The Agarase Gene (dag A) of Streptomyces coelicolor A3(2): Affinity Purification and Characterization of the Cloned Gene Product

Abstract

The coding and regulatory sequences of the agarase gene of Streptomyces coelicolor A3(2) were cloned in Streptomyces lividans 66 on the plasmid vector pIJ61, resulting in a several hundred-fold increase in the production of the secreted protein. Subcloning experiments localized the sequences required for agarase production and for the mediation of carbon catabolite repression to a segment of about 1.2 kb. A simple protein purification procedure that uses affinity binding of agarase to agarose beads was developed. Preliminary characterization of the enzyme, together with the results of in vitro transcription-translation studies, suggest that the intracellular form of agarase (about 34 kDa) possesses a signal sequence that is cleaved upon secretion across the cell membrane to produce an extracellular protein of about 29 kDa.