The role of the pro‐sequence in the processing and secretion of the thermolysin‐like neutral protease from Bacillus cereus

Summary The Bacillus cereus cnp gene coding for the thermolysin‐like neutral protease (TNP) has been cloned, sequenced, and expressed in Bacillus subtilis. The protease is first produced as a pre‐pro‐protein (Mr= 61000); the pro‐peptide is approximately two‐thirds of the size of the mature protein. The pro‐sequence has been compared with those of six other TNPs, and significant homologies have been found. Additionally, the TNP pro‐sequences are shown to be homologous to the pro‐sequence of Pseudomonas aeruginosa elastase. A mutant has been constructed from cnp, in which 23 amino acids upstream from the pro‐protein processing site have been deleted. This region has no homologous analogue in any of the other TNP pro‐sequences. The detection results in a delay of six to eight hours in detection of active protease in the growth medium, as well as a 75% decrease in maximum protease production. N‐terminal analysis of the mutant mature protein demonstrates that the processing site is unaltered by the pro‐sequence deletion. The deletion must, therefore, modulate the kinetics of processing and/or secretion of the pro‐protein.