Construction of a modified penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus and purification by immobilized metal affinity chromatography.
The mecA-27r gene, which encodes PBP2a-27r, was modified by site-specific mutagenesis, resulting in replacement of the N-terminal membrane anchor with a short chelating peptide (CP-PBP2a-27r). CP-PBP2a-27r retained the same binding affinity for beta-lactam antibiotics as the wild-type enzyme. Approximately 95% pure CP-PBP2a-27r was recovered in a single step by use of chelating-peptide-immobilized metal ion affinity chromatography.
Wu
C Y
CY
Infectious Disease Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
Blaszczak
L C
LC
Smith
M C
MC
Skatrud
P L
PL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D002435:Cefamandole / Q000378:metabolism; D002846:Chromatography, Affinity; D017931:DNA Primers; D005798:Genes, Bacterial; D006602:Hexosyltransferases; D016106:Methicillin Resistance / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D002435:Cefamandole; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase