PMID:8898387
| Citation |
Thomas, JG and Baneyx, F (1996) Protein folding in the cytoplasm of Escherichia coli: requirements for the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines. Mol. Microbiol. 21:1185-96 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Escherichia coli/metabolism; Gene Expression Regulation, Bacterial; Molecular Chaperones; Mutation; Protein Folding; beta-Galactosidase/metabolism |
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