35] SeeB: A chaperone from Escherichia coli

Abstract

Publisher Summary The chapter presents a study related to SecB, a chaperone from Escherichia coli (E.coli). SecB is a molecular chaperone in E.coli that is dedicated to the facilitation of the export of a number of proteins destined for the periplasmic space or the outer membrane. This role in export is demonstrated in vivo by the accumulation of pulse-labeled precursor species in a strain that is devoid of SecB and in vitro by showing that SecB is required for translocation of precursors into inverted vesicles of cytoplasmic membrane. The binding frame for SecB within two physiologic ligands, maltose-binding protein, and galactose-binding protein has been determined. SecB has the ability to bind selectively and with high affinity to polypeptides that are in a nonnative state. SecB binds precursor polypeptides and maintains them in a state competent for translocation through the cytoplasmic membrane. SecB is purified from strains of E. coli harboring plasmids that contain the secB gene under strong, regulated promoters. The purification involves passage of the sample over a Q-Sepharose ion-exchange column, followed by chromatography using a molecular sieve resin, Sephacryl S-300, and finally a Mono Q ion-exchange column.