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We have systematically investigated the influence of mutations in the sigma(32) heat-shock transcription factor and the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines on the folding of preS2-beta-galactosidase. This 120 kDa fusion protein between the hepatitis B surface antigen preS2 sequence and beta-galactosidase was synthesized in a highly soluble and enzymatically active form in wild-type Escherichia coli cells cultured at temperatures between 30 degrees C and 42 degrees 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 degrees C and 37 degrees C. At 42 degrees 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-beta-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 degrees C or 42 degrees 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-beta-galactosidase at temperatures up to 42 degrees 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.

PreS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively in the cytoplasm of Escherichia coli, was used to systematically investigate the effects of heat-shock protein (hsp) overproduction on protein misfolding and inclusion body formation. While the co-overexpression of the DnaK and DnaJ molecular chaperones led to a 3-6 fold increase in the recovery of enzymatically active preS2-S'-beta-galactosidase over a wide range of growth temperatures (30-42 degrees C), an increase in the concentration of the GroEL and GroES chaperonins had a significant effect at 30 degrees C only. Co-immunoprecipitation experiments confirmed that preS2-S'-beta-galactosidase formed a stable complex with DnaK, but not with GroEL, at 42 degrees C. When the intracellular concentration of chromosomal heat-shock proteins was increased by overproduction of the heat-shock transcription factor sigma 32, or by addition of 3% ethanol (v/v) to the growth medium, a 2-3 fold higher recovery of active enzyme was observed at 30 and 42 degrees C, but not at 37 degrees C. The overexpression of all heat-shock proteins or specific chaperone operons did not significantly affect the synthesis rates or stability of preS2-S'-beta-galactosidase and did not lead to the disaggregation of preformed inclusion bodies. Rather, the improvements in the recovery of soluble and active fusion protein resulted primarily from facilitated folding and assembly. Our findings suggest that titration of the DnaK-DnaJ early folding factors leads to the formation of preS2-S'-beta-galactosidase inclusion bodies.

Chloroplasts contain a 21-kDa co-chaperonin polypeptide (cpn21) formed by two GroES-like domains fused together in tandem. Expression of a double-domain spinach cpn21 in Escherichia coli groES mutant strains supports growth of bacteriophages lambda and T5, and will also suppress a temperature-sensitive growth phenotype of a groES619 strain. Each domain of cpn21 expressed separately can function independently to support bacteriophage lambda growth, and the N-terminal domain will additionally suppress the temperature-sensitive growth phenotype. These results indicate that chloroplast cpn21 has two functional domains, either of which can interact with GroEL in vivo to facilitate bacteriophage morphogenesis. Purified spinach cpn21 has a ring-like toroidal structure and forms a stable complex with E. coli GroEL in the presence of ADP and is functionally interchangeable with bacterial GroES in the chaperonin-facilitated refolding of denatured ribulose-1,5-bisphosphate carboxylase. Cpn21 also inhibits the ATPase activity of GroEL. Cpn21 binds with similar efficiency to both the alpha and beta subunits of spinach cpn60 in the presence of adenine nucleotides, with ATP being more effective than ADP. The tandemly fused domains of cpn21 evolved early and are present in a wide range of photosynthetic eukaryotes examined, indicating a high degree of conservation of this structure in chloroplasts.

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Direct-write nanomanufacturing with scanning beams and probes is flexible and can produce high quality products, but it is normally slow and expensive to raster point-by-point over a pattern. We demonstrate the use of an accelerated direct-write nanomanufacturing method called 'orchestrated structure evolution' (OSE), where a direct-write tool patterns a small number of growth 'seeds' that subsequently grow into the final thin film pattern. Through control of seed size and spacing, it is possible to vary the ratio of 'top-down' to 'bottom-up' character of the patterning processes, ranging from conventional top-down raster patterning to nearly pure bottom-up space-filling via seed growth. Electron beam lithography (EBL) and copper electrodeposition were used to demonstrate trade-offs between process time and product quality over nano- to microlength scales. OSE can reduce process times for high-cost EBL patterning by orders of magnitude, at the expense of longer (but inexpensive) copper electrodeposition processing times. We quantify the degradation of pattern quality that accompanies fast OSE patterning by measuring deviations from the desired patterned area and perimeter. We also show that the density of OSE-induced grain boundaries depends upon the seed separation and size. As the seed size is reduced, the uniformity of an OSE film becomes more dependent on details of seed nucleation processes than normally seen for conventionally patterned films.

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The Escherichia coli outer membrane protease OmpT (protease VII) has been shown to degrade several proteins in vitro, but its function in vivo is uncertain. We demonstrate that OmpT participates in the degradation of a fusion protein secreted into the periplasmic space. A strain with mutations in degP (K.L. Strauch and J. Beckwith, Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988) and ompT exhibits a cumulative decrease in protein degradation and should be useful for the expression of proteolytically sensitive secreted proteins.

Protein folding and misfolding in the cellular environment has emerged as a major research theme over the past fifteen years. The implications are broad, ranging from the production of recombinant proteins in a soluble and bioactive form to the understanding of the etiology of neurodegenerative diseases. The basic principle enunciated by Afinsen – that all the information required for a polypeptide to reach a proper conformation is contained within its amino acid sequence [1] – remains unchallenged. However, it is now well established that many proteins require the assistance of folding modulators to adopt a native structure as they emerge from ribosomes, regain their original conformation following exposure to stress, or traffic to their ultimate destination [2]. One of the most extensively characterized group of folding modulators are molecular chaperones, a ubiquitous class of proteins that help other polypeptides reach a proper conformation or cellular location without affecting folding rates or becoming part of the final structure. Molecular chaperones function by transiently binding hydrophobic stretches of amino acids that should normally be buried within the core of their substrates but have become solvent-exposed due to improper de novo folding or imposition of cellular stress (e.g., temperature increase). As a result of the formation of chaperone-substrate complexes, interactive species are shielded from each other and from the solvent and "on-pathway" folding is facilitated. Three functionally distinct sets of chaperones cooperate in the conformational quality control of the proteome. At the heart of chaperone networks are "folding chaperones" which use conformational changes fueled by ATP hydrolysis to promote the net refolding/unfolding of bound substrates. Archetypal folding chaperones are the DnaK-DnaJ-GrpE (Hsp70-Hsp40) and GroEL-GroES (Hsp60-Hsp10) systems of E. coli. "Holding chaperones" (e.g., small heat shock proteins) passively stabilize partially folded species until folding chaperones become available for their refolding. In doing so, they alleviate titration of folding chaperones by unfolding intermediates. Finally, "disaggregating chaperones" such as E. coli ClpB and S. cerevisiae Hsp104 employ ATP-driven conformational changes to solubilize protein aggregates and transfer partially folded species to folding chaperones for subsequent refolding. Foldases embody the second group of folding modulators. These enzymes accelerate rate-limiting steps along the folding pathway and include peptidyl-prolyl cis/trans isomerases (PPIases), which catalyze the trans to cis isomerization of peptidyl-prolyl bonds, and thiol-disulfide oxidoreductases, which promote the formation and isomerization of disulfide bridges (e.g., the Dsb proteins of the E. coli periplasm). The final participants in the protein quality control machinery are proteases which degrade irreversibly damaged polypeptides that cannot be rescued by the above pathways. Nevertheless, nature loves to frustrate reductionist efforts and it has recently become apparent that functional lines are often blurred. For instance, PPIases and oxidoreductases can combine chaperone and foldase functions, certain protease components remodel polypeptide substrates and can thus be viewed as chaperones, some proteases have the ability to switch from hydrolase to chaperone function, and there is evidence that certain proteins classified as chaperones may exhibit protease activity (see for instance [3-10]). To keep readers abreast of the fast moving field of protein folding, Microbial Cell Factories has commissioned a series of reviews by experts in the topic. The first of these already appeared in January [11]. In "Unscrambling and egg: protein disaggregation by AAA+ proteins", Weibezahn, Bukau and Mogk discuss the discovery, functional characterization and mechanism of action of Hsp104/ClpB, two orthologous ATPases that have the unique ability to fragment protein aggregates. The remodeling activity of other AAA+ proteins (e.g., ClpA, ClpX and ClpC) which are best known more for their role in proteolysis is also examined. The authors close their thorough review by presenting enticing evidence for the roles of AAA+ proteins in the propagation of neurodegenerative diseases. In the second paper of the series [12], Miot and Betton review the mechanisms of protein quality control in the periplasmic space of E. coli. After a brief discussion of the biogenesis of proteins destined for export from the bacterial cytoplasm, the authors describe the players involved in the periplasmic folding-degradation decision and how cells sense and respond to extracytoplasmic stress. The second half of the review provides a historical perspective on how the maltose binding protein has served as a useful model to understand the mechanisms of conformational quality control in the periplasm. With forthcoming reviews on disulfide bridge formation and in vitro refolding, it is a great time to turn to Microbial Cell Factories to keep up with protein folding.

The genes for the enzyme TEM-β-lactamase and Staphylococcus aureus protein A were fused in-frame and expressed in E. coli. The resulting bifunctional polypeptide was secreted through the inner membrane but remained partially associated with the cell envelope. The hybrid protein was very susceptible to degradation, and, as a result, production in strain HB101 was low. It was demonstrated that proteolytic degradation occurs primarily within the β-lactamase domain of the hybrid protein. Expression in E. coli strain KS474, which is deficient in periplasmic proteases, resulted in 40-fold higher production. The hybrid protein was purified from strain KS474 and its catalytic characteristics were compared with β-lactamase. The latter was purified to apparent homogeneity using a new procedure based on the refolding of inclusion body protein. The hybrid protein and β-lactamase exhibited very similar kinetic parameters for the hydrolysis of benzylpenicillin and cephaloridine, indicating that the catalytic properties were essentially unperturbed by the fusion of protein A. The binding constant for the association of the hybrid protein with rabbit IgG in solution was equal to 1.4 × 10−7 M. This value is comparable to that obtained with the intact protein A. It was also demonstrated that protein A-β-lactamase can be immobilized on IgG sepharose with retention of the enzymatic activity.

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