Cloning, Expression and Release of a Vibrio alginolyticus SDS-resistant Ca2+-dependent Exoprotease in Escherichia coli

SUMMARY: A Vibrio alginolyticus SDS-resistant, Ca2+-dependent serine exoprotease gene, cloned on a 7-1 kb DNA fragment in the recombinant plasmid pVP100, was expressed from its own promoter in Escherichia coli. Although active exoprotease was produced by late stationary phase E. coli(pVP100) cultures after 15 h incubation in proteinaceous medium containing Ca2+, transcription and translation of the exoprotease occurred before 6 h, during exponential growth. The cloned exoprotease was synthesized as a pool of inactive precursor molecules during exponential growth, and released as active exoprotease 8 h later by a process which did not require protein synthesis or involve cell lysis. Release of the exoprotease by E. coli(pVP100) was not inhibited by o-phenanthroline, quinacrine or cerulenin. Supernatant samples from E. coli(pVP100) cultures contained two SDS-resistant exoproteases with apparent M r values of approximately 54000 and 39000. The cloned exoprotease activity was inhibited by EDTA and a serine protease inhibitor, but was not affected by an inhibitor of trypsin-like enzymes.