HIGH-YIELD EXPRESSION, REFOLDING, AND PURIFICATION OF PENICILLIN-BINDING PROTEIN 2A FROM METHICILLIN-RESISTANT STAPHYLOCOCCUS-AUREUS STRAIN27R

The mecA-27R gene, which encodes PBP2a from methicillin-resistant Stap hylococcus,; aureus strain 27R, was modified to remove the putative N- terminal membrane-spanning region, cloned into the T7 RNA polymerase e xpression vector pET11d, and used to transform Escherichia coli strain BL21(DE3). The majority of PBP2a was expressed in the form of inclusi on bodies, which were extracted, denatured, and refolded, The protein was then purified by anion-exchange and size-exclusion chromatography, A B-liter culture of induced E. coli provided 37 mg of purified PBP2a which was greater than 99% pure, finding affinities for [H-3]benzylpe nicillin, imipenem, and L-695,256 (a beta-lactam with high affinity fo r PBP2a) were shown to be comparable to PEP2a found in membrane prepar ations of S. aureus strain 27R, A direct binding assay, using C-14-lab eled L-695,256 was developed and used to show stoichiometric binding t o the refolded, soluble PBP2a, In addition, electrospray mass spectrom etry showed that 100% of the refolded PBP2a was covalently bound to th e beta-lactam in a stoichiometric fashion. Finally, two mutations of t he putative active-site serine showed the predicted loss of covalent b inding of the beta-lactam to the PBP2a, demonstrating the high specifi city of the soluble binding assay. (C) 1995 Academic Press, Inc.