Display of Functional β-Lactamase Inhibitory Protein on the Surface of M13 Bacteriophage

The display of proteins on the surface of filamentous phage has been shown to be a powerful method to select variants of a protein with altered binding properties from large combinatorial libraries of mutants. The β-lactamase inhibitory protein (BLIP) is a 165-amino-acid protein that binds and inhibits TEM-1 β-lactamase-catalyzed hydrolysis of the penicillin and cephalosporin antibiotics. Here we describe the construction of a new phagemid vector and the use of this vector to display BLIP on the surface of filamentous phage. It is shown that BLIP-displaying phage bind to immobilized β-lactamase and that the binding can be competed off by the addition of soluble β-lactamase. In addition, a two-step phage enzyme-linked immunosorbent assay procedure was used to demonstrate that the BLIP-displaying phage bind β-lactamase with a 50% inhibitory concentration of 1 nM, which compares favorably with a previously published K(i) of 0.6 nM. A system has therefore been established for protein engineering of BLIP to expand its range of binding to other β-lactamases and penicillin-binding proteins.