A truncated soluble Bacillus signal peptidase produced in Escherichia coliis subject to self-cleavage at its active site

Soluble forms of Bacillus signal peptidases which lack their unique amino-t erminal membrane anchor are prone to degradation, which precludes their hig h-level production in the cytoplasm of Escherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms o f this signal peptidase were not prone to degradation; in fact, these mutan t proteins were produced at very high levels in E. coli. Second, the purifi ed active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-clea vage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter positi on results in complete inactivation of the enzyme, as Ser15 forms a catalyt ic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 canno t be prevented by mutagenesis of Gly13 and Ser15, which conform to the -1, -3 rule for signal peptidase recognition, because these residues are critic al for signal peptidase activity.