High-yield expression, refolding, and purification of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus strain 27R.
The mecA-27R gene, which encodes PBP2a from methicillin-resistant Staphylococcus aureus strain 27R, was modified to remove the putative N-terminal membrane-spanning region, cloned into the T7 RNA polymerase expression vector pET11d, and used to transform Escherichia coli strain BL21(DE3). The majority of PBP2a was expressed in the form of inclusion bodies, which were extracted, denatured, and refolded. The protein was then purified by anion-exchange and size-exclusion chromatography. A 6-liter culture of induced E. coli provided 37 mg of purified PBP2a which was greater than 99% pure. Binding affinities for [3H]benzylpenicillin, imipenem, and L-695,256 (a beta-lactam with high affinity for PBP2a) were shown to be comparable to PBP2a found in membrane preparations of S. aureus strain 27R. A direct binding assay, using 14C-labeled L-695,256 was developed and used to show stoichiometric binding to the refolded, soluble PBP2a. In addition, electrospray mass spectrometry showed that 100% of the refolded PBP2a was covalently bound to the beta-lactam in a stoichiometric fashion. Finally, two mutations of the putative active-site serine showed the predicted loss of covalent binding of the beta-lactam to the PBP2a, demonstrating the high specificity of the soluble binding assay.
Department of Biophysical Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065-0900, USA.
L J
LJ
Frank
D
D
Wisniewski
G G
GG
Hammond
J
J
Hermes
A
A
Marcy
P M
PM
Cameron
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006602:Hexosyltransferases; D007769:Lactams / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D016106:Methicillin Resistance / Q000235:genetics*; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013211:Staphylococcus aureus / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007769:Lactams; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase