Cloning and expression of the ccdA-associated thiol-disulfide oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production

摘要

Brevibacillus choshinensis (Bacillus brevis) is a protein-hyperproducing bacterium with a useful host-vector system for the production of recombinant proteins. Here, we cloned the ccdA-catA (CcdA associated thioredoxin-like thioldisulfide oxidoreductase) locus of B. choshinensis HPD31-S5. CatA protein (molecular weight, 19664) contains a thioredoxin-like motif, Cys-Gly-Pro-Cys. It was successfully expressed in B. choshinensis extracellularly (similar to 100 mug ml(-1) culture) using the secretion vector pNCMO2, and in Escherichia coli intracellularly (similar to 350 mug ml(-1) culture) with an amino-terminal His-tag. Both recombinant proteins showed thiol-disulfide oxidoreductase activity. Incubation of non-native human epidermal growth factor (hEGF) containing incorrect disulfide bonds with B. choshinensis cells secreting CatA protein resulted in the stimulation of the conversion of non-native hEGF to the native form. Furthermore, co-expression of CatA protein with recombinant hEGF in the B. choshinensis production system increased the yield of native hEGF.