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

Authors: Thomas, Jeffrey G.; Baneyx, Fraņois

Source: Molecular Microbiology, Volume 21, Number 6, October 1996 , pp. 1185-1196(12)

Buy & download fulltext article:

Price: $48.00 plus tax (Refund Policy)

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 120 kDa 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 Escheri- chia 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.

Document Type: Research article

DOI: http://dx.doi.org/10.1046/j.1365-2958.1996.651436.x

Affiliations: 1: Department of Chemical Engineering, University of Washington, Box 351750, Seattle, Washington 98195-1750, USA.

Publication date: 1996-10-01

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

Buy & download fulltext article:

Tools

Key

Text size:

Useful pages