates are characterized by decreased ability to colonize host cells relative to human isolates. Our study also provides gene markers that can distinguish human isolates from those of warm-blooded animal and environmental origins, and thus can be used to more reliably assess fecal contamination in natural ecosystems.

Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing.

Science.gov (United States)

Khaki, Mohsen; Salmanian, Ali Hatef; Mosayebi, Ghasem; Baazm, Maryam; Babaei, Saeed; Molaee, Neda; Abtahi, Hamid

2017-07-01

Vascular endothelial growth factor (VEGF) is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs) differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli ( E. coli ) system and then biological activity of this protein was evaluated in animal wound healing. E. coli BL21 (DE3) competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG). The recombinant protein was purified by affinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w) was used for external wound (25×15mm thickness) healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. The recombinant protein with molecular weight of 45 kilodaltons (kDa) and concentration of 0.8 mg/ml was produced. Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing. Recombinant VEGF-A produced by pET32a in E. coli , possesses acceptable structure and has wound healing capability.

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

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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.

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,

Uji Aktivitas Antibakteri Jamur Endofit Akar Bakau Avicennia Marina Terhadap Bakteri Staphylococcus Aureus Dan Escherichia Coli

OpenAIRE

Liwang, Firdy

2014-01-01

: In this his study we used endophytic fungi isolated from the roots of mangrove Avicennia marina growing on tidal zone around Tasik Ria Minahasa, North Sulawesi. The fungi were isolated and then tested the antibacterial effect against Staphylococcus aureus and Escherichia coli. Potato Dextrose agar was used in order to isolate the target fungi. The fungi began to grow on the second day after inoculation. Differentiation and purification processes to isolate the fungus obtained by observing f...

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.

A simple and effective method for construction of Escherichia coli strains proficient for genome engineering.

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Young Shin Ryu

Full Text Available Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.

Mimicking a New 2-Phenylethanol Production Pathway from Proteus mirabilis JN458 in Escherichia coli.

Science.gov (United States)

Liu, Jinbin; Jiang, Jing; Bai, Yajun; Fan, Tai-Ping; Zhao, Ye; Zheng, Xiaohui; Cai, Yujie

2018-04-04

Bacteria rarely produce natural 2-phenylethanol. We verified a new pathway from Proteus mirabilis JN458 to produce 2-phenylethanol using Escherichia coli to coexpress l-amino acid deaminase, α-keto acid decarboxylase, and alcohol dehydrogenase from P. mirabilis. Based on this pathway, a glucose dehydrogenase coenzyme regeneration system was constructed. The optimal conditions of biotransformation by the recombinant strain E-pAEAKaG were at 40 °C and pH 7.0. Finally, the recombinant strain E-pAEAKaG produced 3.21 ± 0.10 g/L 2-phenylethanol in M9 medium containing 10 g/L l-phenylalanine after a 16 h transformation. Furthermore, when the concentration of l-phenylalanine was 4 g/L (24 mM), the production of 2-phenylethanol reached 2.88 ± 0.18 g/L and displayed a higher conversion rate of 97.38 mol %.

Advanced control of dissolved oxygen concentration in fed batch cultures during recombinant protein production.

Science.gov (United States)

Kuprijanov, A; Gnoth, S; Simutis, R; Lübbert, A

2009-02-01

Design and experimental validation of advanced pO(2) controllers for fermentation processes operated in the fed-batch mode are described. In most situations, the presented controllers are able to keep the pO(2) in fermentations for recombinant protein productions exactly on the desired value. The controllers are based on the gain-scheduling approach to parameter-adaptive proportional-integral controllers. In order to cope with the most often appearing distortions, the basic gain-scheduling feedback controller was complemented with a feedforward control component. This feedforward/feedback controller significantly improved pO(2) control. By means of numerical simulations, the controller behavior was tested and its parameters were determined. Validation runs were performed with three Escherichia coli strains producing different recombinant proteins. It is finally shown that the new controller leads to significant improvements in the signal-to-noise ratio of other key process variables and, thus, to a higher process quality.

INACTIVITY OF RECOMBINANT ELA2B PROVIDES A NEW EXAMPLE OF EVOLUTIONARY ELASTASE SILENCING IN HUMANS

OpenAIRE

Szepessy, Edit; Sahin-Tóth, Miklós

2005-01-01

BACKGROUND. The archetypal mammalian elastase (ELA1) is not expressed in the human pancreas, because evolutionary mutations suppressed transcription of the ELA1 gene. AIMS. In this study we tested the theory that the unique duplication of the ELA2 gene in humans might compensate for the loss of ELA1. METHODS. Recombinant ELA2A and ELA2B were expressed in Escherichia coli, and their activity was tested on Glt-Ala-Ala-Pro-Leu-p-nitroanilide, DQ elastin and bovine milk protein. RESULTS. Surprisi...

[Construction and characterization of enterohemorrhagic Escherichia coli O157:H7 ppk- deleted strain].

Science.gov (United States)

Han, Peng; Sun, Qi; Zhao, Suhui; Zhang, Qiwei; Wan, Chengsong

2014-06-01

To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics. The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining. We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

UV irradiation alters deoxynucleoside triphosphate pools in Escherichia coli

International Nuclear Information System (INIS)

Das, S.K.; Loeb, L.A.

1984-01-01

UV irradiation of exponentially growing Escherichia coli increased intracellular concentration of dATP and dTTP without significantly changing the concentrations of dGTP and dCTP. These selective increases in dATP and dTTP pools are seen in wild-type E. coli K12 and AB1157, as well as in recA and umuC strains, and are proportional to UV dose. The possible significance of these findings with respect to induction of the SOS response and nontargeted mutagenesis are discussed. (orig.)

Photoionization and Recombination

Science.gov (United States)

Nahar, Sultana N.

2000-01-01

Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

Crystallization and preliminary X-ray analysis of Escherichia coli RNase G

International Nuclear Information System (INIS)

Fang, Pengfei; Wang, Jing; Li, Xu; Guo, Min; Xing, Li; Cao, Xu; Zhu, Yi; Gao, Yan; Niu, Liwen; Teng, Maikun

2009-01-01

Full-length E. coli RNase G was overexpressed, purified and crystallized. Diffraction data were collected to a resolution of 3.4 Å. The homologous RNases RNase E and RNase G are widely distributed in bacteria and function in many important physiological processes, including mRNA degradation, rRNA maturation and so on. In this study, the crystallization and preliminary X-ray analysis of RNase G from Escherichia coli is described. Purified recombinant E. coli RNase G, which has 497 amino acids, was crystallized in the cubic space group F432, with unit-cell parameters a = b = c = 219.84 Å. X-ray diffraction data were collected to a resolution of 3.4 Å

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

Contribution of viral recombinants to the study of the immune response against the Epstein-Barr virus.

Science.gov (United States)

Delecluse, Henri-Jacques; Feederle, Regina; Behrends, Uta; Mautner, Josef

2008-12-01

Over the past two decades, Epstein-Barr virus (EBV) mutants have become valuable tools for the analysis of viral functions. Several experimental strategies are currently used to generate recombinant mutant genomes that carry alterations in one or several viral genes. The probably most versatile approach utilizes bacterial artificial chromosomes (BAC) carrying parts or the whole EBV genome, which permits extensive genetic manipulations in Escherichia coli cells. The 'mini-EBVs', for example, which contain roughly half of the wild type viral information, efficiently transform primary B cells and have been used as gene vectors for foreign antigens. After expression in lymphoblastoid cell lines (LCLs), these antigens are efficiently presented on MHC molecules and recognized by antigen-specific T cells. These vectors, however, cannot undergo lytic replication and require a helper cell line for efficient replication and DNA packaging. Further experimental systems include the complete viral genome cloned onto a BAC. These mutants can typically be complemented by expression plasmids, some of which are expressed on EBV-derived vectors and can be propagated without requirement of a helper cell line. Over the last years, these viral recombinants have been utilized increasingly to analyse different aspects of the immune response against EBV. Immunological applications are manifold and steadily growing and include crude screening of T cell clones for their specificity towards latent versus lytic antigens, or more detailed analyses in which the exact specificity of T cells is determined using EBV mutants that lack a single viral antigen. Other applications include detailed analysis of protein domains important for immune recognition, e.g. Gly-Ala repeats in the EBV nuclear antigen 1 (EBNA1) protein, expansion of T cell clones directed against virion structures using virus-like particles and phenotypic analysis of virus mutants defective in infection. Future developments might

Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

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Kamala Boonyodying

2012-06-01

Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

Anti-Neuroblastoma Properties of a Recombinant Sunflower Lectin

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Marcela Pinedo

2017-01-01

Full Text Available According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in Escherichia coli representing Helja coupled to thioredoxin (Trx. The identity of this protein was verified using anti-Helja antibodies. This chimera, named Trx-rHelja, was enriched in the soluble bacterial extracts and was purified using Ni+2-Sepharose and d-mannose-agarose chromatography. Trx-rHelja and the enterokinase-released recombinant Helja (rHelja both displayed toxicity on human SH-SY5Y neuroblastomas. rHelja decreased the viability of these tumor cells by 75% according to the tetrazolium reduction assay, and microscopic analyses revealed that the cell morphology was disturbed. Thus, the stellate cells of the monolayer became spheroids and were isolated. Our results indicate that rHelja is a promising tool for the development of diagnostic or therapeutic methods for neuroblastoma cells, the most common solid tumors in childhood.

Expression and Evaluation of Recombinant Plasmodium knowlesi Merozoite Surface Protein-3 (MSP-3 for Detection of Human Malaria.

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Jeremy Ryan De Silva

Full Text Available Malaria remains a major health threat in many parts of the globe and causes high mortality and morbidity with 214 million cases of malaria occurring globally in 2015. Recent studies have outlined potential diagnostic markers and vaccine candidates one of which is the merozoite surface protein (MSP-3. In this study, novel recombinant Plasmodium knowlesi MSP-3 was cloned, expressed and purified in an Escherichia coli system. Subsequently, the recombinant protein was evaluated for its sensitivity and specificity. The recombinant pkMSP-3 protein reacted with sera from patients with P. knowlesi infection in both Western blot (61% and ELISA (100%. Specificity-wise, pkMSP-3 did not react with healthy donor sera in either assay and only reacted with a few non-malarial parasitic patient sera in the ELISA assay (3 of 49. In conclusion, sensitivity and specificity of pkMSP-3 was found to be high in the ELISA and Western Blot assay and thus utilising both assays in tandem would provide the best sero-diagnostic result for P. knowlesi infection.

Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco.

Science.gov (United States)

Garcia, I; Rodgers, M; Pepin, R; Hsieh, T F; Matringe, M

1999-04-01

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

Construction of Escherichia coli Mutant with Decreased Endotoxic Activity by Modifying Lipid A Structure

Directory of Open Access Journals (Sweden)

Qiong Liu

2015-05-01

Full Text Available Escherichia coli BL21 (DE3 and its derivatives are widely used for the production of recombinant proteins, but these purified proteins are always contaminated with lipopolysaccharide (LPS. LPS is recognized by the toll-like receptor 4 and myeloid differentiation factor 2 complex of mammalian immune cells and leads to release of pro-inflammatory cytokines. It is a vital step to remove LPS from the proteins before use for therapeutic purpose. In this study, we constructed BL21 (DE3 ∆msbB28 ∆pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity. The plasmids harboring pagL and/or lpxE were then introduced into this mutant to further modify the LPS. The new strain (S004 carrying plasmid pQK004 (pagL and lpxE produced mono-phosphoryated tetra-acylated lipid A, which induces markedly less production of tumor necrosis factor-α in the RAW264.7 and IL-12 in the THP1, but still retains ability to produce recombinant proteins. This study provides a strategy to decrease endotoxic activity of recombinant proteins purified from E. coli BL21 backgrounds and a feasible approach to modify lipid A structure for alternative purposes such as mono-phosphoryl lipid A (MPL as vaccine adjuvants.

Choreography of recombination proteins during the DNA damage response

DEFF Research Database (Denmark)

Lisby, Michael; Rothstein, Rodney

2009-01-01

Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such...... research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells....

Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing

Directory of Open Access Journals (Sweden)

Mohsen Khaki

2017-07-01

Full Text Available Objective(s: Vascular endothelial growth factor (VEGF is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli (E. coli system and then biological activity of this protein was evaluated in animal wound healing. Materials and Methods: E. coli BL21 (DE3 competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG. The recombinant protein was purified byaffinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w was used for external wound (25×15mm thickness healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. Results: The recombinant protein with molecular weight of 45 kilodaltons (kDa and concentration of 0.8 mg/ml was produced.Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing. Conclusion: Recombinant VEGF-A produced by pET32a in E. coli, possesses acceptable structure and has wound healing capability.

Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

Science.gov (United States)

Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

2010-10-13

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

Science.gov (United States)

Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

1995-08-01

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

Directory of Open Access Journals (Sweden)

Masaki Odahara

2015-03-01

Full Text Available Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology.

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Chen, Po Ting; Chiang, Chung-Jen; Chao, Yun-Peng

2007-01-01

Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007, 23, 808-813). With this developed B. subtilis strain, the design of an optimum but cost-effective medium for high-level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett-Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box-Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake-flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 degrees C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost-effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high-level production of recombinant nattokinase in a fermenter.

Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

DEFF Research Database (Denmark)

Olavarria, K.; De Ingeniis, J.; Zielinski, D. C.

2014-01-01

In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Δpgi). In this work......PDH(R46E,Q47E). Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Δpgi E. coli strains were replaced by DNA encoding LmG6PDH(R46E,Q47E). Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect...

Inhibition of Escherichia coli precursor-16S rRNA processing by mouse intestinal contents

DEFF Research Database (Denmark)

Licht, Tine Rask; Tolker-Nielsen, Tim; Holmstrøm, Kim

1999-01-01

. We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coli cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial...... content of pre-16S rRNA than cultures of the same strain growing rapidly in rich media. We present results suggesting that the mouse intestinal contents contain an agent that inhibits the growth of E. coli by disturbing its ability to process pre-16S rRNA....

Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide

International Nuclear Information System (INIS)

Lai Yuping; Peng Yifei; Zuo Yi; Li Jun; Huang Jing; Wang Linfa; Wu Zirong

2005-01-01

Antimicrobial peptides from human skin are an important component of the innate immune response and play a key role as a first line of defense against infections. One such peptide is the recently discovered dermcidin-1L. To better understand its mechanism and to further investigate its antimicrobial spectrum, recombinant dermcidin-1L was expressed in Escherichia coli as a fusion protein and purified by affinity chromatography. The fusion protein was cleaved by factor Xa protease to produce recombinant dermcidin-1L. Antimicrobial and hemolytic assays demonstrated that dermcidin-1L displayed microbicidal activity against several opportunistic nosocomial pathogens, but no hemolytic activity against human erythrocytes even at concentrations up to 100 μM. Structural studies performed by circular dichroism spectroscopy indicated that the secondary structure of dermcidin-1L was very flexible, and both α-helix and β-sheet structures might be required for the antimicrobial activity. Our results confirmed previous findings indicating that dermcidin-1L could have promising therapeutic potentials and shed new light on the structure-function relationship of dermcidin-1L

Recombinant ACHT1 from Arabidopsis thaliana: crystallization and X-ray crystallographic analysis.

Science.gov (United States)

Pan, Weimin; Wang, Junchao; Yang, Ye; Liu, Lin; Zhang, Min

2017-07-01

Thioredoxins (Trxs) play important roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. They execute their function by regulating the oxidation and reduction of disulfide bonds. ACHT1 (atypical cysteine/histidine-rich Trx1) is a thylakoid-associated thioredoxin-type protein found in the Arabidopsis thaliana chloroplast. Recombinant ACHT1 protein was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal diffracted to 1.7 Å resolution and a complete X-ray data set was collected. Preliminary crystallographic analysis suggested that the crystals belonged to space group C222 1 , with unit-cell parameters a = 102.7, b = 100.6, c = 92.8 Å.

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....

Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses.

Science.gov (United States)

Rasala, Beth A; Mayfield, Stephen P

2015-03-01

Recombinant proteins are widely used for industrial, nutritional, and medical applications. Green microalgae have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins for a number of reasons. These photosynthetic eukaryotic microorganisms are safe, scalable, easy to genetically modify through transformation, mutagenesis, or breeding, and inexpensive to grow. Many microalgae species are genetically transformable, but the green alga Chlamydomonas reinhardtii is the most widely used host for recombinant protein expression. An extensive suite of molecular genetic tools has been developed for C. reinhardtii over the last 25 years, including a fully sequenced genome, well-established methods for transformation, mutagenesis and breeding, and transformation vectors for high levels of recombinant protein accumulation and secretion. Here, we review recent successes in the development of C. reinhardtii as a biomanufacturing host for recombinant proteins, including antibodies and immunotoxins, hormones, industrial enzymes, an orally-active colostral protein for gastrointestinal health, and subunit vaccines. In addition, we review the biomanufacturing potential of other green algae from the genera Dunaliella and Chlorella.

Production of Recombinant Antimicrobial Polymeric Protein Beta Casein-E 50-52 and Its Antimicrobial Synergistic Effects Assessment with Thymol

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Shohreh Fahimirad

2017-05-01

Full Text Available Accelerating emergence of antimicrobial resistance among food pathogens and consumers’ increasing demands for preservative-free foods are two contemporary challenging aspects within the food industry. Antimicrobial packaging and the use of natural preservatives are promising solutions. In the present study, we used beta-casein—one of the primary self-assembly proteins in milk with a high polymeric film production capability—as a fusion partner for the recombinant expression of E 50-52 antimicrobial peptide in Escherichia coli. The pET21a-BCN-E 50-52 construct was transformed to E. coli BL21 (DE3, and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg/mL fusion protein by ultrafiltration. Antimicrobial activities of recombinant BCN-E 50-52 performed against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus, and Candida albicans. Subsequently, the synergistic effects of BCN-E 50-52 and thymol were assayed. Results of checkerboard tests showed strong synergistic activity between two compounds. Time–kill and growth kinetic studies indicated a sharp reduction of cell viability during the first period of exposure, and SEM (scanning electron microscope results validated the severe destructive effects of BCN E 50-52 and thymol in combination on bacterial cells.

Recombiner

International Nuclear Information System (INIS)

Osumi, Morimichi.

1979-01-01

Purpose: To provide a recombiner which is capable of converting hydrogen gas into water by use of high-frequency heating at comparatively low temperatures and is safe and cheap in cost. Constitution: Hydrogen gas is introduced from an outer pipeline to the main structure of a recombiner, and when it passes through the vicinity of the central part of the recombiner, it is reacted with copper oxide (CuO 2 ) heated to a temperature more than 300 0 C by a high-frequency heater, and converted gently into water by reduction operation (2H 2 + CuO 2 → Cu + 2H 2 O). The thus prepared water is exhausted through the outer pipeline to a suppression pool. A part of hydrogen gas which has not been converted completely into water by the reaction and is remaining as hydrogen is recovered through exhaust nozzles and again introduced into the main structure of the recombiner. (Yoshino, Y.)

Mutants of Escherichia coli K-12 with enhanced resistance to ionizing radiation. 4. Peculiarities of recombination in Gamsup(r) mutants

International Nuclear Information System (INIS)

Bresler, S.E.; Kalinin, V.L.; Laneeva, N.I.

1984-01-01

Radioresistant mutant Gam sup(r) 444 differs from a wild type and from Gam sup(r) 445 mutant in decreased frequency of long episome heritage ORF 1 (pur E + -tsx + -proC + -lac + ) and F 14 (ilv + -argE + ), containing hot points of RecRecF - depending recombination and in increased frequency of chromosome mobilization and integrative suppression of temperature sensitive dna A46 mutation by sexual factor F. In this respect Gam sup(r) 444 mutant resembles rec BC sbs B mutant with RecF - recombination type

Recombinant egg drop syndrome subunit vaccine offers an alternative to virus propagation in duck eggs.

Science.gov (United States)

Gutter, B; Fingerut, E; Gallili, G; Eliahu, D; Perelman, B; Finger, A; Pitcovski, J

2008-02-01

Egg drop syndrome (EDS) virus vaccines are routinely produced in embryonated duck eggs (Solyom et al., 1982). This procedure poses the risk of dissemination of pathogens, such as avian influenza virus, as the eggs used are not from specific pathogen free birds. To address this problem, the knob and part of the shaft domain of the fibre protein of the EDS virus (termed knob-s) were expressed in Escherichia coli and assessed as a subunit vaccine. A single vaccination with the recombinant protein induced the production of anti-EDS virus antibodies, as detected by haemagglutination inhibition, enzyme-linked immunosorbent assay and virus neutralization tests, for at least 20 weeks. A positive correlation was demonstrated between these three assays. A dose-response assessment showed that the vaccine was effective over the range of 2 to 64 microg protein per dose. Two vaccinations with the recombinant protein, administered before the onset of lay, induced high haemagglutination inhibition antibody titres, comparable with those induced by an inactivated whole-virus vaccine. The vaccine did not have any adverse effects on egg production, quality or weight. The present study has shown that two vaccinations with the recombinant knob-s protein elicited high neutralizing antibody titres that persisted for more than 50 weeks of lay.

A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale.

Science.gov (United States)

Schiffer, Lina; Anderko, Simone; Hobler, Anna; Hannemann, Frank; Kagawa, Norio; Bernhardt, Rita

2015-02-25

Human mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11β-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). We established an Escherichia coli based whole-cell system for selective cortisol production from 11-deoxycortisol by recombinant co-expression of the demanded 3 proteins. For the subsequent optimization of the whole-cell activity 3 different approaches were pursued: Firstly, CYP11B1 expression was enhanced 3.3-fold to 257 nmol∗L(-1) by site-directed mutagenesis of position 23 from glycine to arginine, which was accompanied by a 2.6-fold increase in cortisol yield. Secondly, the electron transfer chain was engineered in a quantitative manner by introducing additional copies of the Adx cDNA in order to enhance Adx expression on transcriptional level. In the presence of 2 and 3 copies the initial linear conversion rate was greatly accelerated and the final product concentration was improved 1.4-fold. Thirdly, we developed a screening system for directed evolution of CYP11B1 towards higher hydroxylation activity. A culture down-scale to microtiter plates was performed and a robot-assisted, fluorescence-based conversion assay was applied for the selection of more efficient mutants from a random library. Under optimized conditions a maximum productivity of 0.84 g cortisol∗L(-1)∗d(-1) was achieved, which

Transcriptional and metabolic response of recombinant Escherichia coli to spatial dissolved oxygen tension gradients simulated in a scale-down system.

Science.gov (United States)

Lara, Alvaro R; Leal, Lidia; Flores, Noemí; Gosset, Guillermo; Bolívar, Francisco; Ramírez, Octavio T

2006-02-05

Escherichia coli, expressing recombinant green fluorescent protein (GFP), was subjected to dissolved oxygen tension (DOT) oscillations in a two-compartment system for simulating gradients that can occur in large-scale bioreactors. Cells were continuously circulated between the anaerobic (0% DOT) and aerobic (10% DOT) vessels of the scale-down system to mimic an overall circulation time of 50 s, and a mean residence time in the anaerobic and aerobic compartments of 33 and 17 s, respectively. Transcription levels of mixed acid fermentation genes (ldhA, poxB, frdD, ackA, adhE, pflD, and fdhF), measured by quantitative RT-PCR, increased between 1.5- to over 6-fold under oscillatory DOT compared to aerobic cultures (constant 10% DOT). In addition, the transcription level of fumB increased whereas it decreased for sucA and sucB, suggesting that the tricarboxylic acid cycle was functioning as two open branches. Gene transcription levels revealed that cytrochrome bd, which has higher affinity to oxygen but lower energy efficiency, was preferred over cytochrome bO3 in oscillatory DOT cultures. Post-transcriptional processing limited heterologous protein production in the scale-down system, as inferred from similar gfp transcription but 19% lower GFP concentration compared to aerobic cultures. Simulated DOT gradients also affected the transcription of genes of the glyoxylate shunt (aceA), of global regulators of aerobic and anaerobic metabolism (fnr, arcA, and arcB), and other relevant genes (luxS, sodA, fumA, and sdhB). Transcriptional changes explained the observed alterations in overall stoichiometric and kinetic parameters, and production of ethanol and organic acids. Differences in transcription levels between aerobic and anaerobic compartments were also observed, indicating that E. coli can respond very fast to intermittent DOT conditions. The transcriptional responses of E. coli to DOT gradients reported here are useful for establishing rational scale-up criteria and

Clinical trial to evaluate safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid protein.

Science.gov (United States)

Lundgren, A; Leach, S; Tobias, J; Carlin, N; Gustafsson, B; Jertborn, M; Bourgeois, L; Walker, R; Holmgren, J; Svennerholm, A-M

2013-02-06

We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea containing killed recombinant E. coli bacteria expressing increased levels of ETEC colonization factors (CFs) and a recombinant protein (LCTBA), i.e. a hybrid between the binding subunits of E. coli heat labile toxin (LTB) and cholera toxin (CTB). We describe a randomized, comparator controlled, double-blind phase I trial in 60 adult Swedish volunteers of a prototype of this vaccine. The safety and immunogenicity of the prototype vaccine, containing LCTBA and an E. coli strain overexpressing the colonization factor CFA/I, was compared to a previously developed oral ETEC vaccine, consisting of CTB and inactivated wild type ETEC bacteria expressing CFA/I (reference vaccine). Groups of volunteers were given two oral doses of either the prototype or the reference vaccine; the prototype vaccine was administered at the same or a fourfold higher dosage than the reference vaccine. The prototype vaccine was found to be safe and equally well-tolerated as the reference vaccine at either dosage tested. The prototype vaccine induced mucosal IgA (fecal secretory IgA and intestine-derived IgA antibody secreting cell) responses to both LTB and CFA/I, as well as serum IgA and IgG antibody responses to LTB. Immunization with LCTBA resulted in about twofold higher mucosal and systemic IgA responses against LTB than a comparable dose of CTB. The higher dose of the prototype vaccine induced significantly higher fecal and systemic IgA responses to LTB and fecal IgA responses to CFA/I than the reference vaccine. These results demonstrate that CF over-expression and inclusion of the LCTBA hybrid protein in an oral inactivated ETEC vaccine does not change the safety profile when compared to a previous generation of such a vaccine and that the prototype vaccine induces significant dose dependent mucosal immune responses against CFA/I and LTB. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Study on nanocomposite construction based on the multi-functional biotemplate self-assembled by the recombinant TMGMV coat protein for potential biomedical applications.

Science.gov (United States)

Song, Lei; Wang, Shiwen; Wang, Haina; Zhang, Hua; Cong, Haolong; Jiang, Xingyu; Tien, Po

2015-02-01

Nowadays there is a growing interest in bio-scaffolded nanoarchitectures. Rapid progress in nanobiotechnology and molecular biology has allowed the engineering of inorganic-binding peptides termed as genetically engineered polypeptides for inorganics (GEPIs) into self-assembling biological structures to facilitate the design of novel biomedical or bioimaging devices. Here we introduce a novel nanocomposite comprising a self-assembled protein scaffold based on a recombinant tobacco mild green mosaic tobamovirus (TMGMV) coat protein (CP) and the photocatalytic TiO2 nanoparticles attached to it, which may provide a generic method for materials engineering. A template containing a modified TMGMV CP (mCP) gene, with the first six C-terminal amino acid residues deleted to accommodate more foreign peptides and expressing a site-directed mutation of A123C for bioconjugation utility, and two genetically engineered mutants, Escherichia coli-based P-mCP-Ti7 containing a C-terminal TiO2 GEPI sequence of seven peptides (Ti7) and Hi5 insect cells-derived E-CP-Ti7-His6 C-terminally fused with Ti7+His6 tag were created. Expression vectors and protocols for enriching of the two CP variants were established and the resultant proteins were identified by western blot analysis. Their RNA-free self-assembling structures were analyzed by transmission electron microscopy (TEM) and immuno-gold labeling TEM analysis. Adherence of nanoparticles to the P-mCP-Ti7 induced protein scaffold was visualized by TEM analysis. Also discussed is the Cysteine thiol reactivity in bioconjugation reactions with the maleimide-functionalized porphyrin photosensitizers which can function as clinical photodynamic therapy agents. This study introduced a novel approach to producing an assembly-competent recombinant TMGMV CP, examined its ability to serve as a novel platform for the multivalent display of surface ligands and demonstrated an alternative method for nanodevice synthesis for nanobiotechnological

Genetic Transfer of Salmonella typhimurium and Escherichia coli Lipopolysaccharide Antigens to Escherichia coli K-12

Science.gov (United States)

Jones, Randall T.; Koeltzow, Donald E.; Stocker, B. A. D.

1972-01-01

Escherichia coli K-12 ϰ971 was crossed with a smooth Salmonella typhimurium donor, HfrK6, which transfers early the ilv-linked rfa region determining lipopolysaccharide (LPS) core structure. Two ilv+ hybrids differing in their response to the LPS-specific phages FO and C21 were then crossed with S. typhimurium HfrK9, which transfers early the rfb gene cluster determining O repeat unit structure. Most recombinants selected for his+ (near rfb) were agglutinated by Salmonella factor 4 antiserum. Transfer of an F′ factor (FS400) carrying the rfb–his region of S. typhimurium to the same two ilv+ hybrids gave similar results. LPS extracted from two ilv+,his+, factor 4-positive hybrids contained abequose, the immunodominant sugar for factor 4 specificity. By contrast, his+ hybrids obtained from ϰ971 itself by similar HfrK9 and F′FS400 crosses were not agglutinated by factor 4 antiserum, indicating that the parental E. coli ϰ971 does not have the capacity to attach Salmonella O repeat units to its LPS core. It is concluded that the Salmonella rfb genes are expressed only in E. coli ϰ971 hybrids which have also acquired ilv-linked genes (presumably rfa genes affecting core structure or O-translocase ability, or both) from a S. typhimurium donor. When E. coli ϰ971 was crossed with a smooth E. coli donor, Hfr59, of serotype O8, which transfers his early, most his+ recombinants were agglutinated by E. coli O8 antiserum and lysed by the O8-specific phage, Ω8. This suggests that, although the parental E. coli K-12 strain ϰ971 cannot attach Salmonella-specific repeat units to its LPS core, it does have the capacity to attach E. coli O8-specific repeat units. PMID:4559827

Comparison of the spectral emission of lux recombinant and bioluminescent marine bacteria.

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Thouand, Gérald; Daniel, Philippe; Horry, Habib; Picart, Pascal; Durand, Marie José; Killham, Ken; Knox, Oliver G G; DuBow, Michael S; Rousseau, Michel

2003-01-01

The purpose of the present paper was to study the influence of bacteria harbouring the luciferase-encoding Vibrio harveyi luxAB genes upon the spectral emission during growth in batch-culture conditions. In vivo bioluminescence spectra were compared from several bioluminescent strains, either naturally luminescent (Vibrio fischeri and Vibrio harveyi) or in recombinant strains (two Gram-negative Escherichia coli::luxAB strains and a Gram-positive Bacillus subtilis::luxAB strain). Spectral emission was recorded from 400 nm to 750 nm using a highly sensitive spectrometer initially devoted to Raman scattering. Two peaks were clearly identified, one at 491-500 nm (+/- 5 nm) and a second peak at 585-595 (+/- 5 nm) with the Raman CCD. The former peak was the only one detected with traditional spectrometers with a photomultiplier detector commonly used for spectral emission measurement, due to their lack of sensitivity and low resolution in the 550-650 nm window. When spectra were compared between all the studied bacteria, no difference was observed between natural or recombinant cells, between Gram-positive and Gram-negative strains, and growth conditions and growth medium were not found to modify the spectrum of light emission. Copyright 2003 John Wiley & Sons, Ltd.

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.

Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis.

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Chen, Po Ting; Chiang, Chung-Jen; Chao, Yun-Peng

2007-01-01

Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K-based plasmid displayed full structural stability, leading to a high-level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2-fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAMbeta1-derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.

Recombinant bovine uteroglobin at 1.6 Å resolution: a preliminary X-ray crystallographic analysis

International Nuclear Information System (INIS)

Decken, Victoria von der; Delbrück, Heinrich; Herrler, Andreas; Beier, Henning M.; Fischer, Rainer; Hoffmann, Kurt M. V.

2005-01-01

The crystallization of recombinant bovine uteroglobin. Uteroglobin (UG) is a conserved protein which is induced by progesterone and secreted by the epithelia of various mammalian reproductive and respiratory organs. Recombinant bovine uteroglobin (recbUG), consisting of 80 amino acids with a C-terminal His 6 tag, was overexpressed in Escherichia coli and purified. The protein was crystallized in two geometric forms, rhomboid and cuneate (wedge-shaped), by the hanging-drop vapour-diffusion method at 295 K. The rhomboid crystals diffracted to a maximum resolution of 1.6 Å using synchrotron radiation. These crystals belong to space group P2 1 2 1 2, with unit-cell parameters a = 81.42, b = 82.82, c = 45.26 Å, and contain four monomers per asymmetric unit. The cuneate crystals diffracted to 2.35 Å resolution using a rotating-anode generator. These crystals belong to space group C222 1 , with unit-cell parameters a = 43.39, b = 93.94, c = 77.30 Å, and contain two molecules per asymmetric unit

Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

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Zang, Xiaonan; Zhang, Xuecheng; Mu, Xiaosheng; Liu, Bin

2013-03-01

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mg L-1) than that from soluble protein (1.964 mg L-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

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

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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.

Iron control of the Vibrio fischeri luminescence system in Escherichia coli.

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Dunlap, P V

1992-01-01

Iron influences luminescence in Vibrio fischeri; cultures iron-restricted for growth rate induce luminescence at a lower optical density (OD) than faster growing, iron-replete cultures. An iron restriction effect analogous to that in V. fischeri (slower growth, induction of luminescence at a lower OD) was established using Escherichia coli tonB and tonB+ strains transformed with recombinant plasmids containing the V. fischeri lux genes (luxR luxICDABEG) and grown in the presence and absence of the iron chelator ethylenediamine-di(o-hydroxylphenyl acetic acid) (EDDHA). This permitted the mechanism of iron control of luminescence to be examined. A fur mutant and its parent strain containing the intact lux genes exhibited no difference in the OD at induction of luminescence. Therefore, an iron-binding repressor protein apparently is not involved in iron control of luminescence. Furthermore, in the tonB and in tonB+ strains containing lux plasmids with Mu dI(lacZ) fusions in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the luxICDABEG promoter) both increased by a similar amount (8-9 fold each for tonB, 2-3 fold each for tonB+) in the presence of EDDHA. Similar results were obtained with the luxR gene present on a complementing plasmid. The previously identified regulatory factors that control the lux system (autoinducer-LuxR protein, cyclic AMP-cAMP receptor protein) differentially control expression from the luxR and luxICDABEG promoters, increasing expression from one while decreasing expression from the other. Consequently, these results suggest that the effect of iron on the V. fischeri luminescence system is indirect.

Cloning-free genome engineering in Sinorhizobium meliloti advances applications of Cre/loxP site-specific recombination.

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Döhlemann, Johannes; Brennecke, Meike; Becker, Anke

2016-09-10

The soil-dwelling α-proteobacterium Sinorhizobium meliloti serves as model for studies of symbiotic nitrogen fixation, a highly important process in sustainable agriculture. Here, we report advancements of the genetic toolbox accelerating genome editing in S. meliloti. The hsdMSR operon encodes a type-I restriction-modification (R-M) system. Transformation of S. meliloti is counteracted by the restriction endonuclease HsdR degrading DNA which lacks the appropriate methylation pattern. We provide a stable S. meliloti hsdR deletion mutant showing enhanced transformation with Escherichia coli-derived plasmid DNA and demonstrate that using an E. coli plasmid donor, expressing S. meliloti methyl transferase genes, is an alternative strategy of increasing the transformation efficiency of S. meliloti. Furthermore, we devise a novel cloning-free genome editing (CFGE) method for S. meliloti, Agrobacterium tumefaciens and Xanthomonas campestris, and demonstrate the applicability of this method for intricate applications of the Cre/lox recombination system in S. meliloti. An enhanced Cre/lox system, allowing for serial deletions of large genomic regions, was established. An assay of lox spacer mutants identified a set of lox sites mediating specific recombination. The availability of several non-promiscuous Cre recognition sites enables simultaneous specific Cre/lox recombination events. CFGE combined with Cre/lox recombination is put forward as powerful approach for targeted genome editing, involving serial steps of manipulation to expedite the genetic accessibility of S. meliloti as chassis. Copyright © 2016 Elsevier B.V. All rights reserved.

Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins.

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Hoffmann, Daniel; Ebrahimi, Mehrdad; Gerlach, Doreen; Salzig, Denise; Czermak, Peter

2017-11-10

The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.

Suppression of colorectal tumorigenesis by recombinant Bacteroides fragilis enterotoxin-2 in vivo.

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Lv, You; Ye, Tao; Wang, Hui-Peng; Zhao, Jia-Ying; Chen, Wen-Jie; Wang, Xin; Shen, Chen-Xia; Wu, Yi-Bin; Cai, Yuan-Kun

2017-01-28

To evaluate the impact of recombinant Bacteroides fragilis enterotoxin-2 (BFT-2, or Fragilysin) on colorectal tumorigenesis in mice induced by azoxymethane/dextran sulfate sodium (AOM/DSS). Recombinant proBFT-2 was expressed in Escherichia coli strain Rosetta (DE3) and BFT-2 was obtained and tested for its biological activity via colorectal adenocarcinoma cell strains SW-480. Seventy C57BL/6J mice were randomly divided into a blank (BC; n = 10), model (AD; n = 20), model + low-dose toxin (ADLT; n = 20, 10 μg), and a model + high-dose toxin (ADHT; n = 20, 20 μg) group. Mice weight, tumor formation and pathology were analyzed. Immunohistochemistry determined Ki-67 and Caspase-3 expression in normal and tumor tissues of colorectal mucosa. Recombinant BFT-2 was successfully obtained, along with its biological activity. The most obvious weight loss occurred in the AD group compared with the ADLT group (21.82 ± 0.68 vs 23.23 ± 0.91, P ADHT group (21.82 ± 0.68 vs 23.57 ± 1.06, P ADHT groups (19.75 ± 3.30 vs 6.50 ± 1.73, P ADHT group. The incidence of colorectal adenocarcinoma in both the ADHT group and the ADHT group was reduced compared to that in the AD group ( P ADHT group was 50% and 40%, respectively, both of which were lower than that found in the AD group (94.44%, P ADHT group was 45% and 55%, both of which were higher than that found in the BC group (16.67%, P < 0.05, P < 0.05). Oral administration with lower-dose biologically active recombinant BFT-2 inhibited colorectal tumorigenesis in mice.

Recombinant allergen-based IgE testing to distinguish bee and wasp allergy.

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Mittermann, Irene; Zidarn, Mihaela; Silar, Mira; Markovic-Housley, Zora; Aberer, Werner; Korosec, Peter; Kosnik, Mitja; Valenta, Rudolf

2010-06-01

The identification of the disease-causing insect in venom allergy is often difficult. To establish recombinant allergen-based IgE tests to diagnose bee and yellow jacket wasp allergy. Sera from patients with bee and/or wasp allergy (n = 43) and patients with pollen allergy with false-positive IgE serology to venom extracts were tested for IgE reactivity in allergen extract-based tests or with purified allergens, including nonglycosylated Escherichia coli-expressed recombinant (r) Api m 1, rApi m 2, rVes v 5, and insect cell-expressed, glycosylated rApi m 2 as well as 2 natural plant glycoproteins (Phl p 4, bromelain). The patients with venom allergy could be diagnosed with a combination of E coli-expressed rApi m 1, rApi m 2, and rVes v 5 whereas patients with pollen allergy remained negative. For a group of 29 patients for whom the sensitizing venom could not be identified with natural allergen extracts, testing with nonglycosylated allergens allowed identification of the sensitizing venom. Recombinant nonglycosylated allergens also allowed definition of the sensitizing venom for those 14 patients who had reacted either with bee or wasp venom extracts. By IgE inhibition studies, it is shown that glycosylated Api m 2 contains carbohydrate epitopes that cross-react with natural Api m 1, Ves v 2, natural Phl p 4, and bromelain, thus identifying cross-reactive structures responsible for serologic false-positive test results or double-positivity to bee and wasp extracts. Nonglycosylated recombinant bee and wasp venom allergens allow the identification of patients with bee and wasp allergy and should facilitate accurate prescription of venom immunotherapy. Copyright (c) 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Wurtzite-Phased InP Micropillars Grown on Silicon with Low Surface Recombination Velocity.

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Li, Kun; Ng, Kar Wei; Tran, Thai-Truong D; Sun, Hao; Lu, Fanglu; Chang-Hasnain, Connie J

2015-11-11

The direct growth of III-V nanostructures on silicon has shown great promise in the integration of optoelectronics with silicon-based technologies. Our previous work showed that scaling up nanostructures to microsize while maintaining high quality heterogeneous integration opens a pathway toward a complete photonic integrated circuit and high-efficiency cost-effective solar cells. In this paper, we present a thorough material study of novel metastable InP micropillars monolithically grown on silicon, focusing on two enabling aspects of this technology-the stress relaxation mechanism at the heterogeneous interface and the microstructure surface quality. Aberration-corrected transmission electron microscopy studies show that InP grows directly on silicon without any amorphous layer in between. A set of periodic dislocations was found at the heterointerface, relaxing the 8% lattice mismatch between InP and Si. Single crystalline InP therefore can grow on top of the fully relaxed template, yielding high-quality micropillars with diameters expanding beyond 1 μm. An interesting power-dependence trend of carrier recombination lifetimes was captured for these InP micropillars at room temperature, for the first time for micro/nanostructures. By simply combining internal quantum efficiency with carrier lifetime, we revealed the recombination dynamics of nonradiative and radiative portions separately. A very low surface recombination velocity of 1.1 × 10(3) cm/sec was obtained. In addition, we experimentally estimated the radiative recombination B coefficient of 2.0 × 10(-10) cm(3)/sec for pure wurtzite-phased InP. These values are comparable with those obtained from InP bulk. Exceeding the limits of conventional nanowires, our InP micropillars combine the strengths of both nanostructures and bulk materials and will provide an avenue in heterogeneous integration of III-V semiconductor materials onto silicon platforms.

Genome-wide recombination rate variation in a recombination map of cotton.

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Shen, Chao; Li, Ximei; Zhang, Ruiting; Lin, Zhongxu

2017-01-01

Recombination is crucial for genetic evolution, which not only provides new allele combinations but also influences the biological evolution and efficacy of natural selection. However, recombination variation is not well understood outside of the complex species' genomes, and it is particularly unclear in Gossypium. Cotton is the most important natural fibre crop and the second largest oil-seed crop. Here, we found that the genetic and physical maps distances did not have a simple linear relationship. Recombination rates were unevenly distributed throughout the cotton genome, which showed marked changes along the chromosome lengths and recombination was completely suppressed in the centromeric regions. Recombination rates significantly varied between A-subgenome (At) (range = 1.60 to 3.26 centimorgan/megabase [cM/Mb]) and D-subgenome (Dt) (range = 2.17 to 4.97 cM/Mb), which explained why the genetic maps of At and Dt are similar but the physical map of Dt is only half that of At. The translocation regions between A02 and A03 and between A04 and A05, and the inversion regions on A10, D10, A07 and D07 indicated relatively high recombination rates in the distal regions of the chromosomes. Recombination rates were positively correlated with the densities of genes, markers and the distance from the centromere, and negatively correlated with transposable elements (TEs). The gene ontology (GO) categories showed that genes in high recombination regions may tend to response to environmental stimuli, and genes in low recombination regions are related to mitosis and meiosis, which suggested that they may provide the primary driving force in adaptive evolution and assure the stability of basic cell cycle in a rapidly changing environment. Global knowledge of recombination rates will facilitate genetics and breeding in cotton.

Fine-Scale Recombination Maps of Fungal Plant Pathogens Reveal Dynamic Recombination Landscapes and Intragenic Hotspots.

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Stukenbrock, Eva H; Dutheil, Julien Y

2018-03-01

Meiotic recombination is an important driver of evolution. Variability in the intensity of recombination across chromosomes can affect sequence composition, nucleotide variation, and rates of adaptation. In many organisms, recombination events are concentrated within short segments termed recombination hotspots. The variation in recombination rate and positions of recombination hotspot can be studied using population genomics data and statistical methods. In this study, we conducted population genomics analyses to address the evolution of recombination in two closely related fungal plant pathogens: the prominent wheat pathogen Zymoseptoria tritici and a sister species infecting wild grasses Z. ardabiliae We specifically addressed whether recombination landscapes, including hotspot positions, are conserved in the two recently diverged species and if recombination contributes to rapid evolution of pathogenicity traits. We conducted a detailed simulation analysis to assess the performance of methods of recombination rate estimation based on patterns of linkage disequilibrium, in particular in the context of high nucleotide diversity. Our analyses reveal overall high recombination rates, a lack of suppressed recombination in centromeres, and significantly lower recombination rates on chromosomes that are known to be accessory. The comparison of the recombination landscapes of the two species reveals a strong correlation of recombination rate at the megabase scale, but little correlation at smaller scales. The recombination landscapes in both pathogen species are dominated by frequent recombination hotspots across the genome including coding regions, suggesting a strong impact of recombination on gene evolution. A significant but small fraction of these hotspots colocalize between the two species, suggesting that hotspot dynamics contribute to the overall pattern of fast evolving recombination in these species. Copyright © 2018 Stukenbrock and Dutheil.

DNA degradation and reduced recombination following UV irradiation during meiosis in yeast (Saccharomyces cerevisiae)

International Nuclear Information System (INIS)

Salts, Y.; Pinon, R.; Simchen, G.

1976-01-01

Irradiation of meiotic yeast cells with moderate doses of ultraviolet irradiation (1,600 erg/mm 2 ) leads to the arrest of premeiotic DNA synthesis, massive (5-40%) DNA degradation, and a 40-50% loss of cell viability. In contrast, such doses of UV irradiation had a minor effect on viability (15-20% loss) of logarithmically growing cells, and no comparable DNA degradation was observed in irradiated synchronized vegetative cells. Meiotic recombination is also affected by UV irradiation. When administered at a stage comparable to meiotic prophase, low doses of irradiation result in a reduction in recombination frequency without significantly affecting cell viability. (orig.) [de

Recombinant VP1 protein as a potential marker for the diagnosis of acute hepatitis A virus infection.

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da Silva Junior, Haroldo Cid; da Silva, Edimilson Domingos; Lewis-Ximenez de Souza Rodrigues, Lia Laura; Medeiros, Marco Alberto

2017-07-01

Since hepatitis A virus (HAV) production is time-consuming and expensive, the use of recombinant proteins may represent an alternative source of antigens for diagnostic purposes. The present study aimed to express, purify and evaluate the potential of recombinant VP1 protein (rVP1) as a marker for the diagnosis of acute HAV infection. The rVP1 was expressed and purified successfully from Escherichia coli. The purified rVP1 was used to establish an in-house enzyme-linked immunosorbent assay (ELISA-rVP1) for detection of IgM antibodies in sera from HAV-positive patients. For a cut-off point of 0.351, the sensitivity and specificity of ELISA-rVP1 were 100.0% and 95.0%, respectively. These results indicate that rVP1 may be a useful antigen for detection of IgM antibodies against HAV. Copyright © 2017 Elsevier B.V. All rights reserved.

Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis.

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Gomez, F J; Allendoerfer, R; Deepe, G S

1995-07-01

HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated approximately 70 and approximately 50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice.

Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae

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M Boustanshenas

2013-01-01

Full Text Available Purpose: The aim of this study was to assess the production of recombinant cholera toxin B subunit (rCTB protein in two different expression systems (pAE_ctxB and pQE_ctxB constructs in Escherichia coli BL21 (DE3. Materials and Methods: The ctxB fragment was amplified from Vibrio cholerae O 1 ATCC14035 and cloned in pGETM-T easy vector after which it was transformed to E. coli Top 10F′ and grown on LB-ampicillin agar medium. Sequence analysis confirmed the complete ctxB gene sequence in the construct which was further subcloned to pQE-30 vector. The construct was subsequently transformed to E. coli M15 (pREP4. The recombinant pAE_ctxB and pQE_ctxB were transformed to competent E. coli BL21 (DE3 cells to express CTB protein. Result: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 h after induction and western blot analysis confirmed the presence of recombinant CTB in blotting membranes. Conclusion: Expression of rCTB in pAE_ctxB construct was more efficient (15-fold than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3 is more compatible with the former construct. This expression system can be used to produce recombinant CTB in high yield which may enable us to study the oral tolerance or mucosal adjuvant properties of rCTB using animal models.

Characterisation of recombinant immunoreactive antigens of the scab mite Sarcoptes scabiei.

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Kuhn, C; Lucius, R; Matthes, H F; Meusel, G; Reich, B; Kalinna, B H

2008-05-31

Sarcoptic mange (or scabies) is an important skin disease which can affect a variety of species including humans, cattle, goats, sheep, horses, pigs, rabbits, and dogs. Approximately 300 million people are affected worldwide and in lifestock animals the infestation may lead to substantial economic losses caused by depression in growth and feed conversion rates. Diagnosis of Sarcoptes infestation is difficult and only a few serological tests have been developed using whole mite antigen for diagnosis of mange in animals. Here we describe the isolation and characterisation of cDNAs of several immunoreactive clones and their recombinant expression in Escherichia coli. Three of the proteins contain repetitive sequences which suggests that they might be involved in immune evasion. The application of these antigens in serodiagnosis and the suitability for diagnosis is discussed.

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

Expression by Streptomyces lividans of the Rat α Integrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein

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Dorra Zouari Ayadi

2007-01-01

Full Text Available We already reported the use of a long synthetic signal peptide (LSSP to secrete the Streptomyces sp. TO1 amylase by Streptomyces lividans strain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP and S. lividans as host strain. We have used the Escherichia coli/Streptomyces shuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutive ermE-up promoter of Streptomyces erythraeus. Using this construct and S. lividans as a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyte β2 integrin CD11/CD18 alpha M subunit (CD11b. This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability of Streptomyces to produce heterologous recombinant proteins as soluble secreted form using the “LSSP” synthetic signal peptide.

Cloning of Bacteroides fragilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli

International Nuclear Information System (INIS)

Wehnert, G.U.; Abratt, V.R.; Goodman, H.J.; Woods, D.R.

1990-01-01

Since reduced metronidazole causes DNA damage, resistance to metronidazole was used as a selection method for the cloning of Bacteroides fragilis genes affecting DNA repair mechanisms in Escherichia coli. Genes from B. fragilis Bf-2 were cloned on a recombinant plasmid pMT100 which made E. coli AB1157 and uvrA, B, and C mutant strains more resistant to metronidazole, but more sensitive to far uv irradiation under aerobic conditions. The loci affecting metronidazole resistance and uv sensitivity were linked and located on a 5-kb DNA fragment which originated from the small 6-kb cryptic plasmid pBFC1 present in B. fragilis Bf-2 cells

Biotransformation of rutin to isoquercitrin using recombinant α-L-rhamnosidase from Bifidobacterium breve.

Science.gov (United States)

Zhang, Ru; Zhang, Bian-Ling; Xie, Tao; Li, Gu-Cai; Tuo, Yi; Xiang, Yu-Ting

2015-06-01

To biotransform rutin into isoquercitrin. A α-L-rhamnosidase from Bifidobacterium breve was produced by using Escherichia coli BL21 for biotransformation of rutin to isoquercitrin. The enzyme was purified by Ni(2+)-NTA chromatography to yield a soluble protein with a specific activity of 56 U protein mg(-1). The maximum enzyme activities were at pH 6.5, 55 °C, 20 mM rutin, and 1.2 U enzyme ml(-1). Under optimal conditions, the half-life of the enzyme was 96 h. The K m and V max values were 2.2 mM, 56.4 μmol mg(-1) min(-1) and 2.1 mM, 57.5 μmol mg(-1) min(-1) using pNP-Rha and rutin as substrates, respectively. The kinetic behavior indicated that the recombinant α-L-rhamnosidase has good catalytic performance for producing isoquercitrin. 20 mM rutin was biotransformed into 18.25 and 19.87 mM isoquercitrin after 60 and 240 min. The specific biotransformation of rutin to isoquercitrin using recombinant α-L-rhamnosidase from B. breve is a feasible method for use in industrial processes.

Recombinant Tyrosinase from Polyporus arcularius: Overproduction in Escherichia coli, Characterization, and Use in a Study of Aurones as Tyrosinase Effectors

Czech Academy of Sciences Publication Activity Database

Marková, Eva; Kotík, Michael; Křenková, Alena; Man, Petr; Haudecoeur, R.; Boumendjel, A.; Hardré, R.; Mekmouche, Y.; Dezord-Courvoisier, E.; Réglier, M.; Martínková, Ludmila

2016-01-01

Roč. 64, č. 14 (2016), s. 2925-2931 ISSN 0021-8561 R&D Projects: GA MŠk LD12049; GA MŠk LO1509; GA TA ČR TA04021212 Institutional support: RVO:61388971 Keywords : tyrosinase * Polyporus arcularius * Escherichia coli Subject RIV: CE - Biochemistry Impact factor: 3.154, year: 2016

Genetic Recombination

Science.gov (United States)

Whitehouse, H. L. K.

1973-01-01

Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

A termite symbiotic mushroom maximizing sexual activity at growing tips of vegetative hyphae.

Science.gov (United States)

Hsieh, Huei-Mei; Chung, Mei-Chu; Chen, Pao-Yang; Hsu, Fei-Man; Liao, Wen-Wei; Sung, Ai-Ning; Lin, Chun-Ru; Wang, Chung-Ju Rachel; Kao, Yu-Hsin; Fang, Mei-Jane; Lai, Chi-Yung; Huang, Chieh-Chen; Chou, Jyh-Ching; Chou, Wen-Neng; Chang, Bill Chia-Han; Ju, Yu-Ming

2017-09-19

Termitomyces mushrooms are mutualistically associated with fungus-growing termites, which are widely considered to cultivate a monogenotypic Termitomyces symbiont within a colony. Termitomyces cultures isolated directly from termite colonies are heterokaryotic, likely through mating between compatible homokaryons. After pairing homokaryons carrying different haplotypes at marker gene loci MIP and RCB from a Termitomyces fruiting body associated with Odontotermes formosanus, we observed nuclear fusion and division, which greatly resembled meiosis, during each hyphal cell division and conidial formation in the resulting heterokaryons. Surprisingly, nuclei in homokaryons also behaved similarly. To confirm if meiotic-like recombination occurred within mycelia, we constructed whole-genome sequencing libraries from mycelia of two homokaryons and a heterokaryon resulting from mating of the two homokaryons. Obtained reads were aligned to the reference genome of Termitomyces sp. J132 for haplotype reconstruction. After removal of the recombinant haplotypes shared between the heterokaryon and either homokaryons, we inferred that 5.04% of the haplotypes from the heterokaryon were the recombinants resulting from homologous recombination distributed genome-wide. With RNA transcripts of four meiosis-specific genes, including SPO11, DMC1, MSH4, and MLH1, detected from a mycelial sample by real-time quantitative PCR, the nuclear behavior in mycelia was reconfirmed meiotic-like. Unlike other basidiomycetes where sex is largely restricted to basidia, Termitomyces maximizes sexuality at somatic stage, resulting in an ever-changing genotype composed of a myriad of coexisting heterogeneous nuclei in a heterokaryon. Somatic meiotic-like recombination may endow Termitomyces with agility to cope with termite consumption by maximized genetic variability.

In vitro Ca(2+)-dependent maturation of milk-clotting recombinant Epr: minor extracellular protease: from Bacillus licheniformis.

Science.gov (United States)

Ageitos, José Manuel; Vallejo, Juan Andrés; Serrat, Manuel; Sánchez-Pérez, Angeles; Villa, Tomás G

2013-06-01

The minor extracellular protease (Epr) is secreted into the culture medium during Bacillus licheniformis, strain USC13, stationary phase of growth. Whereas, B. subtilis Epr has been reported to be involved in swarming; the B. licheniformis protease is also involved in milk-clotting as shown by the curd forming ability of culture broths expressing this protein. The objectives of this study are the characterization of recombinant B. licheniformis Epr (minor extracellular protease) and the determination of its calcium-dependent activation process. In this work, we have cloned and expressed B. licheniformis Epr in Escherichia coli. We were also able to construct a tridimensional model for Epr based on its homology to Thermococcus kodakarensis pro-tk-subtilisin 2e1p, fervidolysin from Fervidobacterium pennivorans 1rv6, and B. lentus 1GCI subtilisin. Recombinant Epr was accumulated into inclusion bodies; after protein renaturation, Epr undergoes an in vitro calcium-dependent activation, similar to that described for tk protease. The recombinant Epr is capable of producing milk curds with the same clotting activity previously described for the native B. licheniformis Epr enzyme although further rheological and industrial studies should be carried out to confirm its real applicability. This work represents for the first time that Epr may be successfully expressed in a non-bacilli microorganism.

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

Science.gov (United States)

Galli, Vanessa; Simionatto, Simone; Marchioro, Silvana Beutinger; Klabunde, Gustavo Henrique Ferrero; Conceição, Fabricio Rochedo

2013-01-01

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), which is a respiratory disease responsible for huge economic losses in the pig industry worldwide. The commercially available vaccines provide only partial protection and are expensive. Thus, the development of alternatives for the prophylaxis of EP is critical for improving pig health. The use of multiple antigens in the same immunization may represent a promising alternative. In the present study, seven secreted proteins of M. hyopneumoniae were cloned, expressed in Escherichia coli, and evaluated for antigenicity using serum from naturally and experimentally infected pigs. In addition, the immunogenicity of the seven recombinant proteins delivered individually or in protein cocktail vaccines was evaluated in mice. In Western blot assays and enzyme-linked immunosorbent assays, most of the recombinant proteins evaluated were recognized by convalescent-phase serum from the animals, indicating that they are expressed during the infectious process. The recombinant proteins were also immunogenic, and most induced a mixed IgG1/IgG2a humoral immune response. The use of these proteins in a cocktail vaccine formulation enhanced the immune response compared to their use as antigens delivered individually, providing evidence of the efficacy of the multiple-antigen administration strategy for the induction of an immune response against M. hyopneumoniae. PMID:23803903

A stochastic killing system for biological containment of Escherichia coli

DEFF Research Database (Denmark)

Klemm, P.; Jensen, Lars Bogø; Molin, Søren

1995-01-01

Bacteria with a stochastic conditional lethal containment system have been constructed. The invertible switch promoter located upstream of the fimA gene from Escherichia coli was inserted as expression cassette in front of the Lethal gef gene deleted of its own natural promoter. The resulting...... fusion was placed on a plasmid and transformed to E. coli. The phenotype connected with the presence of such a plasmid was to reduce the population growth rate with increasing significance as the cell growth rate was reduced. In very fast growing cells, there was no measurable effect on growth rate. When...

New techniques for growing anaerobic bacteria: experiments with Clostridium butyricum and Clostridium acetobutylicum

International Nuclear Information System (INIS)

Adler, H.I.; Crow, W.D.; Hadden, C.T.; Hall, J.; Machanoff, R.

1983-01-01

Stable membrane fragments derived from Escherichia coli produce and maintain strict anaerobic conditions when added to liquid or solid bacteriological media. Techniques for growing Clostridium butyricum and Clostridium acetobutylicum in membrane-containing media are described. Liquid cultures initiated by very small inocula can be grown in direct contact with air. In solid media, colonies develop rapidly from individual cells even without incubation in anaerobic jars or similar devices. Observations on growth rates, spontaneous mutations, radiation, and oxygen sensitivity of anaerobic bacteria have been made using these new techniques

Diagnostic value of recombinant Tp0821 protein in serodiagnosis for syphilis.

Science.gov (United States)

Xie, Y; Xu, M; Wang, C; Xiao, J; Xiao, Y; Jiang, C; You, X; Zhao, F; Zeng, T; Liu, S; Kuang, X; Wu, Y

2016-04-01

Syphilis is a multistage sexually transmitted disease that remains a serious public health concern worldwide. The coexistence of Treponema pallidum with other closely related members of spirochaeta, such as Leptospira spp. and Borrelia burgdorferi, has complicated the serodiagnosis due to cross-reactive antigens. In this study, recombinant Tp0821 protein was expressed in Escherichia coli and purified by metal affinity chromatography. Then enzyme-linked immunosorbent assays (ELISAs) based on Tp0821 for the detection of specific antibodies were established. The relative positive rates of the IgM ELISA and the IgG ELISA were found to be 91·0 and 98·3%, respectively, when screening 578 syphilis specimens. The specificities were 94·3 and 100%, respectively, when cross-checking with serum samples obtained from 30 patients with Lyme disease, five patients with leptospirosis, and 52 uninfected controls. In addition, relative positive rates and specificities of Tp0821 for human sera were all 100% in Western blotting. When compared to the syphilis diagnostic tests commonly used in clinical settings, we found that the results of Tp0821-based ELISAs correlated well with the results of the treponemal tests, specifically the T. pallidum particle agglutination (TP-PA) test and the chemiluminescent immunoassay (CIA). Thus, these findings identify Tp0821 as a novel serodiagnostic candidate for syphilis. In this study, we expressed and purified the Treponema pallidum protein Tp0821 and developed Tp0821-based enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies. The serodiagnostic performance of the recombinant protein was then evaluated. When compared to the results of syphilis diagnostic tests commonly used in clinical settings, we found that the reactivities of syphilitic sera with the recombinant antigen correlated well with the results of the treponemal tests, specifically the T. pallidum particle agglutination (TP-PA) test and the

Effect of the mutations recB21, recD1013 and recJ284 of Escherichia coli on the indirect recombinogenesis of the lambda bacteriophage

International Nuclear Information System (INIS)

Alcantara D, D.

1994-01-01

The protein RecBCD of Escherichia coli is of those more important, so much in the genetic recombination as in the repair of the genetic damage. Due to their importance in the genetic recombination in general and to that the enzyme is not completely inactivated in cells infected with lambda, it was decided to prove their participation in the indirect viral recombinogenesis. The obtained data indicated that RecBCD plays a central role in the indirect recombinogenesis of lambda, since in the mutants recB21 and recD1013 the response is not presented or it decreases drastically. Also it was proven that RecBCD cannot be substituted by the double band DNA exonuclease, coded by the recJ gene. (Author)

Modeling the contamination of lettuce with Escherichia coli 157:H7 from manure-amended soil and the effect of intervention strategies

NARCIS (Netherlands)

Franz, E.; Semenov, A.V.; Bruggen, van A.H.C.

2008-01-01

Aims: A growing number of foodborne illnesses has been associated with the consumption of fresh produce. In this study, the probability of lettuce contamination with Escherichia coli O157:H7 from manure-amended soil and the effect of intervention strategies was determined. Methods and Results:

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

Science.gov (United States)

Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

2014-11-01

Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

One-step production of biocommodities from lignocellulosic biomass by recombinant cellulolytic bacillus subtilis: opportunities and challenges

Energy Technology Data Exchange (ETDEWEB)

Zhang, Xiao-Zhou [Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA (United States); Zhang, Yi-Heng P. [Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA (United States); Institute for Critical Technology and Applied Science, Virginia Polytechnic Institute and State University, Blacksburg, VA (United States); BioEnergy Science Center of Department of Energy, Oak Ridge, TN (United States)

2010-10-15

One-step consolidated bioprocessing that integrates cellulase production, cellulose hydrolysis, and product fermentation into a single step for decreasing costly cellulase use, increasing volumetric productivity, and reducing capital investment is widely accepted for low-cost production of biofuels or other value-added biochemicals. Considering the narrow margins between biomass and low-value biocommodities, good physiological performance of industrial microbes is crucial for economically viable production. Bacillus subtilis, the best-characterized Gram-positive microorganism, is a major industrial microorganism with numerous valuable features such as hexose and pentose utilization, low-nutrient needs, fast growth rate, high protein secretion capacity, industrial safety, etc. As compared with other potential consolidated bioprocessing microorganisms such as Clostridium spp., Escherichia coli, and the yeast Saccharomyces cerevisiae, recombinant cellulolytic B. subtilis strains would be a potential platform for biocommodity production from nonfood biomass. Here, we review the advances in recombinant cellulolytic B. subtilis development and metabolic engineering for biocommodity production, and discuss the opportunities and challenges of cellulolytic B. subtilis for biocommodity production. (Copyright copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

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.

Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum.

Science.gov (United States)

Heiss, Silvia; Hörmann, Angelika; Tauer, Christopher; Sonnleitner, Margot; Egger, Esther; Grabherr, Reingard; Heinl, Stefan

2016-03-10

Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector(®) micro-fermentation system. Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector(®) micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems.

Science.gov (United States)

Kemper, Katarina; Hirte, Max; Reinbold, Markus; Fuchs, Monika; Brück, Thomas

2017-01-01

With over 50.000 identified compounds terpenes are the largest and most structurally diverse group of natural products. They are ubiquitous in bacteria, plants, animals and fungi, conducting several biological functions such as cell wall components or defense mechanisms. Industrial applications entail among others pharmaceuticals, food additives, vitamins, fragrances, fuels and fuel additives. Central building blocks of all terpenes are the isoprenoid compounds isopentenyl diphosphate and dimethylallyl diphosphate. Bacteria like Escherichia coli harbor a native metabolic pathway for these isoprenoids that is quite amenable for genetic engineering. Together with recombinant terpene biosynthesis modules, they are very suitable hosts for heterologous production of high value terpenes. Yet, in contrast to the number of extracted and characterized terpenes, little is known about the specific biosynthetic enzymes that are involved especially in the formation of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized terpene producing Escherichia coli , this review shall give an insight in recent progresses regarding manipulation of mostly diterpene synthases.

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems

Directory of Open Access Journals (Sweden)

Katarina Kemper

2017-05-01

Full Text Available With over 50.000 identified compounds terpenes are the largest and most structurally diverse group of natural products. They are ubiquitous in bacteria, plants, animals and fungi, conducting several biological functions such as cell wall components or defense mechanisms. Industrial applications entail among others pharmaceuticals, food additives, vitamins, fragrances, fuels and fuel additives. Central building blocks of all terpenes are the isoprenoid compounds isopentenyl diphosphate and dimethylallyl diphosphate. Bacteria like Escherichia coli harbor a native metabolic pathway for these isoprenoids that is quite amenable for genetic engineering. Together with recombinant terpene biosynthesis modules, they are very suitable hosts for heterologous production of high value terpenes. Yet, in contrast to the number of extracted and characterized terpenes, little is known about the specific biosynthetic enzymes that are involved especially in the formation of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized terpene producing Escherichia coli, this review shall give an insight in recent progresses regarding manipulation of mostly diterpene synthases.

Cre-loxP-Mediated Recombination: General Principles and Experimental Considerations.

Science.gov (United States)

McLellan, Micheal A; Rosenthal, Nadia A; Pinto, Alexander R

2017-03-02

The cre-loxP-mediated recombination system (the "cre-loxP system") is an integral experimental tool for mammalian genetics and cell biology. Use of the system has greatly expanded our ability to precisely interrogate gene function in the mouse, providing both spatial and temporal control of gene expression. This has been largely due to the simplicity of its use and its adaptability to address diverse biological questions. While the use of the cre-loxP system is becoming increasingly widespread, in particular because of growing availability of conditional mouse mutants, many considerations need to be taken into account when utilizing the cre-loxP system. This review provides an overview of the cre-loxP system and its various permutations. It addresses the limitations of cre-loxP technology and related considerations for experimental design, and it discusses alternative strategies for site-specific genetic recombination and integration. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Cloning, expression, and immunological characterization of recombinant Lolium perenne allergen Lol p II.

Science.gov (United States)

Sidoli, A; Tamborini, E; Giuntini, I; Levi, S; Volonté, G; Paini, C; De Lalla, C; Siccardi, A G; Baralle, F E; Galliani, S

1993-10-15

The molecular cloning of the cDNA encoding for an isoallergenic form of Lol p II, a major rye grass (Lolium perenne) pollen allergen, was performed by polymerase chain reaction amplification on mRNA extracted from pollen. The amino acid sequence derived from the cDNA was truncated by 4 and 5 residues at the NH2- and COOH-terminal ends, respectively, and differed only in one position from that previously reported. This cDNA was expressed in Escherichia coli by fusion to the carboxyl terminus of the human ferritin H-chain. The molecule was produced in high yields as a soluble protein and was easily purified. The protein retains the multimeric quaternary structure of ferritin, and it exposes on the surface the allergenic moiety, which can be recognized in Western blotting and in enzyme-linked immunosorbent assay experiments by specific IgE from allergic patients. The recombinant allergen was used to analyze the sera of 26 patients allergic to L. perenne compared with control sera. The results were in good agreement with the values obtained with the radioallergosorbent test assay. In addition, histamine release experiments in whole blood from an allergic patient and skin prick tests showed that the recombinant allergen retains some of the biological properties of the natural compound. These findings indicate that the availability of homogeneous recombinant allergens may be useful for the development of more specific diagnostic and therapeutic procedures. Moreover, this expression system may be of more general interest for producing large amounts of soluble protein domains in E. coli.

Immunogenicity of recombinant class 1 protein from Neisseria meningitidis refolded into phospholipid vesicles and detergent.

Science.gov (United States)

Niebla, O; Alvarez, A; Martín, A; Rodríguez, A; Delgado, M; Falcón, V; Guillén, G

2001-05-14

The possibility of eliciting bactericidal antibodies against a recombinant class 1 protein (P1) from Neisseria meningitidis, joined to the first 45 amino acids of the neisserial LpdA protein (PM82), was examined. P1 was produced in Escherichia coli as intracellular inclusion bodies, from which it was purified and reconstituted by (a) inclusion into phospholipid vesicles and detergent and (b) refolding in 0.1% SDS. When Balb/c mice were immunised, high titres of subtype-specific bactericidal antibodies against P1 were obtained in both cases. These results suggest that in spite of being a denaturing agent, it is possible to use SDS to reconstitute the P1 protein in a conformation that exposes the immunodominat regions.

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....

Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv from Escherichia coli

Directory of Open Access Journals (Sweden)

Ishii Marina

2002-04-01

Full Text Available Abstract Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside, express the green fluorescent protein (gfpuv during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1 of transformed (pGFP Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm were sonicated in successive intervals of sonication (25 vibrations/pulse to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations. The intracellular permeate with gfpuv in extraction buffer (TE solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF was subjected to the three-phase partitioning (TPP method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0. Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA, after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP

Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs

International Nuclear Information System (INIS)

Singh, Deepti; Rawat, Surender; Waseem, Mohd; Gupta, Sunita; Lynn, Andrew; Nitin, Mukesh; Ramchiary, Nirala; Sharma, Krishna Kant

2016-01-01

The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, K m values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative property against BSA and Cu 2+ /H 2 O 2 model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies. - Highlights: • Laccase from Yersinia enterocolitica strain 7 was expressed in Escherichia coli BL21 (DE3). • Recombinant laccase was found to be thermostable and alkali tolerant. • The in silico and experimental studied proves the biotransformation of NSAIDs. • Laccase binds to ligands differentially under different protonation state. • Laccase also possesses free radical scavenging property.

Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs

Energy Technology Data Exchange (ETDEWEB)

Singh, Deepti; Rawat, Surender [Laboratory of Enzymology and Recombinant DNA Technology, Department of Microbiology, Maharshi Dayanand University, Rohtak 124001, Haryana (India); Waseem, Mohd; Gupta, Sunita; Lynn, Andrew [School of Computational & Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067 (India); Nitin, Mukesh; Ramchiary, Nirala [School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067 (India); Sharma, Krishna Kant, E-mail: kekulsharma@gmail.com [Laboratory of Enzymology and Recombinant DNA Technology, Department of Microbiology, Maharshi Dayanand University, Rohtak 124001, Haryana (India)

2016-01-08

The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, K{sub m} values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative property against BSA and Cu{sup 2+}/H{sub 2}O{sub 2} model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies. - Highlights: • Laccase from Yersinia enterocolitica strain 7 was expressed in Escherichia coli BL21 (DE3). • Recombinant laccase was found to be thermostable and alkali tolerant. • The in silico and experimental studied proves the biotransformation of NSAIDs. • Laccase binds to ligands differentially under different protonation state. • Laccase also possesses free radical scavenging property.

Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens.

Science.gov (United States)

Wikman, Maria; Friedman, Mikaela; Pinitkiatisakul, Sunan; Hemphill, Andrew; Lövgren-Bengtsson, Karin; Lundén, Anna; Ståhl, Stefan

2005-04-01

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K(D) approximately 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni(2+)-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M5), derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native

Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

Science.gov (United States)

Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

2015-01-01

Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

Laser-induced electron--ion recombination used to study enhanced spontaneous recombination during electron cooling

International Nuclear Information System (INIS)

Schramm, U.; Wolf, A.; Schuess ler, T.; Habs, D.; Schwalm, D.; Uwira, O.; Linkemann, J.; Mueller, A.

1997-01-01

Spontaneous recombination of highly charged ions with free electrons in merged velocity matched electron and ion beams has been observed in earlier experiments to occur at rates significantly higher than predicted by theoretical estimates. To study this enhanced spontaneous recombination, laser induced recombination spectra were measured both in velocity matched beams and in beams with well defined relative velocities, corresponding to relative electron-ion detuning energies ranging from 1 meV up to 6.5 meV where the spontaneous recombination enhancement was found to be strongly reduced. Based on a comparison with simplified calculations, the development of the recombination spectra for decreasing detuning energies indicates additional contributions at matched velocities which could be related to the energy distribution of electrons causing the spontaneous recombination rate enhancement

Effects of nuclear mutations for recombination and repair functions and of caffeine on mitochondrial recombination

International Nuclear Information System (INIS)

Fraenkel, A.H.M.

1974-01-01

Studies of both prokaryotic and eukaryotic organisms indicate that pathways governing repair of damage to nuclear DNA caused by x-ray or ultraviolet irradiation overlap with those controlling recombination. Fourteen nuclear mutants of Saccharomyces cerevisiae were tested in order to determine whether these mutant genes affected mitochondrial recombination. None of the mutations studied significantly affected mitochondrial recombination. The nuclear recombination and repair pathways studied do not overlap with the nuclear pathway which controls recombination of mitochondrial DNA. A second set of experiments was designed to test the effect of caffeine on both nuclear and mitochondrial recombination in Saccharomyces cerevisiae. (U.S.)

Induced immune response of Escherichia coli BL21 expressing recombinant MSP1a and MSP1b proteins of Anaplasma marginale

Directory of Open Access Journals (Sweden)

Katia Tamekuni

2009-11-01

Full Text Available This work aims to evaluate the potential of immunization with E. coli BL21 expressing the recombinant rMSP1a and rMSP1b proteins of Anaplasma marginale. E. coli BL21 was transformed with recombinant plasmids pET102/msp1α and pET101/msp1β, and rMSP1a and rMSP1b were expressed after induction by IPTG. BALB/c mice were vaccinated with formolized BL21/rMSP1a and BL21/rMSP1b, and the production in mice sera of whole IgG was determined by ELISA. The mice immunized with BL21/rMSP1a showed a better humoral response for whole IgG when compared to the mice immunized with BL21/rMSP1b; these mice exhibited a small response after the second vaccination. Sera of mice immunized with BL21/rMSP1a reacted via western blot with BL21 and rMSP1a, with molecular masses varying from 70 to 105 kDa. Sera of mice immunized with BL21/rMSP1b reacted with BL21 and rMSP1b with a molecular mass of 100 kDa. These results demonstrate that BL21 containing rMSP1a and rMSP1b in the outer membrane were able to produce an immune response in mice, reinforcing its use in vaccine models against bovine anaplasmosis.Esse trabalho avaliou o potencial de imunização de Escherichia coli BL21 expressando as proteínas recombinantes rMSP1a e rMSP1b de Anaplasma marginale. A E. coli BL21 foi transformada com os plasmídios recombinantes pET102/msp1α e pET101/msp1β e as proteínas rMSP1a e rMSP1b foram expressas após indução com IPTG. Camundongos BALB/c foram vacinados com BL21/rMSP1a e BL21/rMSP1b formolisadas, e a produção de IgG total foi determinada pelo teste de ELISA nos soros dos camundongos imunizados. Os camundongos imunizados com a BL21/rMSP1a mostraram uma melhor resposta humoral para IgG total, comparada à resposta apresentada pelos camundongos imunizados com BL21/rMSP1b; estes camundongos exibiram uma menor resposta após a segunda vacinação. Soros de camundongos imunizados BL21/rMSP1a reagiram pelo western blot com BL21 e rMSP1a, com massa molecular variando de 70 a

Novel sequence types of extended-spectrum and acquired AmpC beta-lactamase producing Escherichia coli and Escherichia clade V isolated from wild mammals.

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Alonso, Carla Andrea; Alcalá, Leticia; Simón, Carmen; Torres, Carmen

2017-08-01

The closer contact with wildlife due to the growing human population and the destruction of natural habitats emphasizes the need of gaining insight into the role of animals as source of antimicrobial resistance. Here, we aim at characterizing the antimicrobial resistance genes and phylogenetic distribution of commensal Escherichia coli from 62 wild mammals. Isolates exhibiting resistance to ≥1 antibiotic were detected in 25.8% of the animals and 6.4% carried an extended-spectrum beta-lactamase (ESBL)/AmpC-producing E. coli. Genetic mechanisms involved in third-generation cephalosporin resistance were as follows: (i) hyperproduction of chromosomal AmpC (hedgehog), (ii) production of acquired CMY-2 β-lactamase (hedgehog), (iii) production of SHV-12 and CTX-M-14 ESBLs (n = 2, mink and roe-deer). ESBL genes were transferable by conjugation, and blaCMY-2 was mobilized by a 95kb IncI1 plasmid. The distribution of the phylogenetic groups in the E. coli collection studied was B1 (44.6%), B2 (24.6%), E (15.4%), A (4.6%) and F (3.1%). Five isolates (7.7%) were cryptic Escherichia clades (clade IV, 4 mice; clade V, 1 mink). ESBL/AmpC-E. coli isolates showed different sequence types (STs): ST1128/B1, ST4564/B1 (new), ST4996/B1 (new) and a non-registered ST. This study contributes to better understand the E. coli population and antimicrobial resistance flow in wildlife and reports new AmpC-E. coli STs and a first described ESBL-producing Escherichia clade V isolate. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Construction and characterization of human rotavirus recombinant VP8* subunit parenteral vaccine candidates.

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Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka

2012-09-21

Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines. Published by Elsevier Ltd.

The crystal structure of galactitol-1-phosphate 5-dehydrogenase from Escherichia coli K12 provides insights into its anomalous behavior on IMAC processes.

Science.gov (United States)

Esteban-Torres, María; Alvarez, Yanaisis; Acebrón, Iván; de las Rivas, Blanca; Muñoz, Rosario; Kohring, Gert-Wieland; Roa, Ana María; Sobrino, Mónica; Mancheño, José M

2012-09-21

Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni(2+)-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn(2+)- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Repair by genetic recombination in bacteria: overview

International Nuclear Information System (INIS)

Howard-Flanders, P.

1975-01-01

DNA molecules that have been damaged in both strands at the same level are not subject to repair by excision but instead can be repaired through recombination with homologous molecules. Examples of two-strand damage include postreplication gaps opposite pyrimidine dimers, two-strand breaks produced by x-rays, and chemically induced interstrand cross-links. In ultraviolet-irradiated bacteria, and newly synthesized DNA is of length equal to the interdimer spacing. With continued incubation, this low-molecular-weight DNA is joined into high-molecular-weight chains (postreplication repair), a process associated with sister exchanges in bacteria. Recombination is initiated by pyrimidine dimers opposite postreplication gaps and by interstrand cross-links that have been cut by excision enzymes. The free ends at the resulting gaps presumably initiate the exchanges. Postreplication repair in Escherichia coli occurs in recB - and recC - but is greatly slowed in recF - mutants. RecB and recC are the structural genes for exonuclease V, which digests two-stranded DNA by releasing oligonucleotides first from one strand and then from the other. The postreplication sister exchanges in ultraviolet-irradiated bacteria result in the distribution of pyrimidine dimers between parental and daughter strands, indicating that long exchanges involving both strands of each duplex occur. The R1 restriction endonuclease from E. coli has been used to cut the DNA of a bacterial drug-resistance transfer factor with one nuclease-sensitive site, and also DNA from the frog Xenopus enriched for ribosomal 18S and 28S genes. The fragments were annealed with the cut plasmid DNA and ligated, producing a new larger plasmid carrying the eukaryotic rDNA and able to infect and replicate in E. coli

Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila.

Science.gov (United States)

Smukowski Heil, Caiti S; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A F

2015-10-01

Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human-chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Therapeutic Recombinant Monoclonal Antibodies

Science.gov (United States)

Bakhtiar, Ray

2012-01-01

During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

Creating leptin-like biofunctions by active immunization against chicken leptin receptor in growing chickens.

Science.gov (United States)

Lei, M M; Wu, S Q; Shao, X B; Li, X W; Chen, Z; Ying, S J; Shi, Z D

2015-01-01

In this study, immunization against chicken leptin receptor (cLEPR) extracellular domain (ECD) was applied to investigate leptin regulation and LEPR biofunction in growing chicken pullets. A recombinant protein (cLEPR ECD) based on the cLEPR complemenary DNA sequence corresponding to the 582nd to 796th amino acid residues of cLEPR mature peptide was prepared and used as antigen. Immunization against cLEPR ECD in growing chickens increased anti-cLEPR ECD antibody titers in blood, enhanced proportions of phosphorylated janus kinase 2 (JAK2) and served as signal transducer and activator of transcription 3 (STAT3) protein in liver tissue. Chicken live weight gain and abdominal fat mass were significantly decreased (P chickens. Copyright © 2015 Elsevier Inc. All rights reserved.

Applications of recombinant antibodies in plant pathology.

Science.gov (United States)

Ziegler, Angelika; Torrance, Lesley

2002-09-01

Summary Advances in molecular biology have made it possible to produce antibody fragments comprising the binding domains of antibody molecules in diverse heterologous systems, such as Escherichia coli, insect cells, or plants. Antibody fragments specific for a wide range of antigens, including plant pathogens, have been obtained by cloning V-genes from lymphoid tissue, or by selection from large naive phage display libraries, thus avoiding the need for immunization. The antibody fragments have been expressed as fusion proteins to create different functional molecules, and fully recombinant assays have been devised to detect plant viruses. The defined binding properties and unlimited cheap supply of antibody fusion proteins make them useful components of standardized immunoassays. The expression of antibody fragments in plants was shown to confer resistance to several plant pathogens. However, the antibodies usually only slowed the progress of infection and durable 'plantibody' resistance has yet to be demonstrated. In future, it is anticipated that antibody fragments from large libraries will be essential tools in high-throughput approaches to post-genomics research, such as the assignment of gene function, characterization of spatio-temporal patterns of protein expression, and elucidation of protein-protein interactions.

Mini review: Recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives.

Science.gov (United States)

Lakowitz, Antonia; Godard, Thibault; Biedendieck, Rebekka; Krull, Rainer

2018-05-01

Bio-pharmaceuticals like antibodies, hormones and growth factors represent about one-fifth of commercial pharmaceuticals. Host candidates of growing interest for recombinant production of these proteins are strains of the genus Bacillus, long being established for biotechnological production of homologous and heterologous proteins. Bacillus strains benefit from development of efficient expression systems in the last decades and emerge as major industrial workhorses for recombinant proteins due to easy cultivation, non-pathogenicity and their ability to secrete recombinant proteins directly into extracellular medium allowing cost-effective downstream processing. Their broad product portfolio of pharmaceutically relevant recombinant proteins described in research include antibody fragments, growth factors, interferons and interleukins, insulin, penicillin G acylase, streptavidin and different kinases produced in various cultivation systems like microtiter plates, shake flasks and bioreactor systems in batch, fed-batch and continuous mode. To further improve production and secretion performance of Bacillus, bottlenecks and limiting factors concerning proteases, chaperones, secretion machinery or feedback mechanisms can be identified on different cell levels from genomics and transcriptomics via proteomics to metabolomics and fluxomics. For systematical identification of recurring patterns characteristic of given regulatory systems and key genetic targets, systems biology and omics-technology provide suitable and promising approaches, pushing Bacillus further towards industrial application for recombinant pharmaceutical protein production. Copyright © 2017. Published by Elsevier B.V.

Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum.

Science.gov (United States)

Gupta, Ruchi; Hogan, Campbell J; Perugini, Matthew A; Soares da Costa, Tatiana P

2018-05-09

Recombinant wheat DHDPS was produced for the first time in milligram quantities and shown to be an enzymatically active tetramer in solution using analytical ultracentrifugation and small angle X-ray scattering. Wheat is an important cereal crop with an extensive role in global food supply. Given our rapidly growing population, strategies to increase the nutritional value and production of bread wheat are of major significance in agricultural science to satisfy our dietary requirements. Lysine is one of the most limiting essential amino acids in wheat, thus, a thorough understanding of lysine biosynthesis is of upmost importance to improve its nutritional value. Dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7) catalyzes the first committed step in the lysine biosynthesis pathway of plants. Here, we report for the first time the expression and purification of recombinant DHDPS from the bread wheat Triticum aestivum (Ta-DHDPS). The optimized protocol yielded 36 mg of > 98% pure recombinant Ta-DHDPS per liter of culture. Enzyme kinetic studies demonstrate that the recombinant Ta-DHDPS has a K M (pyruvate) of 0.45 mM, K M (l-aspartate-4-semialdehyde) of 0.07 mM, k cat of 56 s -1 , and is inhibited by lysine (IC 50 LYS of 0.033 mM), which agree well with previous studies using labor-intensive purification from wheat suspension cultures. We subsequently employed circular dichroism spectroscopy, analytical ultracentrifugation and small angle X-ray scattering to show that the recombinant enzyme is folded with 60% α/β structure and exists as a 7.5 S tetrameric species with a R g of 33 Å and D max of 118 Å. This study is the first to report the biophysical properties of the recombinant Ta-DHDPS in aqueous solution and offers an excellent platform for future studies aimed at improving nutritional value and primary production of bread wheat.

[Improvement of thermal adaptability and fermentation of industrial ethanologenic yeast by genomic DNA mutagenesis-based genetic recombination].

Science.gov (United States)

Liu, Xiuying; He, Xiuping; Lu, Ying; Zhang, Borun

2011-07-01

Ethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, in the process of industrial production of ethanol, both cell growth and fermentation of ethanologenic S. cerevisiae are dramatically affected by environmental stresses, such as thermal stress. In this study, we improved both the thermotolerance and fermentation performance of industrial ethanologenic S. cerevisiae by combined usage of chemical mutagenesis and genomic DNA mutagenesis-based genetic recombination method. The recombinant S. cerevisiae strain T44-2 could grow at 44 degrees C, 3 degrees C higher than that of the original strain CE6. The survival rate of T44-2 was 1.84 and 1.87-fold of that of CE6 when heat shock at 48 degrees C and 52 degrees C for 1 h respectively. At temperature higher than 37 degrees C, recombinant strain T44-2 always gave higher cell growth and ethanol production than those of strain CE6. Meanwhile, from 30 degrees C to 40 degrees C, recombinant strain T44-2 produces 91.2-83.8 g/L of ethanol from 200 g/L of glucose, which indicated that the recombinant strain T44-2 had both thermotolerance and broad thermal adaptability. The work offers a novel method, called genomic DNA mutagenesis-based genetic recombination, to improve the physiological functions of S. cerevisiae.

Ethanol production from marine algal hydrolysates using Escherichia coli KO11.

Science.gov (United States)

Kim, Nag-Jong; Li, Hui; Jung, Kwonsu; Chang, Ho Nam; Lee, Pyung Cheon

2011-08-01

Algae biomass is a potential raw material for the production of biofuels and other chemicals. In this study, biomass of the marine algae, Ulva lactuca, Gelidium amansii,Laminaria japonica, and Sargassum fulvellum, was treated with acid and commercially available hydrolytic enzymes. The hydrolysates contained glucose, mannose, galactose, and mannitol, among other sugars, at different ratios. The Laminaria japonica hydrolysate contained up to 30.5% mannitol and 6.98% glucose in the hydrolysate solids. Ethanogenic recombinant Escherichia coli KO11 was able to utilize both mannitol and glucose and produced 0.4g ethanol per g of carbohydrate when cultured in L. japonica hydrolysate supplemented with Luria-Bertani medium and hydrolytic enzymes. The strategy of acid hydrolysis followed by simultaneous enzyme treatment and inoculation with E. coli KO11 could be a viable strategy to produce ethanol from marine alga biomass. Copyright © 2011 Elsevier Ltd. All rights reserved.

High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

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Ramos-Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

2017-09-01

Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP) n , wherein n = 10 or 20]. The yields of the (SP) n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP) n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP) n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP) n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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

Directory of Open Access Journals (Sweden)

Torres Leticia L

2012-08-01

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

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

Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli.

Science.gov (United States)

Grönlund, Hans; Bergman, Tomas; Sandström, Kristofer; Alvelius, Gunvor; Reininger, Renate; Verdino, Petra; Hauswirth, Alexander; Liderot, Karin; Valent, Peter; Spitzauer, Susanne; Keller, Walter; Valenta, Rudolf; van Hage-Hamsten, Marianne

2003-10-10

Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.

Biologically active and C-amidated hinnavinII-38-Asn produced from a Trx fusion construct in Escherichia coli.

Science.gov (United States)

Kang, Chang Soo; Son, Seung-Yeol; Bang, In Seok

2008-12-01

The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15-20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.

Pathological And Immunological Study On Infection With Escherichia Coli In ale BALB/c mice

Science.gov (United States)

Ali, Intisar H.; Jabir, Majid S.; Al-Shmgani, Hanady S. A.; Sulaiman, Ghassan M.; Sadoon, Ali H.

2018-05-01

Escherichia coli bacteria is considered as one of the common responsible for the frequency and severity of infections that it hospitalized patients. E. coli simultaneously carries a harmful side in which only a slight genetic recombination can bring about a highly pathogenic strain that most frequently causes the scourge of bacterial infections worldwide including sepsis, neonatal meningitis, pneumonia, bacteremia and traveler’s diarrhea. This study was carried out to assess Escherichia coli infection induced pathologically and immunologically. Following Escherichia coli isolation, identification and counting, the lethal dose (LD-50) was determined before infection. Twenty-two mice were used in this study for 21 days infection, the animals were sacrificed at 3, 6, 9, 12, 15, 18 and 21 days, and tissues of different tissue were collected, examined for bacterial infection. Bacteria and mice immunization and ELISA were used to detect immunoglobulin G level in serum as well. For histological study, different infected organs were used. The results indicated that the LH50 was 1×109 cell; and all organs were infected after 3 days followed by decreased in infection level shown in brain at day 12, lung, kidney and intestine at day 15 and in liver, spleen and heart at day 21. Moreover, ELISA results revealed that concentration 1:200 of serum in positive and negative state and optimum concentration of Ag 1:40 dilution and compact dilution is 1:1000. In addition, diversity of histopathological alteration occurs in tissue on time-depended manner. This study concluded that the ability of activated E.coli to stimulate the intestinal secretory immune system of germ might result from a retardation of immunological maturity.

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.

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

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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.

Sensitivity of strains of Escherichia coli differing in repair capability to far UV, near UV and visible radiations

International Nuclear Information System (INIS)

Webb, R.B.; Brown, M.S.

1976-01-01

In stationary phase, strains of Escherichia coli deficient in excision (B/r Hcr) or recombination repair (K12 AB2463) were more sensitive than a repair proficient strain (B/r) to monochromatic near-ultraviolet (365nm) and visible (460 nm) radiations. The relative increase in sensitivity of mutants deficient in excision or recombination repair in comparison to the wildtype, was less at 365 nm than at 254 nm. However, a strain deficient in both excision and recombination repair (K12 AB2480) showed a large, almost equal, increase in sensitivity over mutants deficient in either excision or recombination repair at 365 nm and 254 nm. All strains tested were highly resistant to 650 nm radiation. Action spectra for lethality of strains B/r and B/r Hcr in stationary phase reveal small peaks or shoulders in the 330 to 340, 400 to 410 and 490 to 510 nm wavelength ranges. The presence of 5 micro g/ml acriflavine (an inhibitor of repair) in the plating medium greatly increased the sensitivity of strain B/r to radiation at 254, 365 and 460 nm, while strains E.coli B/r Hcr and K12 AB2463 were sensitized by small amounts. At each of the wavelengths tested, acriflavine in the plating medium had at most a small effect on E.coli K12 AB2480. Acriflavine failed to sensitize any strain tested at 650 nm. Evidence supports the interpretation that lesions induced in DNA by 365 nm and 460 nm radiations play the major role in the inactivation of E.coli by these wavelengths. Single-strand breaks (or alkali-labile bonds), but not pyrimidine dimers are candidates for the lethal DNA lesions in uvrA and repair proficient strains. At high fluences lethality may be enhanced by damage to the excision and recombination repair systems. (author)

Sensitivity of strains of Escherichia coli differing in repair capability to far uv, near uv and visible radiations

Energy Technology Data Exchange (ETDEWEB)

Webb, R B; Brown, M S [Argonne National Lab., Ill. (USA)

1976-11-01

In stationary phase, strains of Escherichia coli deficient in excision (B/r Hcr) or recombination repair (K12 AB2463) were more sensitive than a repair proficient strain (B/r) to monochromatic near-ultraviolet (365nm) and visible (460 nm) radiations. The relative increase in sensitivity of mutants deficient in excision or recombination repair in comparison to the wildtype, was less at 365 nm than at 254 nm. However, a strain deficient in both excision and recombination repair (K12 AB2480) showed a large, almost equal, increase in sensitivity over mutants deficient in either excision or recombination repair at 365 nm and 254 nm. All strains tested were highly resistant to 650 nm radiation. Action spectra for lethality of strains B/r and B/r Hcr in stationary phase reveal small peaks or shoulders in the 330 to 340, 400 to 410 and 490 to 510 nm wavelength ranges. The presence of 5 micro g/ml acriflavine (an inhibitor of repair) in the plating medium greatly increased the sensitivity of strain B/r to radiation at 254, 365 and 460 nm, while strains E.coli B/r Hcr and K12 AB2463 were sensitized by small amounts. At each of the wavelengths tested, acriflavine in the plating medium had at most a small effect on E.coli K12 AB2480. Acriflavine failed to sensitize any strain tested at 650 nm. Evidence supports the interpretation that lesions induced in DNA by 365 nm and 460 nm radiations play the major role in the inactivation of E.coli by these wavelengths. Single-strand breaks (or alkali-labile bonds), but not pyrimidine dimers are candidates for the lethal DNA lesions in uvrA and repair proficient strains. At high fluences lethality may be enhanced by damage to the excision and recombination repair systems.

[Construction of a recombinant HVT virus expressing the HA gene of avian influenza virus H5N1 via Rde/ET recombination system].

Science.gov (United States)

Lan, Desong; Shi, Xingming; Wang, Yunfeng; Liu, Changjun; Wang, Mei; Cui, Hongyu; Tian, Guobin; Li, Jisong; Tong, Guangzhi

2009-01-01

In recent years,manipulation of large herpesvirus genomes has been facilitated by using bacterial artificial chromosome (BAC) vectors. We have previously reported the construction of the BAC clones (HVT BACs) of herpesvirus of turkey (HVT). With these BAC clones in hand,we manipulated the genome of HVT by utilizing Red/ET recombination system, and developed a biologically safe live vaccine based on the HVT BACs. In this two-step approach, we first transformed the plasmid pRedET into the DH10B competent cells that carried the HVT BACs,and added inducer L-arabinose into the cells. We prepared the cells into competent cells and electroporated the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms into the cells. So the functional cassette was inserted into the U(S)2 locus. Only colonies carrying the modified BAC would survive Kanamycin selection on the agar plates. The successful integration of the rpsL-neo cassette was monitored by PCR and Streptomycin selection, for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive. Secondly, in the same way, we replaced the rpsL-neo cassette with the hemagglutinin (HA) gene of (HPAIV) A/Goose/ Guangdong/1/96(H5N1) flanked by the same homology arms. Only colonies which lost the rpsL-neo cassette will grow on Streptomycin containing plates. Finally, we obtained many colonies of which the HA gene of the AIV was inserted into the U(S)2 locus to be modified of HVT. And we reconstituted one recombinant virus from transfecting one of these BAC clones DNA into chick embryo fibroblasts (CEFs). We achieved one rescued recombinant virus which designated as rHVT-HA3. The H5 subtype HA gene expression in this recombinant virus rHVT-HA3 was confirmed by immunofluorescence assay.

Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

International Nuclear Information System (INIS)

Funk, W.D.; MacGillivray, R.T.A.; Mason, A.B.; Brown, S.A.; Woodworth, R.C.

1990-01-01

A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO 4 -polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, the authors developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. The recombinant hTF/2N appeared to behave identically with the proteolytically derived half-molecule, but to show a higher degree of monodispersity than the latter protein. Addition of m-fluorotyrosine to the culture medium resulted in random incorporation of this amino acid into cellular protein in lieu of tyrosine. Purified recombinant 19 F-Tyr hTF/2N gave four well-resolved 19 F NMR resonances of 20-40 Hz line width, two with suggestions of shoulders

Phylogenetic incongruence in E. coli O104: understanding the evolutionary relationships of emerging pathogens in the face of homologous recombination.

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Weilong Hao

Full Text Available Escherichia coli O104:H4 was identified as an emerging pathogen during the spring and summer of 2011 and was responsible for a widespread outbreak that resulted in the deaths of 50 people and sickened over 4075. Traditional phenotypic and genotypic assays, such as serotyping, pulsed field gel electrophoresis (PFGE, and multilocus sequence typing (MLST, permit identification and classification of bacterial pathogens, but cannot accurately resolve relationships among genotypically similar but pathotypically different isolates. To understand the evolutionary origins of E. coli O104:H4, we sequenced two strains isolated in Ontario, Canada. One was epidemiologically linked to the 2011 outbreak, and the second, unrelated isolate, was obtained in 2010. MLST analysis indicated that both isolates are of the same sequence type (ST678, but whole-genome sequencing revealed differences in chromosomal and plasmid content. Through comprehensive phylogenetic analysis of five O104:H4 ST678 genomes, we identified 167 genes in three gene clusters that have undergone homologous recombination with distantly related E. coli strains. These recombination events have resulted in unexpectedly high sequence diversity within the same sequence type. Failure to recognize or adjust for homologous recombination can result in phylogenetic incongruence. Understanding the extent of homologous recombination among different strains of the same sequence type may explain the pathotypic differences between the ON2010 and ON2011 strains and help shed new light on the emergence of this new pathogen.

Associations between Antimicrobial Resistance Phenotypes, Antimicrobial Resistance Genes, and Virulence Genes of Fecal Escherichia coli Isolates from Healthy Grow-Finish Pigs ▿

OpenAIRE

Rosengren, Leigh B.; Waldner, Cheryl L.; Reid-Smith, Richard J.

2009-01-01

Escherichia coli often carries linked antimicrobial resistance genes on transmissible genetic elements. Through coselection, antimicrobial use may select for unrelated but linked resistance or virulence genes. This study used unconditional statistical associations to investigate the relationships between antimicrobial resistance phenotypes and antimicrobial resistance genes in 151 E. coli isolates from healthy pigs. Phenotypic resistance to each drug was significantly associated with phenotyp...

Recombination every day: abundant recombination in a virus during a single multi-cellular host infection.

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Remy Froissart

2005-03-01

Full Text Available Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10(-5 to 4 x 10(-5. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

Nitrofurantoin and Fosfomycin for Extended Spectrum Beta-lactamases Producing Escherichia coli and Klebsiella pneumoniae

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Neeraj Kumar Tulara

2018-01-01

Full Text Available Urinary tract infection (UTI is a common and painful human illness that, unfortunately not responsive to commonly used antibiotics in current practice. The role of fosfomycin and nitrofurantoin in the era of growing bacteria resistance has been widely discussed. In this study, we aimed to know the local antimicrobial susceptibilities, fosfomycin and nitrofurantoin susceptibility in particular, for urinary extended-spectrum-beta-lactamase-producing Escherichia coli and Escherichia pneumoniae (ESBL-EC and ESBL-KP isolates in our hospital. We collected 464 urine isolates, including 384 ESBL-EC and 80 ESBL-KP isolates. Of 464 urine isolates culture positive ESBL-UTIs, EC caused 384 (82.75%, followed by Klebsiella in 80 (17.24%. Carbapenems and Colistin seems to remain as the first line therapy for the majority of ESBL-UTIs in the local setting. Colistin and fosfomycin remains the most sensitive antibiotic while nitrofurantoin still preserves the good sensitivity against ESBL and found to be an only oral sensitive antibiotic.

Two proline porters in Escherichia coli K-12.

Science.gov (United States)

Stalmach, M E; Grothe, S; Wood, J M

1983-11-01

Escherichia coli mutants defective at putP and putA lack proline transport via proline porter I and proline dehydrogenase activity, respectively. They retain a proline uptake system (proline porter II) that is induced during tryptophan-limited growth and are sensitive to the toxic L-proline analog, 3,4-dehydroproline. 3,4-Dehydroproline-resistant mutants derived from a putP putA mutant lack proline porter II. Auxotrophic derivatives derived from putP+ or putP bacteria can grow if provided with proline at low concentration (25 microM); those derived from the 3,4-dehydroproline-resistant mutants require high proline for growth (2.5 mM). We conclude that E. coli, like Salmonella typhimurium, possesses a second proline porter that is inactivated by mutations at the proP locus.

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

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Yi Ma

2016-01-01

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

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.

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...

Mutations that alter the transport function of the LamB protein in Escherichia coli.

OpenAIRE

Wandersman, C; Schwartz, M

1982-01-01

Some Escherichia coli K-12 lamB mutants, those producing reduced amounts of LamB protein (one-tenth the wild type amount), grow normally on dextrins but transport maltose when present at a concentration of 1 microM at about one-tenth the normal rate. lamB Dex- mutants were found as derivatives of these strains. These Dex- mutants are considerably impaired in the transport of maltose at low concentrations (below 10 microM), and they have a structurally altered LamB protein which is impaired in...

Regulation of Meiotic Recombination

Energy Technology Data Exchange (ETDEWEB)

Gregory p. Copenhaver

2011-11-09

Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

Simplified Method to Produce Human Bioactive Leukemia Inhibitory Factor in Escherichia coli

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Houman Kahroba

2016-07-01

Full Text Available Background Human leukemia inhibitory factor (hLIF is a poly functional cytokine with numerous regulatory effects on different cells. Main application of hLIF is maintaining pluripotency of embryonic stem cells. hLIF indicated effective work in implantation rate of fertilized eggs and multiple sclerosis (MS treatment. Low production of hLIF in eukaryotic cells and prokaryotic host’s problems for human protein production convinced us to develop a simple way to reach high amount of this widely used clinical and research factor. Objectives In this study we want to purify recombinant human leukemia inhibitory factor in single simple method. Materials and Methods This is an experimental study, gene expression: human LIF gene was codon optimized for expression in Escherichia coli and attached his-tag tail to make it extractable. After construction and transformation of vector to E. coli, isopropyl β-D-1-thiogalactopyranoside (IPTG used for induction. Single step immobilized metal affinity chromatography (IMAC used for purification confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE and western blotting. Bioactivity of the hLIF were tested by MTT assay with TF-1 cells and CISH gene stimulation in monocyte and TF-1 by real-time PCR. Induction by 0.4 mM of IPTG in 25°C for 3 hours indicated best result for soluble expression. SPSS indicated P ˂ 0.05 that is significant for our work. Results Cloning, expression, and extraction of bio active rhLIF was successfully achieved according MTT assay and real time PCR after treatment of TF-1 and monocyte cell lines. Conclusions We developed an effective single step purification method to produce bioactive recombinant hLIF in E. coli. For the first time we used CISH gene stimulating for bioactivity test for qualifying of recombinant hLIF for application.

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.

Noncomplementing diploidy resulting from spontaneous zygogenesis in Escherichia coli.

Science.gov (United States)

Gratia, Jean-Pierre

2005-09-01

With the aim of understanding sexual reproduction and phenotypic expression, a novel type of mating recently discovered in Escherichia coli was investigated. Termed spontaneous zygogenesis (or Z-mating), it differs from F-mediated conjugation. Its products proved phenotypically unstable, losing part of the phenotype for which they were selected. Inactivation of a parental chromosome in the zygote is strongly suggested by fluctuation tests, respreading experiments, analysis of reisolates, and segregation of non-viable cells detected by epifluorescence staining. Some phenotypically haploid subclones were interpreted as stable noncomplementing diploids carrying an inactivated co-replicating chromosome. Pedigree analysis indicated that the genetic composition of such cells consisted of parental genomes or one parental plus a recombinant genome. Inactivation of a chromosome carrying a prophage resulted in the disappearance of both the ability to produce phage particles and the immunity to superinfection. Phage production signalled transient reactivation of such a chromosome and constituted a sensitive test for stable noncomplementing diploidy. Chromosome inactivation thus appears to be a spontaneous event in bacteria.

Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds

DEFF Research Database (Denmark)

Ostergaard Pedersen, L; Nissen, Mogens Holst; Hansen, N J

2001-01-01

The expression of major histocompatibility class I (MHC-I) crucially depends upon the binding of appropriate peptides. MHC-I from natural sources are therefore always preoccupied with peptides complicating their purification and analysis. Here, we present an efficient solution to this problem....... Recombinant MHC-I heavy chains were produced in Escherichia coli and subsequently purified under denaturing conditions. In contrast to common practice, the molecules were not reduced during the purification. The oxidized MHC-I heavy chain isoforms were highly active with respect to peptide binding....... This suggests that de novo folding of denatured MHC-I molecules proceed efficiently if directed by preformed disulfide bond(s). Importantly, these molecules express serological epitopes and stain specific T cells; and they bind peptides specifically. Several denatured MHC-I heavy chains were analyzed and shown...

Physical analyses of E. coli heteroduplex recombination products in vivo: on the prevalence of 5' and 3' patches.

Directory of Open Access Journals (Sweden)

Laura M Gumbiner-Russo

Full Text Available BACKGROUND: Homologous recombination in Escherichia coli creates patches (non-crossovers or splices (half crossovers, each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE repair specify that only the 3'-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands. METHODOLOGY/PRINCIPLE FINDINGS: This paper re-examines heteroduplex-patch-strand polarity using phage lambda and the lambdadv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for lambda recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5' or 3' DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation. CONCLUSIONS/SIGNIFICANCE: These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the

Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Setlow, J.K.; Spikes, D.; Griffin, K.

1988-09-01

The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein.

Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli

International Nuclear Information System (INIS)

Setlow, J.K.; Spikes, D.; Griffin, K.

1988-01-01

The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein

Recombination of cluster ions

Science.gov (United States)

Johnsen, Rainer

1993-01-01

Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

Directory of Open Access Journals (Sweden)

Abbas Mohajeri

2017-04-01

Full Text Available Angiogenesis plays an essential role in rapid growing and metastasis of the tumors. Inhibition of angiogenesis is a putative strategy for cancer therapy. Endostatin (Es is an attractive anti-angiogenesis protein with some clinical application challenges including; short half-life, instability in serum and requirement to high dosage. Therefore, production of recombinant endostatin (rEs is necessary in large scale. The production of rEs is difficult because of its structural properties and is high-cost. Therefore, this review focused on the different expression systems that involved in rEs production including; mammalian, baculovirus, yeast, and Escherichia coli (E. coli expression systems. The evaluating of the results of different expression systems declared that none of the mentioned systems can be considered to be generally superior to the other. Meanwhile with considering the advantages and disadvantage of E. coli expression system compared with other systems beside the molecular properties of Es, E. coli expression system can be a preferred expression system for expressing of the Es in large scale. Also, the molecular bioengineering and sustained release formulations that lead to improving of its stability and bioactivity will be discussed. Point mutation (P125A of Es, addition of RGD moiety or an additional zinc biding site to N-terminal of Es , fusing of Es to anti-HER2 IgG or heavy-chain of IgG, and finally loading of the endostar by PLGA and PEG- PLGA nanoparticles and gold nano-shell particles are the effective bioengineering methods to overcome to clinical changes of endostatin.

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

Science.gov (United States)

Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

2017-12-01

Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a

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

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Cashman Kathleen A

2008-06-01

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

Recombination pattern reanalysis of some HIV-1 circulating recombination forms suggest the necessity and difficulty of revision.

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Lei Jia

Full Text Available Recombination is one of the major mechanisms underlying the generation of HIV-1 variability. Currently 61 circulating recombinant forms of HIV-1 have been identified. With the development of recombination detection techniques and accumulation of HIV-1 reference stains, more accurate mosaic structures of circulating recombinant forms (CRFs, like CRF04 and CRF06, have undergone repeated analysis and upgrades. Such revisions may also be necessary for other CRFs. Unlike previous studies, whose results are based primarily on a single recombination detection program, the current study was based on multiple recombination analysis, which may have produced more impartial results.Representative references of 3 categories of intersubtype recombinants were selected, including BC recombinants (CRF07 and CRF08, BG recombinants (CRF23 and CRF24, and BF recombinants (CRF38 and CRF44. They were reanalyzed in detail using both the jumping profile hidden Markov model and RDP3.The results indicate that revisions and upgrades are very necessary and the entire re-analysis suggested 2 types of revision: (i length of inserted fragments; and (ii number of inserted fragments. The reanalysis also indicated that determination of small regions of about 200 bases or fewer should be performed with more caution.Results indicated that the involvement of multiple recombination detection programs is very necessary. Additionally, results suggested two major challenges, one involving the difficulty of accurately determining the locations of breakpoints and the second involving identification of small regions of about 200 bases or fewer with greater caution. Both indicate the complexity of HIV-1 recombination. The resolution would depend critically on development of a recombination analysis algorithm, accumulation of HIV-1 stains, and a higher sequencing quality. With the changes in recombination pattern, phylogenetic relationships of some CRFs may also change. All these results may

Control of Ribosome Synthesis in Escherichia coli

DEFF Research Database (Denmark)

Molin, Søren; Meyenburg, K. von; Måløe, O.

1977-01-01

The rate of ribosome synthesis and accumulation in Escherichia coli during the transition after an energy source shift-down was analyzed. The shift was imposed on cultures of stringent and relaxed strains growing in glucose minimal medium by the addition of the glucose analogue {alpha...... and to estimate the transcription time for the rRNA operon under different conditions. In steady states of growth with growth rates ranging from 0.75 to 2.3 doublings/h, as well as during the transition after a shift-down, the transcription time of the rRNA operon was constant. The rate of synthesis of r......RNA correlated during this transition – in contrast to the rate of accumulation (M. T. Hansen et al., J. Bacteriol. 122: 585-591, 1975) – with the ppGpp pool in the same way as has been observed during partial amino acid starvation....

Structure of recombinant Ves v 2 at 2.0 Angstrom resolution

DEFF Research Database (Denmark)

Skov, Lars K; Seppälä, Ulla; Coen, Jeremy J F

2006-01-01

Wasp venom from Vespula vulgaris contains three major allergens: Ves v 1, Ves v 2 and Ves v 5. Here, the cloning, expression, biochemical characterization and crystal structure determination of the hyaluronidase Ves v 2 from family 56 of the glycoside hydrolases are reported. The allergen...... was expressed in Escherichia coli as an insoluble protein and refolded and purified to obtain full enzymatic activity. Three N-glycosylation sites at Asn79, Asn99 and Asn127 were identified in Ves v 2 from a natural source by enzymatic digestions combined with MALDI-TOF mass spectrometry. The crystal structure...... of recombinant Ves v 2 was determined at 2.0 A resolution and reveals a central (beta/alpha)(7) core that is further stabilized by two disulfide bonds (Cys19-Cys308 and Cys185-Cys197). Based on sequence alignments and structural comparison with the honeybee allergen Api m 2, it is proposed that a conserved...

Characterization of recombinant glyoxylate reductase from thermophile Thermus thermophilus HB27.

Science.gov (United States)

Ogino, Hiroyasu; Nakayama, Hitoshi; China, Hideyasu; Kawata, Takuya; Doukyu, Noriyuki; Yasuda, Masahiro

2008-01-01

A glyoxylate reductase gene from the thermophilic bacterium Thermus thermophilus HB27 (TthGR) was cloned and expressed in Escherichia coli cells. The recombinant enzyme was highly purified to homogeneity and characterized. The purified TthGR showed thermostability up to 70 degrees C. In contrast, the maximum reaction condition was relatively mild (45 degrees C and pH 6.7). Although the kcat values against co-enzyme NADH and NADPH were similar, the Km value against co-enzyme NADH was approximately 18 times higher than that against NADPH. TthGR prefers NADPH rather than NADH as an electron donor. These results indicate that a phosphate group of a co-enzyme affects the binding affinity rather than the reaction efficiency, and TthGR demands appropriate amount of phosphate for a high activity. Furthermore, it was found that the half-lives of TthGR in the presence of 25% dimethyl sulfoxide and diethylene glycol were significantly longer than that in the absence of an organic solvent.

Expression of recombinant antibacterial lactoferricin-related peptides from Pichia pastoris expression system.

Science.gov (United States)

Chen, Gen-Hung; Chen, Wei-Ming; Huang, Guo-Ting; Chen, Yu-Wen; Jiang, Shann-Tzong

2009-10-28

Four recombinant antimicrobial peptide (rAMP) cDNAs, constructed from two goat lactoferricin-related peptide cDNAs (GLFcin and GLFcin II) with/without (His)(6)-Tag, were cloned into pPICZalphaC and transformed into Pichia pastoris SMD1168H. After methanol induction, these rAMPs were expressed and secreted into broth. They were purified after CM-Sepharose (without His-tg), HisTrap (with His-tg) and Sephadex G-25 chromatographies. The yield of purified rAMP was 0.15 mg/mL of broth. These 4 rAMPs were thermal-stable and with high antibacterial activity against Escherichia coli BCRC 11549, Pseudomonas aeruginosa BCRC 12450, Bacillus cereus BCRC 10603, Staphylococcus aureus BCRC 25923, Propioni bacterium acnes BCRC 10723, and Listera monocytogenes BCRC 14845. The minimum inhibitory concentration (MIC) of rAMPs against these indicators ranged from 4.07 to 16.00 mg/mL.

Systematic high-yield production of human secreted proteins in Escherichia coli

International Nuclear Information System (INIS)

Dai Xueyu; Chen Qiang; Lian Min; Zhou Yanfeng; Zhou Mo; Lu Shanyun; Chen Yunjia; Luo Jingchu; Gu Xiaocheng; Jiang Ying; Luo Ming; Zheng Xiaofeng

2005-01-01

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies

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

Electron-ion recombination in merged beams

International Nuclear Information System (INIS)

Wolf, A.; Habs, D.; Lampert, A.; Neumann, R.; Schramm, U.; Schuessler, T.; Schwalm, D.

1993-01-01

Detailed studies of recombination processes between electrons and highly charged ions have become possible by recent improvements of merged-beams experiments. We discuss in particular measurements with stored cooled ion beams at the Test Storage Ring (TSR) in Heidelberg. The cross section of dielectronic recombination was measured with high energy resolution for few-electron systems up to the nuclear charge of Cu at a relative energy up to 2.6 keV. At low energy (∼0.1 eV) total recombination rates of several ions were measured and compared with calculated radiative recombination rates. Laser-stimulated recombination of protons and of C 6+ ions was investigated as a function of the photon energy using visible radiation. Both the total recombination rates and the stimulated recombination spectra indicate that in spite of the short interaction time in merged beams, also collisional capture of electrons into weakly bound levels (related to three-body recombination) could be important

Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

Science.gov (United States)

Richardson, Ruth E.; Suzuki, Yo

2015-01-01

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work. PMID:26348330

Multiple barriers to recombination between divergent HIV-1 variants revealed by a dual-marker recombination assay

DEFF Research Database (Denmark)

Nikolaitchik, Olga A; Galli, Andrea; Moore, Michael D

2011-01-01

Recombination is a major force for generating human immunodeficiency virus type 1 (HIV-1) diversity and produces numerous recombinants circulating in the human population. We previously established a cell-based system using green fluorescent protein gene (gfp) as a reporter to study the mechanisms...... of HIV-1 recombination. We now report an improved system capable of detecting recombination using authentic viral sequences. Frameshift mutations were introduced into the gag gene so that parental viruses do not express full-length Gag; however, recombination can generate a progeny virus that expresses...

Rapid customised operon assembly by yeast recombinational cloning.

Science.gov (United States)

Liu, Michael A; Kenyon, Johanna J; Lee, Jason; Reeves, Peter R

2017-06-01

We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons. We demonstrated the utility of the OAP by assembling and expressing a series of E. coli O1A O-antigen gene cluster clones containing various gene deletions or replacements. We then used these constructs to assess the substrate preferences of several Wzx flippases, which are responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. We were able to identify several O unit structural features that appear to be important determinants of Wzx substrate preference. The OAP system should be broadly applicable for the genetic manipulation of any bacterial operon and can be modified for use in other host species. It could also have potential uses in fields such as glycoengineering.

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

Oxygen-hydrogen recombination system

International Nuclear Information System (INIS)

Sato, Shuichiro; Takejima, Masaki.

1981-01-01

Purpose: To avoid reduction in the performance of catalyst used for an oxygen-hydrogen recombiner in the off gas processing system of a nuclear reactor. Constitution: A thermometer is provided for the detection of temperature in an oxygen-hydrogen recombiner. A cooling pipe is provided in the recombiner and cooling medium is introduced externally. The cooling medium may be water or air. In accordance with the detection value from the thermometer, ON-OFF control is carried out for a valve to control the flow rate of the cooling medium thereby rendering the temperature in the recombiner to a predetermined value. This can prevent the catalyst from being exposed to high temperature and avoid the reduction in the performance of the catalyst. (Ikeda, J.)

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Directory of Open Access Journals (Sweden)

Ken Motohashi

2017-03-01

Full Text Available Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. In vivo Escherichia coli cloning (iVEC can directly transform a mixture of insert and vector DNA fragments into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp end homology regions. However, colony formation was 30–60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbcA gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo. Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli recA− strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

International Nuclear Information System (INIS)

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

Transformation and characterization of an arsenic gene operon from urease-positive thermophilic Campylobacter (UPTC) in Escherichia coli.

Science.gov (United States)

Matsuda, M; Kuribayashi, T; Yamamoto, S; Millar, B C; Moore, J E

2016-01-01

An arsenate susceptibility test was performed with transformed and cultured Escherichia coli DH5α cells, which carried recombinant DNA of full-length arsenic (ars) operon, namely a putative membrane permease, ArsP; a transcriptional repressor, ArsR; an arsenate reductase, ArsC; and an arsenical-resistance membrane transporter, Acr3, from the Japanese urease-positive thermophilic Campylobacter lari (UPTC) CF89-12. The E. coli DH5α transformant showed reduced susceptibility to arsenate (~1536 μg/mL), compared to the control. Thus, these ars four-genes from the UPTC CF89-12 strain cells could confer a reduced susceptibility to arsenate in the transformed and E. coli DH5α cells. E. coli transformants with truncated ars operons, acr3 (acr3) and arsC-acr3 (∆arsC-acr3), of the ars operon, showed an MIC value of 384 μg/mL (~384 μg/mL), similar to the E. coli cells which carried the pGEM-T vector (control). Reverse transcription PCR confirmed in vivo transcription of recombinant full-length ars operon and deletion variants (∆acr3 and ∆arsC-acr3) in the transformed E. coli cells.

Expression of an enzymatically active Yb3 glutathione S-transferase in Escherichia coli and identification of its natural form in rat brain.

Science.gov (United States)

Abramovitz, M; Ishigaki, S; Felix, A M; Listowsky, I

1988-11-25

Glutathione S-transferases containing Yb3 subunits are relatively uncommon forms that are expressed in a tissue-specific manner and have not been identified unequivocally or characterized. A cDNA clone containing the entire coding sequence of Yb3 glutathione S-transferase mRNA was incorporated into a pIN-III expression vector used to transform Escherichia coli. A fusion Yb3-protein containing 14 additional amino acid residues at its N terminus was purified to homogeneity. Recombinant Yb3 was enzymatically active with both 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates but lacked glutathione peroxidase activity. Substrate specificity patterns of recombinant Yb3 were more limited than those of glutathione S-transferase isoenzymes containing Yb1- or Yb2-type subunits. Peptides corresponding to unique amino acid sequences of Yb3 as well as a peptide from a region of homology with Yb1 and Yb2 subunits were synthesized. These synthetic peptides were used to raise antibodies specific to Yb3 and others that cross-reacted with all Yb forms. Immunoblotting was utilized to identify the natural counterpart of recombinant Yb3 among rat glutathione transferases. Brain and testis glutathione S-transferases were rich in Yb3 subunits, but very little was found in liver or kidney. Physical properties, substrate specificities, and binding patterns of the recombinant protein paralleled properties of the natural isoenzyme isolated from brain.

Hydrogen recombiner development at AECL

International Nuclear Information System (INIS)

Dewit, W.A.; Koroll, G.W.; Loesel Sitar, J.; Graham, W.R.C.

1997-01-01

Catalytic recombiners have been developed at AECL for the purpose of hydrogen removal in post-accident nuclear containment buildings. The recombiners are based on a particular catalyst designed by AECL which has extraordinary resistance to fouling from water and water vapour and a large thermodynamic range of operation. The catalysts were developed, originally, for the purpose of heavy water manufacturing by way of a catalytic exchange process. Application of these catalyst materials in recombiners for containment applications began in the late 1980's. The first application was a passive recombiner, qualified for use in control of radiolytic hydrogen in the headspace of a pool-type experimental reactor of AECL design in 1988. The passive, or natural convection recombiner concept has continued development to commercial stage for application in power reactor containments. This paper reviews the AECL recombiner development, describes the current model and shows results from tests of full-scale recombiners in the Large Scale Vented Combustion Test Facility at AECL-WL. The AECL recombiner is designed for compactness and ease of engineering into containment. The design is a simple, open-ended rectangular enclosure with catalyst elements arranged inside to promote optimum convective flow driven by heat of recombination at the catalyst surface. Self start, as evidenced by catalyst heating and initiation of flow, is achieved in less than 1% hydrogen, with available oxygen, at room temperature and 100% relative humidity. This low temperature start-up in condensing atmospheres is viewed as the most challenging condition for wet-proofing effectiveness. Cold start-up is a vital performance requirement in containments, such as CANDU, where engineered air-cooling systems are operating and where long-term hydrogen control is required, after containment atmospheres have cooled. Once started, the removal capacity scales linearly with the inlet cross-section area and the partial

On the relict recombination lines

International Nuclear Information System (INIS)

Bershtejn, I.N.; Bernshtejn, D.N.; Dubrovich, V.K.

1977-01-01

Accurate numerical calculation of intensities and profiles of hydrogen recombination lines of cosmological origin is made. Relie radiation distortions stipulated by recombination quantum release at the irrevocable recombination are investigated. Mean number calculation is given for guantums educing for one irrevocably-lost electron. The account is taken of the educed quantums interraction with matter. The main quantum-matter interrraction mechanisms are considered: electronic blow broadening; free-free, free-bound, bound-bound absorptions Recombination dynamics is investigated depending on hydrogen density and total density of all the matter kinds in the Universe

Multiplex polymerase chain reaction for identification of Escherichia coli, Escherichia albertii and Escherichia fergusonii.

Science.gov (United States)

Lindsey, Rebecca L; Garcia-Toledo, L; Fasulo, D; Gladney, L M; Strockbine, N

2017-09-01

Escherichia coli, Escherichia albertii, and Escherichia fergusonii are closely related bacteria that can cause illness in humans, such as bacteremia, urinary tract infections and diarrhea. Current identification strategies for these three species vary in complexity and typically rely on the use of multiple phenotypic and genetic tests. To facilitate their rapid identification, we developed a multiplex PCR assay targeting conserved, species-specific genes. We used the Daydreamer™ (Pattern Genomics, USA) software platform to concurrently analyze whole genome sequence assemblies (WGS) from 150 Enterobacteriaceae genomes (107 E. coli, 5 Shigella spp., 21 E. albertii, 12 E. fergusonii and 5 other species) and design primers for the following species-specific regions: a 212bp region of the cyclic di-GMP regulator gene (cdgR, AW869_22935 from genome K-12 MG1655, CP014225) for E. coli/Shigella; a 393bp region of the DNA-binding transcriptional activator of cysteine biosynthesis gene (EAKF1_ch4033 from genome KF1, CP007025) for E. albertii; and a 575bp region of the palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase (EFER_0790 from genome ATCC 35469, CU928158) for E. fergusonii. We incorporated the species-specific primers into a conventional multiplex PCR assay and assessed its performance with a collection of 97 Enterobacteriaceae strains. The assay was 100% sensitive and specific for detecting the expected species and offers a quick and accurate strategy for identifying E. coli, E. albertii, and E. fergusonii in either a single reaction or by in silico PCR with sequence assemblies. Published by Elsevier B.V.

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

Science.gov (United States)

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

2017-01-17

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

GENETIC CONTROL OF RESTRICTION AND MODIFICATION IN ESCHERICHIA COLI1

Science.gov (United States)

Boyer, Herbert

1964-01-01

Boyer, Herbert (Yale University, New Haven, Conn.). Genetic control of restriction and modification in Escherichia coli. J. Bacteriol. 88:1652–1660. 1964.—Bacterial crosses with K-12 strains of Escherichia coli as Hfr donors (Hfr Hayes, Hfr Cavalli, and Hfr P4X-6) and B/r strains of E. coli as F− recipients were found to differ from crosses between K-12 Hfr donors and K-12 F− recipients in two ways: (i) recombinants (leu, pro, lac, and gal) did not appear at discrete time intervals but did appear simultaneously 30 min after matings were initiated, and (ii) the linkage of unselected markers to selected markers was reduced. Integration of a genetic region linked to the threonine locus of K-12 into the B/r genome resulted in a hybrid which no longer gave anomalous results in conjugation experiments. A similar region of the B strain was introduced into the K-12 strain, which then behaved as a typical B F− recipient. These observations are interpreted as the manifestation of host-controlled modification and restriction on the E. coli chromosome. This was verified by experiments on the restriction and modification of the bacteriophage lambda, F-lac, F-gal, and sex-factor, F1. It was found that the genetic region that controlled the mating responses of the K-12 and B/r strains also controlled the modification and restriction properties of these two strains. The genes responsible for the restricting and modifying properties of the K-12 and B strains of E. coli were found to be allelic, linked to each other, and linked to the threonine locus. PMID:14240953

Late replicating domains are highly recombining in females but have low male recombination rates: implications for isochore evolution.

Directory of Open Access Journals (Sweden)

Catherine J Pink

Full Text Available In mammals sequences that are either late replicating or highly recombining have high rates of evolution at putatively neutral sites. As early replicating domains and highly recombining domains both tend to be GC rich we a priori expect these two variables to covary. If so, the relative contribution of either of these variables to the local neutral substitution rate might have been wrongly estimated owing to covariance with the other. Against our expectations, we find that sex-averaged recombination rates show little or no correlation with replication timing, suggesting that they are independent determinants of substitution rates. However, this result masks significant sex-specific complexity: late replicating domains tend to have high recombination rates in females but low recombination rates in males. That these trends are antagonistic explains why sex-averaged recombination is not correlated with replication timing. This unexpected result has several important implications. First, although both male and female recombination rates covary significantly with intronic substitution rates, the magnitude of this correlation is moderately underestimated for male recombination and slightly overestimated for female recombination, owing to covariance with replicating timing. Second, the result could explain why male recombination is strongly correlated with GC content but female recombination is not. If to explain the correlation between GC content and replication timing we suppose that late replication forces reduced GC content, then GC promotion by biased gene conversion during female recombination is partly countered by the antagonistic effect of later replicating sequence tending increase AT content. Indeed, the strength of the correlation between female recombination rate and local GC content is more than doubled by control for replication timing. Our results underpin the need to consider sex-specific recombination rates and potential covariates in

Construction of Methanol-Sensing Escherichia coli by the Introduction of a Paracoccus denitrificans MxaY-Based Chimeric Two-Component System.

Science.gov (United States)

Ganesh, Irisappan; Vidhya, Selvamani; Eom, Gyeong Tae; Hong, Soon Ho

2017-06-28

Escherichia coli was engineered to sense methanol by employing a chimeric two-component system (TCS) strategy. A chimeric MxaY/EnvZ (MxaYZ) TCS was constructed by fusing the Paracoccus denitrificans MxaY with the E. coli EnvZ. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed maximum transcription of ompC and the fluorescence at 0.01% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric MxaYZ. By using this strategy, various chimeric TCS-based bacterial biosensors can be constructed and used for the development of biochemical-producing recombinant microorganisms.

Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells

Energy Technology Data Exchange (ETDEWEB)

Lio, Yi-Ching; Schild, David; Brenneman, Mark A.; Redpath, J. Leslie; Chen, David J.

2004-05-01

The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, XRCC3) are expressed in mitotically growing cells, and are thought to play mediating roles in homologous recombination, though their precise functions remain unclear. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA cross-linking agent mitomycin C, and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G{sub 2}/M phases of the cell cycle but not in G{sub 1} phase. Together, these results provide direct cellular evidence for the importance of human Rad51C in homologous recombinational repair.

Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant Bacillus sp. strain BRC1 with increased inulinase activity.

Science.gov (United States)

Park, Jang Min; Oh, Baek-Rock; Kang, In Yeong; Heo, Sun-Yeon; Seo, Jeong-Woo; Park, Seung-Moon; Hong, Won-Kyung; Kim, Chul Ho

2017-07-01

A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L -1 . Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L -1 , showing a high theoretical yield of 92.3%.

Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

International Nuclear Information System (INIS)

Watanabe, Daisuke; Brockman, Mark A.; Ndung'u, Thumbi; Mathews, Lydia; Lucas, William T.; Murphy, Cynthia G.; Felber, Barbara K.; Pavlakis, George N.; Deluca, Neal A.; Knipe, David M.

2007-01-01

Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV gene products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli β-galactosidase induced durable β-gal-specific IgG and CD8 + T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes

Escherichia coli in broiler chickens with airsacculitis

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Leandro S. Machado

2014-09-01

Full Text Available ABSTRACT. Machado L.S., do Nascimento E.R., Pereira V.L.A., Abreu D.L.C., Gouvea R. & Santos L.M.M. 2014. [Escherichia coli in broiler chickens with airsacculitis.] Escherichia coli em frangos de corte com aerossaculite. Revista Brasileira de Medicina Veterinária, 36(3:261-265, 2014. Departamento de Medicina Veterinária Preventiva e Saúde Pública, Faculdade de Veterinária, Universidade Federal Fluminense, Rua Dr. Vital Brazil Filho 64, Vital Brazil, Niterói, RJ 24230-340, Brazil. E-mail: leandromachadovet@yahoo.com.br The Brazilian poultry industry grows each year and becomes increasingly representative in the production and export of products. The health care with poultry have accompanied and favored this evolution, however, respiratory agents that affect the weight and carcass quality, continue to cause great damage to the poultry industry. Airsacculitis is considered the main cause of total and partial condemnation of carcasses of broilers, and has been attributed to Mycoplasmosis mostly caused by Mycoplasma gallisepticum (MG and Mycoplasma synoviae (MS and Escherichia coli. The aim of this study was to relate the positivity of MG / MS and E. coli detected by PCR as a risk factor for airsacculitis in condemnation of broilers in Health Inspection Service. We studied 30 broiler poultry slaughtered in a slaughterhouse under Federal Sanitary Inspection, located in the State of Rio de Janeiro. 30 chickens were randomly collected from different lots and tracheas obtained in each PCR. DNA was extracted by phenol-chloroform method and amplified using pairs of “primer”specific for MG, MS and E. coli. Of the 30 chickens analyzed by PCR, 30% (9/30 had lesions in air sacs. None of the birds showed infection with MG and/or MS PCR, however 33.3% (3/9 birds were positive for airsacculitis iss gene from E.coli. E.coli found in broiler chickens that were negative for mycoplasma airsacculitis, implying the presence of such bacteria may be sufficient

21 CFR 866.3255 - Escherichia coli serological reagents.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from cultured...

Rhesus monkey rhadinovirus (RRV): construction of a RRV-GFP recombinant virus and development of assays to assess viral replication

International Nuclear Information System (INIS)

DeWire, Scott M.; Money, Eric S.; Krall, Stuart P.; Damania, Blossom

2003-01-01

Rhesus monkey rhadinovirus (RRV) is a γ-2-herpesvirus that is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). Lack of an efficient culture system to grow high titers of virus, and the lack of an in vivo animal model system, has hampered the study of KSHV replication and pathogenesis. RRV is capable of replicating to high titers on fibroblasts, thus facilitating the construction of recombinant rhadinoviruses. In addition, the ability to experimentally infect naieve rhesus macaques with RRV makes it an excellent model system to study γ-herpesvirus replication. Our study describes, for the first time, the construction of a GFP-expressing RRV recombinant virus using a traditional homologous recombination strategy. We have also developed two new methods for determining viral titers of RRV including a traditional viral plaque assay and a quantitative real-time PCR assay. We have compared the replication of wild-type RRV with that of the RRV-GFP recombinant virus in one-step growth curves. We have also measured the sensitivity of RRV to a small panel of antiviral drugs. The development of both the recombination strategy and the viral quantitation assays for RRV will lay the foundation for future studies to evaluate the contribution of individual genes to viral replication both in vitro and in vivo

The Walker A motif mutation recA4159 abolishes the SOS response and recombination in a recA730 mutant of Escherichia coli.

Science.gov (United States)

Šimatović, Ana; Mitrikeski, Petar T; Vlašić, Ignacija; Sopta, Mary; Brčić-Kostić, Krunoslav

2016-01-01

In bacteria, the RecA protein forms recombinogenic filaments required for the SOS response and DNA recombination. In order to form a recombinogenic filament, wild type RecA needs to bind ATP and to interact with mediator proteins. The RecA730 protein is a mutant version of RecA with superior catalytic abilities, allowing filament formation without the help of mediator proteins. The mechanism of RecA730 filament formation is not well understood, and the question remains as to whether the RecA730 protein requires ATP binding in order to become competent for filament formation. We examined two mutants, recA730,4159 (presumed to be defective for ATP binding) and recA730,2201 (defective for ATP hydrolysis), and show that they have different properties with respect to SOS induction, conjugational recombination and double-strand break repair. We show that ATP binding is essential for all RecA730 functions, while ATP hydrolysis is required only for double-strand break repair. Our results emphasize the similarity of the SOS response and conjugational recombination, neither of which requires ATP hydrolysis by RecA730. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A Chimeric protein of CFA/I, CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli.

Science.gov (United States)

Zeinalzadeh, Narges; Salmanian, Ali Hatef; Goujani, Goli; Amani, Jafar; Ahangari, Ghasem; Akhavian, Asal; Jafari, Mahyat

2017-07-01

Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti-CF and anti-enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti-CF and antitoxin immunogenicity was then assessed. To achieve high-level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti-CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Auger recombination in sodium iodide

Science.gov (United States)

McAllister, Andrew; Kioupakis, Emmanouil; Åberg, Daniel; Schleife, André

2014-03-01

Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

Regulation of homologous recombination in eukaryotes

OpenAIRE

Heyer, Wolf-Dietrich; Ehmsen, Kirk T.; Liu, Jie

2010-01-01

Homologous recombination is required for accurate chromosome segregation during the first meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage including DNA double-stranded breaks, interstrand crosslinks, and DNA gaps. In addition, recombination and replication are inextricably linked, as recombination recovers stalled and broken replication forks enabling the evolution of larger genomes/replicons. Defects in recombination lead to genomic instability and ...

Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma.

Science.gov (United States)

Lim, Chun Shen; Goh, Siang Ling; Kariapper, Leena; Krishnan, Gopala; Lim, Yat-Yuen; Ng, Ching Ching

2015-08-25

Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC). Thioredoxin fusion VCA p18 (VCA-Trx) and IBs of VCA p18 without fusion tags (VCA-IBs) were purified from E. coli. The diagnostic performances of IgG/VCA-IBs, IgG/VCA-Denat-IBs (using VCA-IBs coated in 8mol/l urea), IgG/VCA-Trx, and IgG/VCA-Peptide assays were compared by screening 100 NPC case-control pairs. The IgG/VCA-Denat-IBs assay showed the best area under the receiver operating characteristic curve (AUC: 0.802; p<0.05), while the AUCs for the IgG/VCA-IBs, IgG/VCA-Trx, and IgG/VCA-Peptide assays were comparable (AUC: 0.740, 0.727, and 0.741, respectively). We improved the diagnostic performance of the ELISA significantly using IBs of recombinant VCA p18. Copyright © 2015 Elsevier B.V. All rights reserved.

Application of dynamic flux balance analysis to an industrial Escherichia coli fermentation.

Science.gov (United States)

Meadows, Adam L; Karnik, Rahi; Lam, Harry; Forestell, Sean; Snedecor, Brad

2010-03-01

We have developed a reactor-scale model of Escherichia coli metabolism and growth in a 1000 L process for the production of a recombinant therapeutic protein. The model consists of two distinct parts: (1) a dynamic, process specific portion that describes the time evolution of 37 process variables of relevance and (2) a flux balance based, 123-reaction metabolic model of E. coli metabolism. This model combines several previously reported modeling approaches including a growth rate-dependent biomass composition, maximum growth rate objective function, and dynamic flux balancing. In addition, we introduce concentration-dependent boundary conditions of transport fluxes, dynamic maintenance demands, and a state-dependent cellular objective. This formulation was able to describe specific runs with high-fidelity over process conditions including rich media, simultaneous acetate and glucose consumption, glucose minimal media, and phosphate depleted media. Furthermore, the model accurately describes the effect of process perturbations--such as glucose overbatching and insufficient aeration--on growth, metabolism, and titer. (c) 2009 Elsevier Inc. All rights reserved.

Efficient production of xylitol from hemicellulosic hydrolysate using engineered Escherichia coli.

Science.gov (United States)

Su, Buli; Wu, Mianbin; Zhang, Zhe; Lin, Jianping; Yang, Lirong

2015-09-01

A metabolically engineered Escherichia coli has been constructed for the production of xylitol, one of the top 12 platform chemicals from agricultural sources identified by the US Department of Energy. An optimal plasmid was constructed to express xylose reductase from Neurospora crassa with almost no inclusion bodies at relatively high temperature. The phosphoenolpyruvate-dependent glucose phosphotransferase system (ptsG) was disrupted to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose. The native pathway for D-xylose catabolism in E. coli W3110 was blocked by deleting the xylose isomerase (xylA) and xylulose kinase (xylB) genes. The putative pathway for xylitol phosphorylation was also blocked by disrupting the phosphoenolpyruvate-dependent fructose phosphotransferase system (ptsF). The xylitol producing recombinant E. coli allowed production of 172.4 g L(-1) xylitol after 110 h of fed-batch cultivation with an average productivity of 1.57 g L(-1) h(-1). The molar yield of xylitol to glucose reached approximately 2.2 (mol xylitol mol(-1) glucose). Furthermore, the recombinant strain also produced about 150 g L(-1) xylitol from hemicellulosic sugars in modified M9 minimal medium and the overall productivity was 1.40 g L(-1) h(-1), representing the highest xylitol concentration and productivity reported to date from hemicellulosic sugars using bacteria. Thus, this engineered E. coli is a candidate for the development of efficient industrial-scale production of xylitol from hemicellulosic hydrolysate. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Continuous treatment process of mercury removal from aqueous solution by growing recombinant E. coli cells and modeling study

International Nuclear Information System (INIS)

Deng, X.; Hu, Z.L.; Yi, X.E.

2008-01-01

A continuous treatment process was developed to investigate the capability of genetically engineered E. coli to simultaneously accumulate mercuric ions and reproduce itself in a continuous stirred tank reactor (CSTR) system. The influence of dilution rate and initial Hg 2+ concentration on continuous process was evaluated. Results indicated that the recombinant E. coli could effectively accumulate Hg 2+ from aqueous solution with Hg 2+ removal ratio up to about 90%, and propagate its cells at the same time in the continuous treatment system under suitable operational conditions. A kinetic model based on mass balance of Hg 2+ was proposed to simulate the continuous process. The modeling results were in good agreement with the experimental data

Production of barley endoprotease B2 in Pichia pastoris and its proteolytic activity against native and recombinant hordeins

DEFF Research Database (Denmark)

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

2014-01-01

-terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni2+-affinity chromatography. Purified protein was evaluated by SDS–PAGE, Western blotting and activity assays...... was 60 °C, thermal stability T50 value was 44 °C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12 h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS–PAGE. The intensities of the B...... and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2 h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were...

Recombination coefficients in extrinsic n-InSb

International Nuclear Information System (INIS)

Schneider, W.; Groh, H.; Huebner, K.

1976-01-01

The bulk recombination coefficients for linear recombination via recombination centers as well as for direct recombination have been determined measuring the conductivity decay after two-photon absorption with a CO 2 laser. The Suhl effect was applied to measure the surface recombination velocity. The corresponding literature is discussed and compared with our results. We conclude that two different kinds of recombination centers are possible in n-InSb, with energy levels (0.1-0.12)eV above the valence band, or (0.14-0.2)eV respectively. (orig.) [de

Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation.

Science.gov (United States)

Jeevan, A; McFarland, C T; Yoshimura, T; Skwor, T; Cho, H; Lasco, T; McMurray, D N

2006-01-01

Gamma interferon (IFN-gamma) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-gamma (gpIFN-gamma) and reported that BCG vaccination induced a significant increase in the IFN-gamma mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-gamma mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-gamma with a histidine tag at the N terminus (His-tagged rgpIFN-gamma) in Escherichia coli. rgpIFN-gamma was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-gamma. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-gamma was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-gamma did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-gamma induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-gamma has been successfully produced and characterized in our laboratory. The study of rgpIFN-gamma will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.

Electron-ion recombination rates for merged-beams experiments

International Nuclear Information System (INIS)

Pajek, M.

1994-01-01

Energy dependence of the electron-ion recombination rates are studied for different recombination processes (radiative recombination, three-body recombination, dissociative recombination) for Maxwellian relative velocity distribution of arbitrary asymmetry. The results are discussed in context of the electron-ion merged beams experiments in cooling ion storage rings. The question of indication of a possible contribution of the three-body recombination to the measured recombination rates versus relative energy is particularly addressed. Its influence on the electron beam temperature derived from the energy dependence of recombination rate is discussed

Intensification of Organophosphorus Hydrolase Synthesis by Using Substances with Gas-Transport Function

Directory of Open Access Journals (Sweden)

Olga Senko

2017-12-01

Full Text Available We have performed studies and comparative analysis of the biosynthesis characteristics of intracellular recombinant enzyme, such as hexahistidine-containing organophosphorus hydrolase (His6-OPH in Escherichia coli SG13009[pREP4] cells when various perfluorocarbon compounds (PFC were introduced into the medium for cell cultivation. The PFC were found to facilitate the biosynthesis of His6-OPH: increased levels of the total OPH-activity (up to 37% were measured upon introduction of 1,1,1,2,2,3,3,4,4,5,5,6,6,6-tetradecafluorohexane (PFH and 4,7,10,13,16,19,22,25,28,31-decaoxaperfluoro-5,8,11,14,17,18,21,24,27,30-decamethyl tetratriacontane (Polyether II into culture medium. We have demonstrated the possibility of effective and multiple (at least five-fold use of PFH for biosynthesis of intracellular recombinant protein His6-OPH, which catalyzes the hydrolysis of organophosphorus pesticides (OP, is widely used in agriculture and can be applied as new antidote for OP-detoxification in vivo. The multiple use of PFH was achieved through recycling of this substance: sediment of Escherichia coli SG13009[pREP4] cell biomass was collected at the end of each culture growing step and disintegrated with ultrasound, and obtained residue containing almost all of the initially introduced PFC was then added to the medium at the start of the following culture growing step.

Production of 7,8-Dihydroxy Unsaturated Fatty Acids from Plant Oils by Whole Recombinant Cells Expressing 7,8-Linoleate Diol Synthase from Glomerella cingulata.

Science.gov (United States)

Seo, Min-Ju; Kang, Woo-Ri; Shin, Kyung-Chul; Oh, Deok-Kun

2016-11-16

The reaction conditions for the production of 7S,8S-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by recombinant Escherichia coli expressing 7,8-linoleate diol synthase from Glomerella cingulata were optimized using response surface methodology. The optimal reaction conditions were pH 7.0, 18.6 °C, 10.8% (v/v) dimethyl sulfoxide, 44.9 g/L cells, and 14.3 g/L linoleic acid, with agitation at 256 rpm. Under these conditions, recombinant cells produced 7,8-dihydroxy unsaturated fatty acids in the range of 7.0-9.8 g/L from 14.3 g/L linoleic acid, 14.3 g/L oleic acid, and plant oil hydrolysates such as waste oil and olive oil containing 14.3 g/L linoleic acid or oleic acid. To the best of the authors' knowledge, this is the first report on the biotechnological production of 7,8-dihydroxy unsaturated fatty acids.

Electron-ion recombination at low energy

International Nuclear Information System (INIS)

Andersen, L.H.

1993-01-01

The work is based on results obtained with a merged-beams experiment. A beam of electronics with a well characterized density and energy distribution was merged with a fast, monoenergetic ion beam. Results have been obtained for radiative recombination and dielectronic recombination at low relative energies (0 to ∼70eV). The obtained energy resolution was improved by about a factor of 30. High vacuum technology was used to suppress interactions with electrons from the environments. The velocity distribution of the electron beam was determined. State-selective dielectronic-recombination measurements were performable. Recombination processes were studied. The theoretical background for radiative recombination and Kramers' theory are reviewed. The quantum mechanical result and its relation to the semiclassical theory is discussed. Radiative recombination was also measured with several different non-bare ions, and the applicability of the semiclassical theory to non-bare ions was investigated. The use of an effective charge is discussed. For dielectronic recombination, the standard theoretical approach in the isolated resonance and independent-processes approximation is debated. The applicability of this method was tested. The theory was able to reproduce most of the experimental data except when the recombination process was sensitive to couplings between different electronic configurations. The influence of external perturbing electrostatic fields is discussed. (AB) (31 refs.)

Protective immunity induced by the vaccination of recombinant Proteus mirabilis OmpA expressed in Pichia pastoris.

Science.gov (United States)

Zhang, Yongbing; Yang, Shifa; Dai, Xiumei; Liu, Liping; Jiang, Xiaodong; Shao, Mingxu; Chi, Shanshan; Wang, Chuanwen; Yu, Cuilian; Wei, Kai; Zhu, Ruiliang

2015-01-01

Proteus mirabilis (P. mirabilis) is a zoonotic pathogen that has recently presented a rising infection rate in the poultry industry. To develop an effective vaccine to protect chickens against P. mirabilis infection, OmpA, one of the major outer membrane proteins of P. mirabilis, was expressed in Pichia pastoris. The concentration of the expressed recombinant OmpA protein reached 8.0μg/mL after induction for 96h with 1.0% methanol in the culture. In addition, OmpA protein was confirmed by SDS-PAGE and Western blot analysis using the antibody against Escherichia coli-expressed OmpA protein. Taishan Pinus massoniana pollen polysaccharide, a known plant-derived adjuvant, was mixed into the recombinant OmpA protein to prepare the OmpA subunit vaccine. We then subcutaneously inoculated this vaccine into chickens to examine the immunoprotective effects. ELISA analysis indicated that an excellent antibody response against OmpA was elicited in the vaccinated chickens. Moreover, a high protection rate of 80.0% was observed in the vaccinated group, which was subsequently challenged with P. mirabilis. The results suggest that the eukaryotic P. mirabilis OmpA was an ideal candidate protein for developing an effective subunit vaccine against P. mirabilis infection. Copyright © 2014 Elsevier Inc. All rights reserved.

Recombinant Innovation and Endogenous Transitions

OpenAIRE

Koen Frenken; Luis R. Izquierdo; Paolo Zeppini

2012-01-01

We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create “short-cuts” which reduce switching costs allowing agents to escape a technological lock-in. As a result, recombinant innovations speed up technological progress allowing transitions that are impossible with only branching ...

Calcium-dependent binding of Escherichia coli alpha-hemolysin to erythrocytes

International Nuclear Information System (INIS)

Boehm, D.F.

1989-01-01

Alpha hemolysin (AH), a protein secreted by certain strains of Escherichia coli, causes lysis of erythrocytes (RBCs) and is cytotoxic for other cells. The primary structure of AH contains an eight amino acid sequence tandemly repeated 13 times near the C-terminus. These repeated sequences are essential for hemolytic activity. AH also requires an unknown modification by an accessory protein, Hly C, for hemolytic activity. The role of calcium in the interaction of Ah with RBCs was investigated using recombinant strains which produced active and inactive forms of the toxin. Hemolytic activity was calcium-dependent. Osmotic protection experiments and immunoblots of SDS-PAGE separated proteins from washed, toxin-treated RBCs showed that the binding of active AH to RBCs was calcium-dependent. Binding of active AH to RBCs increased the calcium permeability of RBC membranes and resulted in changes in membrane protein profiles. The changes in membrane proteins did not cause the lysis of the cells. These results were consistent with a mechanism of lysis involving the formation of cation-selective pores in the membranes of target cells. 45 Ca-autoradiography of the recombinant hemolysins separated by SDS-PAGE and transferred to nitrocellulose showed that active AH bound calcium. The domain involved in binding calcium was identified as the tandemly repeated sequences since a deletion hemolysin missing 11 of the 13 repeated sequences did not bind calcium. This deletion hemolysin was non-hemolytic and did not bind to RBC membranes. Hemolysin lacking the Hly C modification was also non-hemolytic and did not bind to RBC membranes. This unmodified AH contained the repeated sequences and bound calcium as efficiently as active AH

Interface recombination influence on carrier transport

International Nuclear Information System (INIS)

Konin, A

2013-01-01

A theory of interface recombination in the semiconductor–semiconductor junction is developed. The interface recombination rate dependence on the nonequilibrium carrier densities is derived on the basis of a model in which the interface recombination occurs through the mechanism of trapping. The general relation between the interface recombination parameters at small carrier density deviation from the equilibrium ones is obtained. The validity of this relation is proved considering the generation of the Hall electric field in the extrinsic semiconductor sample. The anomalous Hall electromotive force in a weak magnetic field was investigated and interpreted by means of a new interface recombination model. The experimental data corroborate the developed theory. (paper)

Functionally undefined gene, yggE, alleviates oxidative stress generated by monoamine oxidase in recombinant Escherichia coli.

Science.gov (United States)

Ojima, Yoshihiro; Kawase, Daisuke; Nishioka, Motomu; Taya, Masahito

2009-01-01

Real-time PCR analysis showed that yggE gene was about two and three times up-regulated in Escherichia coli cells exposed to UVA irradiation and thermal elevation, respectively, suggesting that this gene is responsive to physiological stress. The yggE gene was introduced into E. coli BL21 cells, together with a monoamine oxidase (MAO) gene as a model source for oxidative stress generation. The distribution of independently isolated transformants (two dozen isolates) was examined in terms of MAO activity and cell vitality. In the case of control strain expressing MAO alone, the largest number of transformants existed in the low range of MAO activity less than 2 units mg(-1) and the number significantly decreased at increased MAO activity. On the other hand, the distribution of MAO/YggE-coexpressing transformants shifted to higher MAO activity with frequent appearance in the activity range of 4-8 units mg(-1). The yggE gene product therefore has a possible function for alleviating the stress generated in the cells.

Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

Science.gov (United States)

Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

2015-11-20

The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol. Copyright © 2015 Elsevier B.V. All rights reserved.

Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination.

Directory of Open Access Journals (Sweden)

Joseph A Ross

2011-07-01

Full Text Available The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.

PATHOGENIC POTENTIALS OF ESCHERICHIA COLI ISOLATED ...

African Journals Online (AJOL)

Electrolyte and haematological parameters in rabbits infected with pathogenic isolates of Escherichia coli from rural water supplies in Rivers State, Nigeria, where monitored. Rabbits were orally infected with suspension containing 3x107 cfu /ml of Escherichia coli to induce diarrhoea, and the electrolyte (sodium, potassium ...

Surface Enhanced Raman Scattering for Quantification of p-Coumaric Acid Produced by Escherichia coli

DEFF Research Database (Denmark)

Morelli, Lidia; Zor, Kinga; Jendresen, Christian Bille

2017-01-01

The number of newly developed genetic variants of microbial cell factories for production of biochemicals has been rapidly growing in recent years, leading to an increased need for new screening techniques. We developed a method based on surface-enhanced Raman scattering (SERS) coupled with liquid......-liquid extraction (LLE) for quantification of p-coumaric acid (pHCA) in the supernatant of genetically engineered Escherichia coli (E. coli) cultures. pHCA was measured in a dynamic range from 1 μM up to 50 μM on highly uniform SERS substrates based on leaning gold-capped nanopillars, which showed an in...

Role of glycolytic intermediate in regulation: Improving lycopene production in Escherichia coli by engineering metabolic control

Energy Technology Data Exchange (ETDEWEB)

Farmer, W.R.; Liao, J.C.

2001-06-01

illustrate an application of this approach for the enhanced production of lycopene in Escherichia coli. The chose lycopene as a model compound because of its potential beneficial effects on human health. Lycopene, being an effective antioxidant, has been proposed as a possible treatment for some cancers and other degenerative human conditions. As a result, the in vivo synthesis of lycopene and related carotenoids has received increasing attention, and a number of reports have described their production in recombinant microorganism.

Molecular requirements for radiation-activated recombination

International Nuclear Information System (INIS)

Stevens, Craig W.; Zeng Ming; Stamato, Thomas; Cerniglia, George

1997-01-01

Purpose/Objective: The major stumbling block to successful gene therapy today is poor gene transfer. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. We further hypothesized that known DNA-damage-repair proteins might also be important in radiation-activated recombination. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human (A549 and 39F) and rodent (NIH/3T3) cell lines. Continuous low dose rate and multiple radiation fractions were also tested. Nuclear extracts were made and the effect of irradiation on inter-plasmid recombination/ligation determined. Multiple DNA damage-repair deficient cell lines were tested for radiation-activated recombination. Results: A significant radiation dose-dependent improvement in stable plasmid transfection (by as much as 1300 fold) is demonstrated in neoplastic and primary cells. An improvement in transient plasmid transfection is also seen, with as much as 85% of cells transiently expressing b-galactosidase (20-50 fold improvement). Stable transfection is only improved for linearized or nicked plasmids. Cells have improved gene transfer for at least 96 hours after irradiation. Both fractionated and continuous low dose rate irradiation are effective at improving stable gene transfer in mammalian cells, thus making relatively high radiation dose delivery clinically feasible. Inter-plasmid recombination is radiation dose dependent in nuclear extract assays, and the type of overhang (3', 5' or blunt end) significantly affects recombination efficiency and the type of product. The most common end-joining activity involves filling-in of the overhang followed by blunt end ligation. Adenovirus is a linear, double stranded DNA virus. We demonstrate that adenoviral infection efficiency is increased by irradiation. The duration of transgene expression is lengthened because the virus integrates with high efficiency (∼10

A cheap, simple high throughput method for screening native Helicobacter pylori urease inhibitors using a recombinant Escherichia coli, its validation and demonstration of Pistacia atlantica methanolic extract effectivity and specificity.

Science.gov (United States)

Amar, Natalie; Peretz, Avi; Gerchman, Yoram

2017-02-01

Helicobacter pylori is the most frequent and persistent bacterial infection worldwide, and a risk factor for active gastritis, peptic ulcers, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Although combined antibiotics treatment is effective cases of antibiotic resistance are reported at an alarming rate. The H. pylori urease enzyme is essential for the bacteria establishment in the gastric mucosa, resulting urease inhibitors being sought after as effective and specific anti- H. pylori treatment. To-date, screening assays are based mostly on the analog plant urease enzyme but difference in properties of the plant and bacterial enzymes hamper these efforts. We have developed a screening assay based on recombinant Escherichia coli expressing native H. pylori urease, and validated this assay using thiourea and a methanolic extract of Pistacia atlantica. The assay demonstrated the thiourea and the extract to be potent urease inhibitors, with the extract having strong bacteriostatic activity against clinical isolates of H. pylori, including such with antibiotic resistance. The extract was also found to be neutral toward common probiotic bacteria, supporting its specificity and compatibility with digestive system desired microflora and suggesting it could be a good source for anti-H. pylori compounds. The assay has proven to be cheap, simple and native alternative to the plant enzyme based assay and could allow for high throughput screening for new urease inhibitors and could expedite screening and development of novel, better H. pylori remedies helping us to combat this infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

Science.gov (United States)

Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

2001-04-03

Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

Population inversion in recombining hydrogen plasma

International Nuclear Information System (INIS)

Furukane, Utaro; Yokota, Toshiaki; Oda, Toshiatsu.

1978-11-01

The collisional-radiative model is applied to a recombining hydrogen plasma in order to investigate the plasma condition in which the population inversion between the energy levels of hydrogen can be generated. The population inversion is expected in a plasma where the three body recombination has a large contribution to the recombining processes and the effective recombination rate is beyond a certain value for a given electron density and temperature. Calculated results are presented in figures and tables. (author)

Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

Science.gov (United States)

Kalscheuer, Rainer; Stöveken, Tim; Luftmann, Heinrich; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

2006-02-01

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

77 FR 9888 - Shiga Toxin-Producing Escherichia coli

Science.gov (United States)

2012-02-21

... Toxin-Producing Escherichia coli in Certain Raw Beef Products AGENCY: Food Safety and Inspection Service... toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145). This new date..., that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121...

In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation.

Science.gov (United States)

Kämpf, Michael M; Braun, Martin; Sirena, Dominique; Ihssen, Julian; Thöny-Meyer, Linda; Ren, Qun

2015-01-23

Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering. In this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production. The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step

Endonuclease IV of Escherichia coli is induced by paraquat

Energy Technology Data Exchange (ETDEWEB)

Chan, E.; Weiss, B.

1987-05-01

The addition of paraquat (methyl viologen) to a growing culture of Escherichia coli K-12 led within 1 hr to a 10- to 20-fold increase in the level of endonuclease IV, a DNase for apurinic/apyrimidinic sites. The induction was blocked by chloramphenicol. Increases of 3-fold or more were also seen with plumbagin, menadione, and phenazine methosulfate. H/sub 2/O/sub 2/ produced no more than a 2-fold increase in endonuclease IV activity. The following agents had no significant effect: streptonigrin, nitrofurantoin, tert-butyl hydroperoxide, ..gamma.. rays, 260-nm UV radiation, methyl methanesulfonate, mitomycin C, and ascorbate. Paraquat, plumbagin, menadione, and phenazine methosulfate are known to generate superoxide radical anions via redox cycling in vivo. A mutant lacking superoxide dismutase was unusually sensitive to induction by paraquat. In addition, endonuclease IV could be induced by merely growing the mutant in pure O/sub 2/. The levels of endonuclease IV in uninduced or paraquat-treated cells were unaffected by mutations of oxyR, a H/sub 2/O/sub 2/-inducible gene that governs an oxidative-stress regulon. The results indicate that endonuclease IV is an inducible DNA-repair enzyme and that its induction can be mediated via the production of superoxide radicals.

Endonuclease IV of Escherichia coli is induced by paraquat

International Nuclear Information System (INIS)

Chan, E.; Weiss, B.

1987-01-01

The addition of paraquat (methyl viologen) to a growing culture of Escherichia coli K-12 led within 1 hr to a 10- to 20-fold increase in the level of endonuclease IV, a DNase for apurinic/apyrimidinic sites. The induction was blocked by chloramphenicol. Increases of 3-fold or more were also seen with plumbagin, menadione, and phenazine methosulfate. H 2 O 2 produced no more than a 2-fold increase in endonuclease IV activity. The following agents had no significant effect: streptonigrin, nitrofurantoin, tert-butyl hydroperoxide, γ rays, 260-nm UV radiation, methyl methanesulfonate, mitomycin C, and ascorbate. Paraquat, plumbagin, menadione, and phenazine methosulfate are known to generate superoxide radical anions via redox cycling in vivo. A mutant lacking superoxide dismutase was unusually sensitive to induction by paraquat. In addition, endonuclease IV could be induced by merely growing the mutant in pure O 2 . The levels of endonuclease IV in uninduced or paraquat-treated cells were unaffected by mutations of oxyR, a H 2 O 2 -inducible gene that governs an oxidative-stress regulon. The results indicate that endonuclease IV is an inducible DNA-repair enzyme and that its induction can be mediated via the production of superoxide radicals

Consequences of recombination on traditional phylogenetic analysis

DEFF Research Database (Denmark)

Schierup, M H; Hein, J

2000-01-01

We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mt......DNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination....... With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may...

Fundamental Studies of Recombinant Hydrogenases

Energy Technology Data Exchange (ETDEWEB)

Adams, Michael W. [Univ. of Georgia, Athens, GA (United States)

2014-01-25

This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

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

Science.gov (United States)

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

2016-03-01

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

Molecular and Immunogenic Properties of Apyrase SP01B and D7-Related SP04 Recombinant Salivary Proteins of Phlebotomus perniciosus from Madrid, Spain

Directory of Open Access Journals (Sweden)

Inés Martín-Martín

2013-01-01

Full Text Available Sand fly salivary proteins are on the spotlight to become vaccine candidates against leishmaniasis and to markers of exposure to sand fly bites due to the host immune responses they elicit. Working with the whole salivary homogenate entails serious drawbacks such as the need for maintaining sand fly colonies and the laborious task of glands dissection. In order to overcome these difficulties, producing recombinant proteins of different vectors has become a major task. In this study, a cDNA library was constructed with the salivary glands of Phlebotomus perniciosus from Madrid, Spain, the most widespread vector of Leishmania infantum in the Mediterranean basin. Analysis of the cDNA sequences showed several polymorphisms among the previously described salivary transcripts. The apyrase SP01B and the D7-related protein SP04 were successfully cloned, expressed in Escherichia coli, and purified. Besides, recombinant proteins were recognized by sera of hamsters and mice previously immunized with saliva through the exposure to uninfected sand fly bites. These results suggest that these two recombinant proteins conserved their immunogenic properties after expression in a prokaryote system. Therefore, this work contributes to expand the knowledge of P. perniciosus saliva that would be eventually used for the development of tools for vector control programs.

Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

Science.gov (United States)

Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W.A.

2012-01-01

The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. PMID:23137753

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli.

Directory of Open Access Journals (Sweden)

Christiane Y Ozaki

Full Text Available Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv that were produced in E. coli against enterotoxins of ETEC strains.Recombinant scFv were developed against ETEC heat-labile toxin (LT and heat-stable toxin (ST, from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coli

International Nuclear Information System (INIS)

Gu, Jiang; Ji, Xiaowei; Qi, Jianxun; Ma, Ying; Mao, Xuhu; Zou, Quanming

2010-01-01

In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a β-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. The crystals belonged to space group I4 1 32, with unit-cell parameter a = 158.99 Å. The Matthews coefficient and solvent content were calculated to be 2.55 Å 3 Da −1 and 51.77%, respectively, for two molecules in the asymmetric unit

Popularity versus similarity in growing networks.

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Papadopoulos, Fragkiskos; Kitsak, Maksim; Serrano, M Ángeles; Boguñá, Marián; Krioukov, Dmitri

2012-09-27

The principle that 'popularity is attractive' underlies preferential attachment, which is a common explanation for the emergence of scaling in growing networks. If new connections are made preferentially to more popular nodes, then the resulting distribution of the number of connections possessed by nodes follows power laws, as observed in many real networks. Preferential attachment has been directly validated for some real networks (including the Internet), and can be a consequence of different underlying processes based on node fitness, ranking, optimization, random walks or duplication. Here we show that popularity is just one dimension of attractiveness; another dimension is similarity. We develop a framework in which new connections optimize certain trade-offs between popularity and similarity, instead of simply preferring popular nodes. The framework has a geometric interpretation in which popularity preference emerges from local optimization. As opposed to preferential attachment, our optimization framework accurately describes the large-scale evolution of technological (the Internet), social (trust relationships between people) and biological (Escherichia coli metabolic) networks, predicting the probability of new links with high precision. The framework that we have developed can thus be used for predicting new links in evolving networks, and provides a different perspective on preferential attachment as an emergent phenomenon.

Density dependence of dielectronic recombination in selenium

International Nuclear Information System (INIS)

Hagelstein, P.L.; Rosen, M.D.; Jacobs, V.L.

1986-01-01

Dielectronic recombination has been found to be the dominant recombination process in the determination of the ionization balance of selenium near the Ne-like sequence under conditions relevant to the exploding-foil EUV laser plasmas. The dielectronic recombination process tends to populate excited levels, and these levels in turn are more susceptible to subsequent excitation and ionization than are the ground-state ions. If one defines an effective recombination rate which includes, in addition to the primary recombination, the subsequent excitation and ionization of the additional excited-state population due to the primary recombination, then this effective recombination rate can be density-sensitive at relatively low electron density. We present results for this effective dielectronic recombination rate at an electron density of 3 x 10/sup 20/ electrons/cm 3 for recombination from Ne-like to Na-like selenium and from F-like to Ne-like selenium. In the former case, the effective recombination rate coefficient is found to be 1.8 x 10/sup -11/ cm 3 /sec at 1.0 keV, which is to be compared with the zero-density value of 2.8 x 10/sup -11/ cm 3 /sec. In the latter case (F-like to Ne-like), the effective recombination rate coefficient is found to be 1.3 x 10/sup -11/ cm 3 /sec, which is substantially reduced from the zero-density result of 3.3 x 10/sup -11/ cm 3 /sec. We have examined the effects of dielectronic recombination on the laser gain of the dominant Ne-like 3p-3s transitions and have compared our results with those presented by Whitten et al. [Phys. Rev. A 33, 2171 (1986)

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...

Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.

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Guillén-Chable, Francisco; Arenas-Sosa, Iván; Islas-Flores, Ignacio; Corzo, Gerardo; Martinez-Liu, Cynthia; Estrada, Georgina

2017-08-01

The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutations at the cysteine codons of the recA gene of Escherichia coli

International Nuclear Information System (INIS)

Weisemann, J.M.; Weinstock, G.M.

1988-01-01

Each of the three cysteine residues in the Escherichia coli RecA protein was replaced with a number of other amino acids. To do this, each cysteine codon was first converted to a chain-terminating amber codon by oligonucleotide-directed mutagenesis. These amber mutants were then either assayed for function in different suppressor strains or reverted by a second round of mutagenesis with oligonucleotides that had random sequences at the amber codon. Thirty-three different amino acid substitutions were obtained. Mutants were tested for three functions of RecA: survival following UV irradiation, homologous recombination, and induction of the SOS response. It was found that although none of the cysteines is essential for activity, mutations at each of these positions can affect one or more of the activities of RecA, depending on the particular amino acid substitution. In addition, the cysteine at position 116 appears to be involved in the RecA-promoted cleavage of the LexA protein

SequenceLDhot: detecting recombination hotspots.

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Fearnhead, Paul

2006-12-15

There is much local variation in recombination rates across the human genome--with the majority of recombination occurring in recombination hotspots--short regions of around approximately 2 kb in length that have much higher recombination rates than neighbouring regions. Knowledge of this local variation is important, e.g. in the design and analysis of association studies for disease genes. Population genetic data, such as that generated by the HapMap project, can be used to infer the location of these hotspots. We present a new, efficient and powerful method for detecting recombination hotspots from population data. We compare our method with four current methods for detecting hotspots. It is orders of magnitude quicker, and has greater power, than two related approaches. It appears to be more powerful than HotspotFisher, though less accurate at inferring the precise positions of the hotspot. It was also more powerful than LDhot in some situations: particularly for weaker hotspots (10-40 times the background rate) when SNP density is lower (maths.lancs.ac.uk/~fearnhea/Hotspot.

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

International Nuclear Information System (INIS)

Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui; Leppla, Stephen H.

2013-01-01

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

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

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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.

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.

Cloning and characterization of GDP-perosamine synthetase (Per) from Escherichia coli O157:H7 and synthesis of GDP-perosamine in vitro

International Nuclear Information System (INIS)

Zhao Guohui; Liu Jun; Liu Xiang; Chen Min; Zhang Houcheng; Wang, Peng George

2007-01-01

GDP-perosamine synthetase (Per, E.C. not yet classified) is important to the synthesis of Escherichia coli O157:H7 O-antigen. The mutant in per gene can disrupt the synthesis of O157 O-antigen. In this study, GDP-perosamine synthetase was cloned from E. coli O157:H7 and over-expressed in E. coli BL21 (DE3). The recombinant His-tagged Per fusion protein was a decamer with molecular weight of 431 kDa. The optimal pH value of this recombinant protein was 7.5. The divalent ions had no significant effect on Per-catalyzed reaction. The K m and K cat /K m for GDP-4-keto-6-deoxy-D-mannose were 0.09 mM and 2.1 x 10 5 M -1 S -1 , and those for L-glutamate were 2 mM and 0.52 x 10 5 M -1 S -1 , respectively. Per was used to synthesize GDP-perosamine from GDP-mannose together with recombinant GDP-mannose dehydratase (GMD, E.C. 4.2.1.47). The purified GDP-perosamine was identified by MS and NMR. In summary, this work provided a feasible approach for the synthesis of GDP-perosamine which can lead to the study of LPS biosynthesis of pathogenic E. coli O157:H7

Experimental evolution across different thermal regimes yields genetic divergence in recombination fraction but no divergence in temperature associated plastic recombination.

Science.gov (United States)

Kohl, Kathryn P; Singh, Nadia D

2018-04-01

Phenotypic plasticity is pervasive in nature. One mechanism underlying the evolution and maintenance of such plasticity is environmental heterogeneity. Indeed, theory indicates that both spatial and temporal variation in the environment should favor the evolution of phenotypic plasticity under a variety of conditions. Cyclical environmental conditions have also been shown to yield evolved increases in recombination frequency. Here, we use a panel of replicated experimental evolution populations of D. melanogaster to test whether variable environments favor enhanced plasticity in recombination rate and/or increased recombination rate in response to temperature. In contrast to expectation, we find no evidence for either enhanced plasticity in recombination or increased rates of recombination in the variable environment lines. Our data confirm a role of temperature in mediating recombination fraction in D. melanogaster, and indicate that recombination is genetically and plastically depressed under lower temperatures. Our data further suggest that the genetic architectures underlying plastic recombination and population-level variation in recombination rate are likely to be distinct. © 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.

Correlation between survival, ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in a rec- mutant of Escherichia coli K12

International Nuclear Information System (INIS)

Pirsel, M.; Slezarikova, V.

1977-01-01

A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA - ) or by chloramphenicol (CAP) treatment prior to UV irradiation (2.5 J m -2 ) increased more than tenfold the resistance of the strain Escherichia coli K12 SR19 to UV radiation. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. In Escherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation, and a higher stability of both parental and newly synthesized DNAs could be demonstrated. (author)

High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery.

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Turchetto, Jeremy; Sequeira, Ana Filipa; Ramond, Laurie; Peysson, Fanny; Brás, Joana L A; Saez, Natalie J; Duhoo, Yoan; Blémont, Marilyne; Guerreiro, Catarina I P D; Quinton, Loic; De Pauw, Edwin; Gilles, Nicolas; Darbon, Hervé; Fontes, Carlos M G A; Vincentelli, Renaud

2017-01-17

Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics. In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active. Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large

The extent and importance of intragenic recombination

Directory of Open Access Journals (Sweden)

de Silva Eric

2004-11-01

Full Text Available Abstract We have studied the recombination rate behaviour of a set of 140 genes which were investigated for their potential importance in inflammatory disease. Each gene was extensively sequenced in 24 individuals of African descent and 23 individuals of European descent, and the recombination process was studied separately in the two population samples. The results obtained from the two populations were highly correlated, suggesting that demographic bias does not affect our population genetic estimation procedure. We found evidence that levels of recombination correlate with levels of nucleotide diversity. High marker density allowed us to study recombination rate variation on a very fine spatial scale. We found that about 40 per cent of genes showed evidence of uniform recombination, while approximately 12 per cent of genes carried distinct signatures of recombination hotspots. On studying the locations of these hotspots, we found that they are not always confined to introns but can also stretch across exons. An investigation of the protein products of these genes suggested that recombination hotspots can sometimes separate exons belonging to different protein domains; however, this occurs much less frequently than might be expected based on evolutionary studies into the origins of recombination. This suggests that evolutionary analysis of the recombination process is greatly aided by considering nucleotide sequences and protein products jointly.

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

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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.

Recombination epoch revisited

International Nuclear Information System (INIS)

Krolik, J.H.

1989-01-01

Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons. 18 references

Recombination of Globally Circulating Varicella-Zoster Virus

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Depledge, Daniel P.; Kundu, Samit; Atkinson, Claire; Brown, Julianne; Haque, Tanzina; Hussaini, Yusuf; MacMahon, Eithne; Molyneaux, Pamela; Papaevangelou, Vassiliki; Sengupta, Nitu; Koay, Evelyn S. C.; Tang, Julian W.; Underhill, Gillian S.; Grahn, Anna; Studahl, Marie; Breuer, Judith; Bergström, Tomas

2015-01-01

ABSTRACT Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the