Structure-function analysis of the Escherichia coli GrpE heat shock protein

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild-type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N-terminal region, most likely through a computer predicted coiled-coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N-terminal and C-terminal regions are important for GrpE function in lambda DNA replication and its co-chaperone activity with DnaK.     

Hypochlorous acid activates the heat shock and soxRS systems of Escherichia coli

A series of plasmids, containing fusions of different stress promoters to lux reporter genes, was used in an attempt to monitor the defense circuits activated upon exposure of Escherichia coli to sublethal doses of free chlorine. A significant level of activation was exhibited by promoters of three heat shock genes (grpE, dnaK, and lon), in an rpoH-dependent manner. The promoter of micF, a gene under the control of the soxRS regulon, was also strongly induced, but not in a soxR mutant. This induction was not affected by sodA and sodB mutations, implying that it did not involve oxygen radical activity. Free-chlorine activation of both heat shock and soxRS regulons required an exposure of less then I s in duration. The oxyR or the SOS regulons were apparently not induced by free chlorine (as judged by lack of activation of katG and recA, respectively), and neither was the universal stress (uspA) protein.     

Induction of heat shock gene expression in colonic epithelial cells after incubation with Escherichia coli or endotoxin

OBJECTIVE: The universal cellular response to stress is the expression of a family of genes known as heat shock or stress proteins. We investigated whether bacteria or bacterial products (endotoxin) can induce heat shock protein expression in human enterocytes. DESIGN: Controlled, in vitro study. SETTING: Cell culture laboratory. SUBJECTS: Human Caco-2 enterocyte cell line. MEASUREMENTS AND MAIN RESULTS: Incubation of confluent monolayers of Caco-2 cells with Escherichia coli C25 (1 x 10(9) bacteria/mL) for 1 hr at 37 degrees C was found to induce the expression of the 72-kilodalton molecular weight heat shock protein gene (heat shock protein-72), the major inducible form of the 70-kilodalton molecular weight heat shock protein family of stress proteins, as detected by Western blot analysis. The level of heat shock protein-72 induction after incubation with E. coli was similar to the response of Caco-2 cells to heat shock at 43 degrees C for 1 hr. The induction of heat shock protein-72 gene expression by E. coli was not purely due to the process of phagocytosis, since incubation of Caco-2 cells with latex beads (1 micron) failed to induce heat shock gene expression. To elucidate the possible mechanism of heat shock protein-72 induction mediated by bacteria, Caco-2 cells were incubated with E. coli endotoxin (200 micrograms/mL) for 1 hr at 37 degrees C. Such treatment was also found to induce the synthesis of heat shock protein-72. CONCLUSIONS: These results demonstrate that bacteria and/or bacterial products induce the heat shock gene expression in Caco-2 cells. Since intestinal epithelial cells are constantly in contact with bacteria and bacterial products, we speculate that the heat shock gene expression may be part of the natural mechanism of protection for these cells in the potentially harmful environment that may be present in the intestinal tract.     

Identification of an Escherichia coli protein impurity in preparations of a recombinant pharmaceutical

A host-cell protein impurity found in preparations of recombinant human acidic fibroblast growth factor (aFGF) was identified. Samples of aFGF examined by western blot analysis employing antiserum raised against an Escherichia coli cell lysate contained an immunoreactive protein with a molecular weight of approximately 26,000. The impurity was chromatographically isolated and the N-terminal sequence was determined. Comparing the sequence to a protein database provisionally identified the isolated impurity as the S3 ribosomal protein of E. coli. Monoclonal antibodies recognizing three separate epitopes of S3 confirmed the identity of the impurity in western blots of aFGF samples. The monoclonal antibodies were also used to estimate S3 levels in various preparations of aFGF.     

Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers

1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated. 2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii. 3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h. 4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals.     

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli

We analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli. We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, sigma24, involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.     

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

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 K(m) = 0.70 microM 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.     

Protein substrates and heat shock reduce the DNA-binding ability of Escherichia coli Lon protease

Interaction between the fusion protein MBP-Lon, formed by maltose-binding protein and Lon protease, and the plasmid pBR322 was studied to clarify the DNA-binding behavior of the Lon protease. Since the MBP-Lon fusion protein that was bound to the plasmid was strongly adsorbed by amylose resin, complex formation and dissociation were determined by quantifying the unadsorbed plasmid using agarose gel electrophoresis. The autolysis of MBP-Lon fusion protein was suppressed when the protein was bound to the plasmid. The plasmid was completely dissociated from MBP-Lon fusion protein by the addition of the protein substrates of Lon protease (i.e. alpha-casein and denatured bovine serum albumin). In addition, at high temperatures, MBP-Lon fusion protein lost its plasmid-binding ability, although it fully retained ATP-dependent protease activity. These results suggest that Lon protease loses DNA-binding ability when cells are exposed to abnormal conditions and the amount of damaged proteins increases. On the other hand, DNA probably plays an important role in controlling the Lon protease activity in cells under normal conditions by entrapping the enzyme.     

Cloning of a cDNA encoding SjIrV1, a Schistosoma japonicum calcium-binding protein similar to calnexin, and expression of the recombinant protein in Escherichia coli

Membrane-associated proteins were isolated from adult Philippine strain Schistosoma japonicum by partitioning into the detergent phase of Triton X-114. A rabbit polyclonal antiserum raised against these proteins was used to screen an S. japonicum expression cDNA library. Positive clones were identified which encoded the species orthologue of SmIrV1, a Schistosoma mansoni protein which was initially identified by screening with sera from mice protectively vaccinated with irradiated cercariae [Hawn et al., J. Biol. Chem. 268 (1993) 7692-7698]. The S. japonicum molecule, which we term SjIrV1, is 83% identical to SmIrV1 at the predicted amino acid level and is a member of the calreticulin family of non-EF-hand, calcium-binding proteins. The Chinese strain S. japonicum orthologue of SjIrV1 was obtained by screening with the radiolabelled insert of the Philippine strain clone. Northern blot analysis revealed a single message of around 2.4 kb and gave no indication of alternative splicing. Southern blot analysis gave a simple pattern, indicating a single-copy gene, and showed a single restriction fragment length polymorphism between the genomes of Chinese and Philippine strains of S. japonicum. Recombinant, full-length SjIrV1 was expressed with a hexahistidine tag in Escherichia coli and the recombinant protein isolated by nickel-chelate chromatography. Recombinant SjIrV1 was shown to exhibit calcium-dependent, differential electrophoretic migration and to bind ruthenium red in the absence but not in the presence of calcium ions. The presence of conserved Ca(2+)-binding motifs predicted from the primary sequence, together with the Ca(2+)-dependent electrophoretic mobility of recombinant SjIrV1, confirmed that SjIrV1 was a functional calcium-binding protein.     

Strategies for optimizing heterologous protein expression in Escherichia coli

The many advantages of Escherichia coli have ensured that it remains a valuable host for the efficient, cost-effective and high-level production of heterologous proteins. Here, we describe the current status of this prokaryotic expression system and focus on strategies designed to maximize the yields of recombinant proteins. Major challenges facing this expression system are also outlined.     

Transfer of plasmids between strains of Escherichia coli under rumen conditions

Strains of Escherichia coli originally isolated from the rumen of sheep were shown to be capable of exchanging a 60kb plasmid, conferring resistance to tetracycline and ampicillin, at low frequencies (below 10(-6) per recipient) under anaerobic conditions in the presence of (a) autoclaved and clarified rumen fluid, (b) raw clarified rumen fluid, or (c) whole rumen fluid. Under anaerobic conditions the two rumen strains showed no inhibition of growth rate when 50 mmol l-1 volatile fatty acids were added to LB medium at pH 7, although significant inhibition resulted with 100 mmol l-1 VFA. The two rumen strains, and four strains from the pig gut, showed less inhibition of anaerobic growth by volatile fatty acids than did three laboratory strains examined for comparison. These findings indicate that plasmid transfer between certain E. coli strains can occur under conditions that closely simulate an anaerobic but environment.     

Construction of a gene encoding the insect bactericidal protein attacin. Studies on its expression in Escherichia coli

Attacin, a bactericidal small protein is produced by the giant silk moth Hyalophora cecropia. This paper deals with our efforts to clone the attacin cDNA in a bacterial vector to express it in Escherichia coli and produce the protein in sufficient amount, for further studies. We chose two inducible expression vector/bacterial cell systems: pPL-lambda/N99cI+ cells which is able to be induced by nalidixic acid, and pET3d/BL21(DE3) cells carrying a T7 RNA polymerase gene which is IPTG-inducible. After cloning in the pPL-lambda system and under no addition of the inducer, isolated transformants carried this plasmid with at least 2 concurrent deletions that drastically affected attacin expression, even though attacin gene seems to be intact as deduced by its PCR amplification. It was concluded that basal attacin expression occurred in this system and bacterial growth was limited. Plasmid deletions may have emerged by selection pressure as a way to avoid bactericidal expression and allow bacteria survival. The second cloning attempt was done in pET3d vector/BL21 cells, that should not express the cloned sequence (they lack T7 RNA polymerase gene). Transformed BL21 cells gave 3 recombinant plasmids, 2 of them presented a C deletion that generated an early stop signal in the attacin coding region. The third clone, pET-ATT18, carrying an intact gene, was transferred to BL21(DE3)-IPTG inducible cells in order to be expressed. Attacin was undetectable in stained gels or by Western blot analysis. However, expression was visualized in grown cells after 30 min of IPTG induction and 5 min of [35S]-methionine labeling, as a 22.5 kDa protein band by using gel electrophoresis and fluorography. This low level of expression drastically affected bacterial growth. Considering that attacin has no lytic activity, these results suggest that this molecule should block bacterial growth directly at the cytoplasm by an unknown mechanism, since no signal peptide coding sequence was incorporated in this gene construction, precluding periplasmic or external destination of this protein.     

Increased heat shock protein expression after stress in Japanese quail

The health care received from first admission to a pediatric rheumatology clinic to 9-year follow-up was assessed in 109 patients with chronic inflammatory rheumatic diseases or chronic idiopathic musculoskeletal pain. Ninety-five of the patients had received hospital care after the first admission, of whom 53 patients > or = 18 years, 21 patients < 18 years, and 33 parents of patients < 18 years rated their degree of satisfaction with the health care from 0 to 10. Mean scores of satisfaction with different aspects of care ranged from 6.0 to 9.6. Among patients > or = 18 years, those with idiopathic pain were less satisfied than those with inflammatory rheumatic diseases on the availability of care (mean 6.4 vs. 8.5, p < 0.001), continuity of care (mean 6.5 vs. 8.4, p < 0.001), and empathy of the health care providers (mean 6.7 vs. 7.9, p <0.05). The diagnostic group and the occurrence of remission predicted the level of global satisfaction in patients > or = 18 years. In patients < 18 years, chronic family difficulties predicted patient satisfaction and physical disability and chronic family difficulties predicted parent satisfaction. In conclusion, most parents and patients with inflammatory rheumatic diseases were satisfied with the health care. However, some patients with idiopathic pain had unmet needs for care.     

Overproduction of the Brucella melitensis heat shock protein DnaK in Escherichia coli and its localization by use of specific monoclonal antibodies in B. melitensis cells and fractions

The Brucella melitensis dnaK gene was amplified by the polymerase chain reaction using primers chosen according to the published sequence of B. ovis and cloned in multiple copy plasmids enabling expression under the control of the Plac promoter. Monoclonal antibodies (mAb) obtained by immunizing mice with B. melitensis B115 cell wall (CW) fraction or by infecting mice with virulent B. melitensis strain H38 and recognizing a 73-kDa band in immunoblotting of the B. melitensis CW fraction reacted with the cloned dnaK gene product and were thus shown to be specific for the heat shock protein DnaK. The anti-Dnak protein mAbs did not react with Escherichia coli control cells or cell lysates and could therefore be specific to Brucella DnaK protein epitopes. These mAbs were further used to study overproduction of the DnaK protein. B. melitensis DnaK overproduction in E. coli resulted in a defect in cell septation and formation of cell filaments. Immunogold labelling with the mAbs and electron microscopy localized the DnaK protein inside as well as outside the E. coli cells, probably resulting from lysis due to toxicity of the overproduced DnaK protein. These results indicated that overproduction of the B. melitensis DnaK protein in E. coli had similar physiological consequences as that of E. coli overproduced in E. coli. The DnaK protein localization in B. melitensis cells was essentially cytoplasmic, as shown by immunoelectron microscopy. Heat shock treatment of these cells resulted in increased binding of mAbs and labelling in the cytoplasm. However, in subcellular fractions the DnaK protein was predominantly found in the cell envelope fraction of B. melitensis, which could perhaps be due to interaction of the DnaK protein with membrane proteins.     

Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant Escherichia coli

Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S-transferase (GST). GST-PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity-purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.