title: Construction and overexpression in Escherichia coli of genetically engineered derivatives of penicillin-binding protein 2' of Staphylococcus epidermidis.
abstract: Removal of the putative amino-terminal membrane spanning region of penicillin-binding protein 2' (PBP-2') of Staphylococcus epidermidis WT55 was carried out by truncating the amino terminus-coding end of the mecA gene. PCR and site directed mutagenesis were used to introduce unique restriction sites at position 68 (HindIII) and at position 80 (NcoI) of the mecA gene, respectively. The coupling of the shortened coding regions to the trc promoter and gene fusion to the lacZ gene, aimed to facilitate subsequent protein purifications, resulted in strong expression in the cytoplasm of Escherichia coli and partial sequestration into insoluble protein granules. The truncated PBP-2' retained its penicillin-binding ability and also bound the monoclonal antibody directed against PBP-2' of Staphylococcus aureus.
authors.affiliation: Department of Biochemistry, University of Cambridge, UK.
authors.forename: M O
authors.initials: MO
authors.lastname: Westendorp
authors.forename: P E
authors.initials: PE
authors.lastname: Reynolds
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006602:Hexosyltransferases; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D013212:Staphylococcus epidermidis / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase