Involvement of NH2-terminal pro-sequence in the production of active aqualysin I (a thermophilic serine protease) in Escherichia coli.

Abstract

Aqualysin I is a heat-stable subtilisin-type protease produced by Thermus aquaticus YT-1. The precursor of aqualysin I consists of four domains: an NH2-terminal signal peptide, an NH2-terminal pro-sequence, a protease domain, and a COOH-terminal pro-sequence. In Escherichia coli cells harboring recombinant plasmid carrying the aqualysin I gene, proteolytic activity is obtained on treatment at 65 degrees C and mature enzyme is detected. In the case of mutant genes containing partial deletions in the NH2-terminal pro-sequence, no proteolytic activity was detected and the precursor protein was found to be unstable in E. coli. These results indicate that the NH2-terminal pro-sequence is required to produce the active enzyme by stabilizing the precursor structure. Amino acid substitutions in the conserved sequence of the NH2-terminal pro-sequence found among subtilisin-type proteases made the processing faster compared with the wild type.