Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli

Protein Expression and Purification
J G Thomas, F Baneyx

Abstract

The proper folding of aggregation-prone recombinant proteins in Escherichia coli can be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps). We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S'-beta-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30 degrees C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S'-beta-galactosidase 1.5- to 2-fold. When cultures overexpressing the dnaKJ operon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S'-beta-galactosidase aggregation was almost entirely abolished. Combining DnaK-DnaJ overexpression and growth of the cells at temperatures lower than 30 degrees C did not result in a comparable increase in activity. Although the individual effects of ethanol supplementation and dnaKJ overproduction were more limited when the culture v...Continue Reading

Citations

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