Jean-Michel Betton

Molecular chaperones and energy-dependent proteases are essential components of cellular protein quality control. Many of these proteins form heteroc

Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process.

Background: Heme is typically a major iron source for bacteria, but little is known about how bacteria of the Leptospira genus, composed of both saprophytic and pathogenic species, access heme. Results: In this study, we analysed a two-component system of the saprophyte Leptospira biflexa. In vitro 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 L. biflexa. The mutants could only grow in medium supplemented with hemin or δ-aminolevulinic acid (ALA). In the pathogen L. interrogans, the hklep and rrlep orthologous genes are located between hemE and hemL genes, which encode proteins involved in heme biosynthesis. The L. biflexa hklep mutant could be complemented with a replicative plasmid harbouring the L. interrogans 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. Conclusion: These findings demonstrate that the Hklep/Rrlep regulatory system is critical for the in vitro growth of L. biflexa, and suggest that this two-component system is involved in a complex mechanism that regulates the heme biosynthetic pathway.

The Escherichia coli Cpx envelope stress system is comprised of three proteins; the periplasmic regulatory CpxP, the inner membrane sensor kinase CpxA, and the cytoplasmic transcriptional activator CpxR. Although misfolded envelope proteins activate the Cpx system, the molecular mechanism by which this signal is sensed remains largely unknown. In an attempt to reconstitute the Cpx system from purified proteins, we failed to produce the small CpxP protein in its natural periplasmic compartment, but a high protein level was achieved when it was produced in the cytoplasm. Silent base mutations in the first codons of the cpxP gene encoding the signal sequence or substitution by two well-characterized signal sequences, those of MalE and DsbA, resulted in a large increase of the CpxP level in the periplasm. Our results support the hypothesis that periplasmic expression could be inhibited by sequence elements in the early coding signal sequence region of cpxP.

Summary: To examine the relationship between folding and aggregation in the periplasm of Escherichia coli, we have analysed the cellular fates of exported proteins fused to either the wild‐type maltose‐binding protein (MalE) or the aggregation‐prone variant MalE31. The propensity of fusion proteins to aggregate in the periplasm was determined by the intrinsic folding characteristics of the upstream protein. When β‐lactamase or alkaline phosphatase was linked to the C‐terminus of MalE31, the resultant fusion proteins accumulated in an insoluble form, but retained their catalytic activity. In addition, these protein aggregates induced an extracytoplasmic stress response, similar to unfused MalE31. However, using a fluorescent substrate, we found that alkaline phosphatase activity was present inside periplasmic aggregates. These results suggest that periplasmic inclusion body formation may result in intermolecular interactions between participating proteins without loss of function of the fused enzymes.

La production de protéines recombinantes est une biotechnologie qui entre dans une phase de maturité, entraînant ainsi un essor de la production industrielle. Cependant, le processus de conversion systématique de l’information génétique en protéine biologiquement active se heurte au problème fondamental du repliement cellulaire des protéines : de nombreuses protéines recombinantes ne sont pas produites dans leur état natif, et s’agrègent dans un état biologiquement inactif. Bien que ce phénomène d’agrégation présente certains avantages pratiques, la renaturation in vitro des protéines recombinantes, après solubilisation des agrégats cellulaires, revêt toujours un caractère empirique et aléatoire. Aussi est-il préférable d’intervenir en amont en optimisant les conditions d’expression de la protéine, afin de minimiser ce problème au sein des cellules. Dans ce domaine, l’alternative la plus prometteuse est certainement le développement des méthodes génétiques permettant le criblage à haut débit des protéines correctement repliées dans la cellule. The biotechnology of recombinant protein production is now entering its most advanced stage, and the growth of indusrial protein pharmaceuticals provides solid proof of this evolution. However, the sytematic 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 pratical 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.

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.