The refolding, purification, and activity analysis of a rice Bowman-Birk inhibitor expressed in Escherichia coli

A putative rice trypsin/chymotrypsin inhibitor of the Bowman-Birk family, R BBI-8 of about 20 kDa, was expressed in Escherichia coli as a fusion protei n bearing an N-terminal (His)(6) purification tag. The expressed recombinan t protein, rRBBI-8, is insoluble and accumulates as inclusion bodies. The i nsoluble protein was solubilized in 8 M urea under reducing environment and then refolded into its active conformation under optimized redox condition s. Strategies used to optimize yield and efficiency include selecting the r edox system, increasing protein concentration during refolding by adding th e denatured protein in a stepwise way, utilizing additives to prevent aggre gation, and selecting buffer-exchanging conditions. A Nichelate affinity co lumn was then employed to purify the renatured protein. rRBBI-8 shows stron g inhibitory activity against trypsin and it can slightly inhibit chymotryp sin. In this study, a refolding and purification system was set up for this cysteine-rich recombinant protein expressed in a prokaryotic system. (C) 1 999 Academic Press.