Secretion of active subtilisin YaB by a simultaneous expression of separate pre-pro and pre-mature polypeptides in Bacillus subtilis

Alkaline elastase YaB, produced by alkalophilic Bacillus YaB, is an extracellular serine protease having 55% homology to subtilisin BPN' and thus could be called as subtilisin YaB. It is synthesized as a 378-amino acid preproenzyme and secreted into the culture medium as a 265-amino acid mature protease. To examine if the pro-peptide of subtilisin YaB functions in trans to guide the folding of secreted subtilisin YaB in vivo, we made genes encoding the pre-pro, pro and pre-mature portions and placed them under the control of the spac-I promoter on a multi-copy plasmid. When simultaneous expression in Bacillus subtilis of both the pre-pro and pre-mature genes was induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG). protease activity was detected in the medium. On the other hand, we could not detect protease activity when the expression of either the pre-mature gene alone or both the pro and pre-mature genes was induced. From these results, we concluded that the pro region functions in tra,ls and outside the cells for the proper folding and activation of the enzyme. (C) 1996 Academic Press, Inc.