title: OmpT proteolysis of E. coli initiation factor IF2. Elimination of a cleavage site by site-directed mutagenesis.
abstract: A serious problem during purification of the E. coli initiation factor IF2 is a significant loss of native IF2 due to partial degradation. We have previously shown that the major fragment, IF2 gamma (65 kDa), is the result of cleavage of IF2 alpha at the peptide bond between lysine 289 and arginine 290. In this paper we demonstrate that the cleavage is a result of proteolysis by outer membrane protease OmpT occurring immediately after cell lysis and in the S30 supernatant. By protein engineering we have constructed an IF2 mutant Lys289-greater than Met and shown that the IF2 gamma cleavage site in this mutant protein is insensitive to cleavage by OmpT. However the mutant protein is cleaved by OmpT between arginine 279 and alanine 280, which is a novel sequence specificity for this protease.
authors.affiliation: Department of Chemistry, Aarhus University, Denmark.
authors.forename: S F
authors.initials: SF
authors.lastname: Lassen
authors.forename: K K
authors.initials: KK
authors.lastname: Mortensen
authors.forename: H U
authors.initials: HU
authors.lastname: Sperling-Petersen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D039881:Prokaryotic Initiation Factor-2; D015202:Protein Engineering; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease