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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.