Use of pIVEX plasmids for protein overproduction in Escherichia coli

BACKGROUND: The pIVEX plasmids are vectors optimized for expression in the Rapid Translation System (RTS) cell-free system under control of bacteriophage T7 transcription elements. Even if these plasmids are intended for use in vitro, it is usually worthwhile to compare both cell-free and bacterial expression from the same genetic construct. However, some RTS users encountered problems when they introcuded these plasmids into Escherichia coli host strains producing the T7 RNA polymerase. RESULTS: We verified that difficulties in transforming the commonly used BL21(λDE3) strain with pIVEX arose from the presence of a strong T7 promoter combined with a high-copy number plasmid, independent of gene expression. When these vectors were introduced into this strain harboring a compatible plasmid carrying the lactose repressor (lacI), we improved the transformation efficiency by 4 orders of magnitude. Moreover, we designed a transformation protocol that allows, after induction, the overproduction of pIVEX-encoded proteins in the BL21(λDE3) strain. CONCLUSION: Using the correct plasmid/host combination and transformation-expression protocol, we could directly compare overproduction of the same pIVEX-encoded proteins from both in vivo and in vitro expression systems

Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Despite advances in expression technologies, the efficient production of heterologous secreted proteins in <it>Escherichia coli </it>remains a challenge. One frequent limitation relies on their inability to be exported to the <it>E. coli </it>periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process.</p> <p>Results</p> <p>In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an <it>E. coli </it>codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.l^-1of purified active LCI.</p> <p>Conclusion</p> <p>These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in <it>E. coli</it>.</p

Heme rescues a two-component system Leptospira biflexa mutant

<p>Abstract</p> <p>Background</p> <p>Heme is typically a major iron source for bacteria, but little is known about how bacteria of the <it>Leptospira </it>genus, composed of both saprophytic and pathogenic species, access heme.</p> <p>Results</p> <p>In this study, we analysed a two-component system of the saprophyte <it>Leptospira biflexa</it>. <it>In vitro </it>phosphorylation and site-directed mutagenesis assays showed that Hklep is a histidine kinase which, after autophosphorylation of a conserved histidine, transfers the phosphate to an essential aspartate of the response regulator Rrlep. Hklep/Rrlep two-component system mutants were generated in <it>L. biflexa</it>. The mutants could only grow in medium supplemented with hemin or δ-aminolevulinic acid (ALA). In the pathogen <it>L. interrogans</it>, the <it>hklep </it>and <it>rrlep </it>orthologous genes are located between <it>hemE </it>and <it>hemL </it>genes, which encode proteins involved in heme biosynthesis. The <it>L. biflexa hklep </it>mutant could be complemented with a replicative plasmid harbouring the <it>L. interrogans </it>orthologous gene, suggesting that these two-component systems are functionally similar. By real-time quantitative reverse transcription-PCR, we also observed that this two-component system might influence the expression of heme biosynthetic genes.</p> <p>Conclusion</p> <p>These findings demonstrate that the Hklep/Rrlep regulatory system is critical for the <it>in vitro </it>growth of <it>L. biflexa</it>, and suggest that this two-component system is involved in a complex mechanism that regulates the heme biosynthetic pathway.</p

Identification, recombinant production and partial biochemical characterization of an extracellular cold-active serine-metalloprotease from an Antarctic Pseudomonas isolate

Cold-adapted enzymes are generally derived from psychrophilic microorganisms and have features that make them very attractive for industrial and biotechnological purposes. In this work, we identified a 50 kDa extracellular protease (MP10) from the Antarctic isolate Pseudomonas sp. AU10. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and partially characterized. MP10 is an alkaline thermosensitive serine-metallo protease with optimal activity at pH 8.0 and 40 ℃, in the presence of 1.5 mM Ca2+. MP10 showed 100% residual activity and stability (up to 60 min) when incubated with 7% of non-ionic surfactants (Triton X-100, Tween-80 and Tween-20) and 1.5% of the oxidizing agent hydrogen peroxide. The 3D MP10 structure was predicted and compared with the crystal structure of mesophilic homologous protease produced by Pseudomonas aeruginosa PA01 (reference strain) and other proteases, showing similarity in surface area and volume of proteins, but a significantly higher surface pocket area and volume of MP10. The observed differences presumably may explain the enhanced activity of MP10 for substrate binding at low temperatures. These results give insight to the potential use of MP10 in developing new biotechnologically processes active at low to moderate temperatures, probably with focus in the detergent industry

Etude de la transition conformationnelle de la phosphoglycerate kinase

SIGLECNRS T 55586 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Repliement et production de proteines recombinantes

The biotechnology of recombinant protein production is now entering its most advanced stage, and the growth of industrial protein pharmaceuticals provides solid proof of this evolution. However, the systematic conversion of genetic information into a biologically active protein is constantly confronted by the fundamental problem of protein folding in cells, and many recombinant proteins are not produced in their native state. Instead, they aggregate into a biologically inactive state. Although this aggregation reaction has some practical advantages, in vitro renaturation of recombinant proteins, after solubilization of cellular aggregates, is still an empiric and random process. Thus, it is better to control cellular expression conditions to minimize this problem inside the cells. The most attractive approach is certainly the development of high throughput genetic screens to monitor efficient protein folding

Etude du repliement incorrect des protéines dans le périplasme d'Escherichia coli

Dans un contexte cellulaire, le repliement et le mauvais repliement dépendent de différents facteurs comme la séquence en acides aminés, la translocation, la présence de chaperons, de catalyseurs du repliement ou encore de protéases. L'objectif de ce travail a consisté à rechercher des facteurs contrôlant le repliement incorrect de MalE31, notre modèle d'étude dans la bactérie Escherichia coli. Les facteurs capables de prévenir l'agrégation cellulaire de MalE31 peuvent être de deux types, les suppresseurs intragéniques et les suppresseurs extragéniques. Les suppresseurs extragéniques désignent les acteurs de la machinerie cellulaire dévouée à éviter l'accumulation et l'agrégation des protéines en cours de repliement. En utilisant MalE31 comme modèle nous avons montré; que la protéine FkpA agit comme un chaperon moléculaire capable d'empêcher la formation de corps d'inclusion; que la protéase DegP possède une structure oligomérique et dégrade les intermédiaires de mauvaises conformations. L'oligomérisation et la dégradation de la protéase dépendent de la présence de domaines PDZ. Dans le but d'éviter l'agrégation de MalE31, nous recherchons aussi des suppresseurs intragéniques du mauvais repliement des protéines. Or, en étudiant le comportement de protéines fusionnées à MalE31, nous avons mis en évidence l'importance de l'ordre d'émergence des partenaires fusionnés dans le périplasme pour la solubilité de la protéine chimère. Nous recherchons à l'heure actuelle, par la technique du phage display, des acides aminés susceptibles de " corriger " les pièges cinétiques à l'origine de l'agrégation de MalE31. Les premières expériences allant dans ce sens sont présentées ici. La nature de ces suppresseurs intragéniques ou extragéniques est susceptible de nous aider à comprendre la compétition cinétique existante entre l'agrégation, la dégradation et le repliement dans le contexte cellulaire, et les mécanismes mis en place par les cellules pour se protéger du mauvais repliement.Protein folding and misfolding both depend of different parameters such amino acid sequence, the protein translocation or the presence of molecular chaperones or proteases. The aim of our work is to identify factors that are able to prevent protein aggregation into the periplasm of Escherichia coli. The nature of these factors can be either extragenic or intragenic. In this study we used as a model protein a periplasmic prone-to-aggregation protein, MalE31. Extragenic suppressors designate proteins that belong to the cellular machinery and involved in the protection against protein aggregation. Checking the MalE31 aggregation we found that; FkpA act as a molecular chaperone and suppresses the inclusion body formation; the DegP protease possesses an oligomeric quaternary structure and degrades misfolded intermediates; both the degradation and the oligomerisation of DegP require the presence of some PDZ domains. In order to investigate the influence of primary structure on folding we have designed some protein fusion that contain MalE31. We have demonstrated that the fusion order is critical for the solubility of the whole fusion and that some correctly folded proteins can be embedded into an inclusion granule. Moreover, using the phage display strategy, we are now trying to found some amino acids that can correct MalE31 misfolding by preventing kinetic traps responsible for the aggregation. The first experiments concerning this topic are presented here. The identification of intragenic and extragenic suppressors could led us understand the kinetic competition that exists in vivo on protein folding, degradation and aggregation, and the way that cells protect themselves against misfolding.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Etude de deux acteurs majeurs des voies de réponse au stress d'enveloppe chez Escherichia coli (la protéase - chaperon périplasmique DegP et l'histidine kinase membranaire CpxA)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF