In vitro selection for catalytic turnover from a library of β-lactamase mutants and penicillin-binding proteins

A library of mutants of the RTEM ß-lactamase displayed on phage was created; it contained penicillin binding proteins (PBPs) as well as a small fraction of active ß-lactamases. The library was submitted to a selection process to extract the ß-lactamases i.e. the enzymes that turnover efficiently. This was achieved by a two steps procedure. In the first step, the ß-lactamases were labelled by reaction with a biotinylated suicide inhibitor while the PBPs were blocked by incubation in the presence of benzylpenicillin. In the second step, the labelled active phage-enzymes were separated by affinity chromatography on streptavidin coated beads.