title: A truncated soluble Bacillus signal peptidase produced in Escherichia coli is subject to self-cleavage at its active site.
abstract: Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-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 of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified 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-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the -1, -3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.
authors.affiliation: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, 9750 AA Haren, The Netherlands.
authors.forename: M L
authors.initials: ML
authors.lastname: van Roosmalen
authors.forename: J D
authors.initials: JD
authors.lastname: Jongbloed
authors.forename: A
authors.initials: A
authors.lastname: Kuipers
authors.forename: G
authors.initials: G
authors.lastname: Venema
authors.forename: S
authors.initials: S
authors.lastname: Bron
authors.forename: J M
authors.initials: JM
authors.lastname: van DijL
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase