High-Yield Expression, Refolding, and Purification of Penicillin-Binding Protein 2a from Methicillin-Resistant Staphylococcus aureus Strain 27R

Soluble Penicillin-Binding Protein 2a: β-Lactam Binding and Inhibition by Non-β-Lactams Using a 96-Well Format

“…PBP2a (aa 23-668) was studied as a refolded protein (17) as well as a maltose-binding protein-His 6 double affinity fusion (MBP-His-tag) (24) intact and cleaved using thrombin. Expression and purification of intact and cleaved MBP-His-tag-PBP2a was carried out as described (24).…”

“…SDS-PAGE assays using MRSA membranes were carried out as described (17). Briefly, samples were preincubated for 10 min at 37°C followed by an additional 10 min incubation in the presence of 3 H-PenG.…”

Reaction of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with β-lactams and acyclic substrates: kinetics in homogeneous solution

“…The expression and purification of recombinant sPBP2a were performed essentially as described by Frank et al [11]. The final PBP2a protein solution was dialysed against 25 mM Hepes (pH 7)\1 M NaCl\0.01 % NaN $ and stored at 4 mC.…”

“…Wu et al [7] first reported the construction of a water-soluble form of PBP2a (sPBP2a) by removal of the putative membrane anchor region. The subsequent purification and initial characterization of sPBP2a by both Roychoudhury et al [10] and Frank et al [11] have shown that homogeneous solutions of sPBP2a retain the ability to bind stoichiometrically to several β-lactams with the same apparent affinity as membrane-bound PBP2a. The affinities for several β-lactams also correlated well with their respective minimum inhibitory concentrations for methicillin-resistant Staph.…”

“…Here we describe a detailed kinetic study of sPBP2a [11] in homogeneous solution. Rate constants were measured for its reaction with a number of penicillins and cephalosporins.…”