Purification of the Escherichia Coli SecB Gene Product and Demonstration of Its Activity in an in Vitro Protein Translocation System

Mutations in the Escherichia coli secB gene lead to protein export defects in vivo (Kumamoto, C.A., and Beckwith, J. (1985) J. Bacteriol. 163, 267-274). To demonstrate directly the participation of the secB gene product (SecB) in protein export, SecB was purified, and its effects on in vitro protein translocation were analyzed. SecB was purified from soluble extracts of a strain that overproduced it, by ammonium sulfate precipitation, DEAE-cellulose chromatography, and differential precipitation at acid pH. The chromatographic behavior on gel filtration columns indicated apparent molecular masses of approximately 90 kDa for both purified SecB and SecB in cytosolic extracts of wild type cells. When added to a translocation mixture, purified SecB stimulated pro-OmpA translocation into membrane vesicles. SecB also suppressed the thermoinduced defect in translocating activity of membranes derived from a secY24 mutant. The results of these in vitro studies and of previous in vivo studies demonstrate that SecB plays a direct role in normal protein export in E. coli.