title: Two-step fast protein liquid chromatographic purification of the Serratia marcescens hemolysin and peptide mapping with mass spectrometry.
abstract: The pore forming toxin of Serratia marcescens (ShlA) is secreted and activated by an outer membrane protein (ShlB). Activation of inactive ShlA (termed ShlA*) by ShlB is dependent on phosphatidylethanolamine (PE). Activation may be a covalent modification of ShlA. To test this hypothesis, the responsible activation domain (in the N-terminal 255 amino acids of ShlA) was isolated from whole bacteria with 8 M urea in an inactive form (ShlA-255*) and from the culture supernatant in an active form (ShlA-255), followed by a two-step purification by anion-exchange chromatography and gel permeation chromatography. Comparison of a tryptic peptide map of both forms with subsequent electrospray mass spectrometry (ES-MS) and sequencing by tandem ES-MS revealed no modification. These data imply that ShlB presumably imposes a conformation on ShlA-255 that triggers activity.
authors.affiliation: Institut für Mikrobiologie II, Universität Tübingen, Germany. ralf.hertle@mikrobio.uni-tuebingen.de
authors.forename: R
authors.initials: R
authors.lastname: Hertle
authors.forename: R
authors.initials: R
authors.lastname: Süssmuth
authors.forename: V
authors.initials: V
authors.lastname: Braun
authors.forename: G
authors.initials: G
authors.lastname: Jung
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002850:Chromatography, Gel / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006460:Hemolysin Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping
chemical_list: D001426:Bacterial Proteins; D006460:Hemolysin Proteins; C089911:ShlA protein, Serratia marcescens