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Protein Expr Purif. 1997 Mar;9(2):211-8.

Expression of aggregation-prone recombinant proteins at low temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems.

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  • 1Department of Chemical Engineering, University of Washington, Seattle 98195, USA.

Abstract

The aggregation-prone fusion protein preS2-S'-beta-galactosidase was used as a model system to compare the efficiencies of the IPTG-inducible tac promoter and the low-temperature-inducible cspA promoter in directing the expression of soluble recombinant polypeptides at reduced growth temperatures in Escherichia coli. At 37 degrees C, the fusion protein was produced at high levels from the tac promoter, but aggregated quantitatively in a biologically inactive form. In contrast, little preS2-S'-beta-galactosidase was synthesized from the cspA promoter at this temperature, presumably due to transcript instability. The highest yields of active enzyme were obtained following temperature downshift from 37 to 30 degrees C for the tac promoter and 25 degrees C for the cspA promoter. At 25 degrees C, the kinetics of accumulation of beta-galactosidase activity, ratios of soluble to insoluble fusion protein, and synthesis rates of preS2-S'-beta-galactosidase were virtually identical for both promoters for a period of 2 h postinduction. Thereafter, the cspA promoter became repressed, whereas synthesis of the fusion protein continued with the tac system. Following transfer to 10 degrees C, the tac promoter was almost completely inhibited while the cspA promoter was able to direct the synthesis of soluble preS2-S'-beta-galactosidase for up to 2 h. However, the levels of active enzyme produced were approximately threefold lower than those measured at 25 degrees C. Overexpression of native CspA had no effect on the accumulation of active preS2-S'-beta-galactosidase from the cspA promoter. It is therefore unlikely that CspA acts as it own positive inducer. Our results indicate that the cspA promoter can efficiently substitute for the tac system at 25 degrees C and may be particularly valuable for the expression of highly aggregation-prone or unstable gene products at 10 degrees C.

PMID:
9056486
[PubMed - indexed for MEDLINE]
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