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 sigma(32) heat-shock transcription factor and the DnaK-DnaJ-GrpE and G roEL-GroES molecular chaperone machines on the folding of preS2-beta-g alactosidase. This 120 kDa fusion protein between the hepatitis B surf ace 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 degre es C, but aggregated extensively in an rpoH165(Am) mutant, Proper fold ing was partially restored upon co-overexpression of the dnaKJ operon, but not when the groE operon or dnaK alone were overproduced, The enz ymatic activities in dnaK103 dnaJ259 and grpE280 mutants were 40-60% l ower relative to a dnaK756 mutant or isogenic wild-type cells at 30 de grees C and 37 degrees C. At 42 degrees C, only 10-40% of the wild-typ e activity was present in each of the early-folding-factor mutants, Al though the synthesis levels of preS2-beta-galactosidase were reduced i n the dnaK103 dnaJ259 and grpE280 genetic backgrounds, aggregation was primarily responsible for the loss of activity when the cells were gr own 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 solubi lity of preS2-beta-galactosidase at temperatures up to 42 degrees C. O ur results are discussed in terms of the current understanding of the E. coli protein-folding cascade, The potential usefulness of heat-shoc k protein mutants for the production of soluble proteins in an inclusi on-body form is addressed.