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

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Abstract
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.