Display of functional beta-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 beta-lactamase inhibitory protein (BLIP) is a 165-amino-acid protein that binds and inhibits TEM-1 beta-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 beta-lactamase and that the binding can be competed off by the addition of soluble beta-lactamase. In addition, a two-step phage enzyme-linked immunosorbent assay procedure was used to demonstrate that the BLIP-displaying phage bind beta-lactamase with a 50% inhibitory concentration of 1 nM, which compares favorably with a previously published Ki of 0.6 nM. A system has therefore been established for protein engineering of BLIP to expand its range of binding to other beta-lactamases and penicillin-binding proteins.
Huang
W
W
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
Petrosino
J
J
Palzkill
T
T
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D015202:Protein Engineering; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases
D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1