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

Abstract

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 ( c̱ cdA a̱ssociated thioredoxin-like ṯhiol-disulfide oxidoreductase) locus of B. choshinensis HPD31-S5. CatA protein (molecular weight, 19 664) contains a thioredoxin-like motif, Cys–Gly–Pro–Cys. It was successfully expressed in B. choshinensis extracellularly (∼100 μg ml −1 culture) using the secretion vector pNCMO2, and in Escherichia coli intracellularly (∼350 μg 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.