HETEROLOGOUS EXPRESSION OF THE MYCOBACTERIUM-TUBERCULOSIS GENE ENCODING ANTIGEN 85A IN CORYNEBACTERIUM-GLUTAMICUM

By using appropriate Corynebacterium glutamicum-Escherichia coli shutt le plasmids, the gene encoding the fibronectin-binding protein 85A (85 A) from Mycobacterium tuberculosis was expressed in C. glutamicum, als o an actinomycete and nonsporulating gram-positive rod bacterium, whic h is widely used in industrial amino acid production. The 85A gene was weakly expressed in C. glutamicum under the control of the ptac promo ter from E. coli, but it was produced efficiently under the control of the promoter of the cspB gene encoding PS2, one of the two major secr eted proteins from C. glutamicum. The 85A protein was produced in vari ous forms, with or without its own signal sequence and with or without the signal sequence and the NH2-terminal (18-amino-acid) mature seque nce of PS2. Western blot analysis with monoclonal antibodies raised ag ainst the M. tuberculosis antigen 85 complex showed that recombinant 8 5A protein was present in the corynebacterial cell wall extract and al so released in extracellular culture medium. NH2-terminal microsequenc ing of recombinant 85A secreted by C. glutamicum shelved that signal p eptide,vas effectively cleaved olf at the predicted site. The recombin ant 85A protein was biologically active in vitro, inducing significant secretion of Th1 T-cell cytokines, particularly interleukin-2 and gam ma interferon, in spleen cell cultures from mice vaccinated with live Mycobacterium bovis BCG. Heterologous expression of mycobacterial anti gens in C. glutamicum now offers a potent tool for further immunologic al characterization and large scale preparation of these recombinant p roteins.