François Baneyx

Recombinant protein expression in Escherichia coli

Escherichia coli is one of the most widely used hosts for the production of heterologous proteins and its genetics are far better characterized than those of any other microorganism. Recent progress in the fundamental understanding of transcription, translation, and protein folding in E. coli, together with serendipitous discoveries and the availability of improved genetic tools are making this bacterium more valuable than ever for the expression of complex eukaryotic proteins.

Recombinant protein folding and misfolding in Escherichia coli

The past 20 years have seen enormous progress in the understanding of the mechanisms used by the enteric bacterium Escherichia coli to promote protein folding, support protein translocation and handle protein misfolding. Insights from these studies have been exploited to tackle the problems of inclusion body formation, proteolytic degradation and disulfide bond generation that have long impeded the production of complex heterologous proteins in a properly folded and biologically active form. The application of this information to industrial processes, together with emerging strategies for creating designer folding modulators and performing glycosylation all but guarantee that E. coli will remain an important host for the production of both commodity and high value added proteins.

Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli. To fold or to refold.

The high-level expression of recombinant gene products in the gramnegative bacteriumEscherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular proteases or its deposition into biologically inactive aggregates known as inclusion bodies. It has recently become clear that in vivo protein folding is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE and GroEL-GroES systems, suppress off-pathway aggregation reactions and facilitate proper folding through ATP-coordinated cycles of binding and release of folding intermediates. On the other hand, folding catalysts (foldases) accelerate rate-limiting steps along the protein folding pathway such as thecis/trans isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges. Manipulating the cytoplasmic folding environment by increasing the intracellular concentration of all or specific folding modulators, or by inactivating genes encoding these proteins, holds great promise in facilitating the production and purification of heterologous proteins. Purified folding modulators and artificial systems that mimic their mode of action have also proven useful in improving the in vitro refolding yields of chemically denatured polypeptides. This review examines the usefulness and limitations of molecular chaperones and folding catalysts in both in vivo and in vitro folding processes.

Biomineralization and Size Control of Stable Calcium Phosphate Core Protein Shell Nanoparticles: Potential for Vaccine Applications

Calcium phosphate (CaP) polymorphs are nontoxic, biocompatible and hold promise in applications ranging from hard tissue regeneration to drug delivery and vaccine design. Yet, simple and robust routes for the synthesis of protein-coated CaP nanoparticles in the sub-100 nm size range remain elusive. Here, we used cell surface display to identify disulfide-constrained CaP binding peptides that, when inserted within the active site loop of Escherichia coli thioredoxin 1 (TrxA), readily and reproducibly drive the production of nanoparticles that are 50-70 nm in hydrodynamic diameter and consist of an approximately 25 nm amorphous calcium phosphate (ACP) core stabilized by the protein shell. Like bone and enamel proteins implicated in biological apatite formation, peptides supporting nanoparticle production were acidic. They also required presentation in a loop for high-affinity ACP binding as elimination of the disulfide bridge caused a nearly 3-fold increase in hydrodynamic diameters. When compared to a commercial aluminum phosphate adjuvant, the small core-shell assemblies led to a 3-fold increase in mice anti-TrxA titers 3 weeks postinjection, suggesting that they might be useful vehicles for adjuvanted antigen delivery to dendritic cells.

ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells

DnaK–DnaJ–GrpE and GroEL–GroES are the best‐characterized molecular chaperone systems in the cytoplasm of Escherichia coli. A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear. Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat‐shock conditions. We show that the absence of either ClpB or HtpG at 42°C leads to increased aggregation of preS2‐β‐galactosidase, a fusion protein whose folding depends on DnaK–DnaJ–GrpE, but not GroEL–GroES. However, only the ΔclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL–GroES chaperonins to reach a proper conformation. Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates. Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation‐prone protein preS2‐S′‐β‐galactosidase in wild‐type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants. By contrast, higher levels of DnaK–DnaJ, but not GroEL–GroES, restore efficient folding in ΔclpB cells. These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E. coli cells, presumably by expanding the ability of the DnaK–DnaJ–GrpE team to interact with newly synthesized polypeptides.

The 1.6-Å crystal structure of the class of chaperones represented by Escherichia coli Hsp31 reveals a putative catalytic triad

Heat shock proteins (Hsps) play essential protective roles under stress conditions by preventing the formation of protein aggregates and degrading misfolded proteins. EcHsp31, the yedU (hchA) gene product, is a representative member of a family of chaperones that alleviates protein misfolding by interacting with early unfolding intermediates. The 1.6-Å crystal structure of the EcHsp31 dimer reveals a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded substrate. The presence of a well conserved, yet buried, triad in each two-domain subunit suggests a still unproven hydrolytic function of the protein. A flexible extended linker between the A and P domains may play a role in conformational flexibility and substrate binding. The α-β sandwich of the EcHsp31 monomer shows structural similarity to PhPI, a protease belonging to the DJ-1 superfamily. The structure-guided sequence alignment indicates that Hsp31 homologs can be divided in three classes based on variations in the P domain that dramatically affect both oligomerization and catalytic triad formation.

Biochemical Characterization of the Small Heat Shock Protein IbpB from Escherichia coli

Escherichia coli IbpB was overexpressed in a strain carrying a deletion in the chromosomalibp operon and purified by refolding. Under our experimental conditions, IbpB exhibited pronounced size heterogeneity. Basic oligomers, roughly spherical and approximately 15 nm in diameter, interacted to form larger particles in the 100–200-nm range, which themselves associated to yield loose aggregates of micrometer size. IbpB suppressed the thermal aggregation of model proteins in a concentration-dependent manner, and its CD spectrum was consistent with a mostly β-pleated secondary structure. Incubation at high temperatures led to a partial loss of secondary structure, the progressive exposure of tryptophan residues to the solvent, the dissociation of high molecular mass aggregates into ≈600-kDa oligomers, and an increase in surface hydrophobicity. Structural changes were reversible between 37 and 55 °C, and, up to 55 °C, hydrophobic sites were reburied upon cooling. IbpB exhibited a biphasic unfolding trend upon guanidine hydrochloride (GdnHCl) treatment and underwent comparable conformational changes upon melting and during the first GdnHCl-induced transition. However, hydrophobicity decreased with increasing GdnHCl concentrations, suggesting that efficient exposure of structured hydrophobic sites involves denaturant-sensitive structural features. By contrast, IbpB hydrophobicity rose at high NaCl concentrations and increased further at high temperatures. Our results support a model in which temperature-driven conformational changes lead to the reversible exposure of normally shielded binding sites for nonnative proteins and suggest that both hydrophobicity and charge context may determine substrate binding to IbpB.

Hyperosmotic shock induces the sigma32 and sigmaE stress regulons of Escherichia coli.

The rise in the levels of σS that accompanies hyperosmotic shock plays an important role in Escherichia coli survival by increasing the transcription of genes involved in the synthesis and transport of osmoprotectants. To determine if other stress regulons collaborate with σS in dealing with high osmolality, we used single copy fusions of lacZ to representative promoters induced by protein misfolding in the cytoplasm (dnaK and ibp ), extracytoplasmic stress [P3rpoH and htrA(degP )] and cold shock (cspA). Both the σ32‐dependent, dnaK and ibp, promoters, and the σE‐dependent, P3rpoH and htrA, promoters were rapidly but transiently induced when mid‐exponential phase cells were treated with 0.464 M sucrose. The cspA promoter, however, did not respond to the same treatment. Overproduction of the cytoplasmic domain of the σE anti‐sigma factor, RseA, reduced the magnitude of osmotic induction in λφ(P3rpoH::lacZ ) lysogens, but had no effect on the activation of the dnaK and ibp promoters. Similarly, induction of the dnaK::lacZ and ibp::lacZ fusions was not altered in either rpoS or ompR genetic backgrounds. Osmotic upshift led to a twofold increase in the enzymatic activity of the λTLF247 rpoH::lacZ translational fusion whether or not the cells were treated with rifampicin, indicating that both heat shock and exposure to high osmolality trigger a transient increase in rpoH translation. Our results suggest that the σ32, σE and σS regulons closely co‐operate in the managment of hyperosmotic stress. Induction of the σ32 and σE regulons appears to be an emergency response required to repair protein misfolding and facilitate the proper folding of proteins that are rapidly synthesized following loss of turgor, while providing a mechanism to increase the activity of σS, the primary stress factor in osmoadaptation.

Stress-based identification and classification of antibacterial agents: Second-generation Escherichia coli reporter strains and optimization of detection

ABSTRACT Escherichia coli strains bearing single-copy fusions between the lacZ reporter gene and the cspA, ibp, or P3rpoH stress promoters offer a simple means to detect sublethal concentrations of antibacterial agents interfering with prokaryotic translation or cell envelope integrity while simultaneously providing information on the mechanism of action of the test compound (A. A. Bianchi and F. Baneyx, Appl. Environ. Microbiol. 65:5023-5027, 1999). Here, we expand the usefulness of this system by (i) demonstrating that a fusion between the SOS-inducible sulA promoter and lacZ is a highly specific probe for the detection of antimicrobial agents that ultimately interfere with DNA replication, (ii) showing that inactivation of the tolC gene allows efficient detection of very low concentrations of model antibiotics (including aminoglycosides) whereas polymyxin B-mediated outer membrane permeabilization facilitates the identification of intermediate concentrations of hydrophobic compounds, and (iii) validating the potential of detector strains and sensitization strategies for high-throughput screening using a reproducible and internally consistent 96-well microplate assay.

Protein folding in the cytoplasm of Escherichia coli: requirements for the DnaK‐DnaJ‐GrpE and GroEL‐GroES molecular chaperone machines

We have systematically investigated the influence of mutations in the σ32 heat‐shock transcription factor and the DnaK‐DnaJ‐GrpE and GroEL‐GroES molecular chaperone machines on the folding of preS2‐β‐galactosidase. This 120kDa fusion protein between the hepatitis B surface antigen preS2 sequence and β‐galactosidase was synthesized in a highly soluble and enzymatically active form in wild‐type Escherichia coli cells cultured at temperatures between 30°C and 42°C, but aggregated extensively in an rpoH165(Am) mutant. Proper folding was partially restored upon co‐overexpression of the dnaKJ operon, but not when the groE operon or dnaK alone were overproduced. The enzymatic activities in dnaK103, dnaJ259 and grpE280 mutants were 40–60% lower relative to a dnaK756 mutant or isogenic wild‐type cells at 30°C and 37°C. At 42°C, only 10–40% of the wild‐type activity was present in each of the early‐folding‐factor mutants. Although the synthesis levels of preS2‐β‐galactosidase were reduced in the dnaK103, dnaJ259 and grpE280 genetic backgrounds, aggregation was primarily responsible for the loss of activity when the cells were grown at 37°C or 42°C. By contrast, the groEL140, groES30 and groES619 mutations, which induced the aggregation of homodimeric ribulose bisphosphate carboxylase (Rubisco), did not affect the solubility of preS2‐β‐galactosidase at temperatures up to 42°C. Our results are discussed in terms of the current understanding of the E. coli protein‐folding cascade. The potential usefulness of heat‐shock protein mutants for the production of soluble proteins in an inclusion‐body form is addressed.