Girja S. Shukla and David N. Krag Pages 75 - 87 ( 13 )
Phage-display selection of combinatorial libraries is a powerful technique for identifying binding ligands against desired targets. Evaluation of target binding capacity of multiple clones recovered from phage display selection to a specific target is laborious, time-consuming, and a rate-limiting step. We constructed phage-display combinatorial peptide libraries in fusion with a β-lactamase enzyme, which acts as a reporter. Linear dodecapeptide and cysteineconstrained decapeptide libraries were created at the amino-terminus of the Enterobacter cloacae P99 cephalosporinase molecule (P99 β-lactamase). The overall and positional diversity of amino acids in both libraries was similar to other phage-display systems. The libraries were selected against the extracellular domain of ErbB2 receptor (ErbB2ECD). The target-selected clones were already conjugated to an enzyme reporter, therefore, did not require subcloning or any other post-panning modifications. We used β-lactamase enzyme activity-based assays for sample normalizations and clone binding evaluation. Clones were identified that bound to purified ErbB2ECD and ErbB2-overexpressing cell-lines. The peptide sequences of the selected binding clones shared significant motifs with several rationally designed peptide mimetics and phage-display derived peptides that have been reported to bind ErbB2ECD. β-Lactamase fusion to peptides saved time and resources otherwise required by the phage-ELISA of a typical phage display screening protocol. The β- lactamase enzyme assay protocols is a one-step process that does not require secondary proteins, several steps of lengthy incubations, or washings and can be finished in a few minutes instead of hours. The clone screening protocol can be adopted for a high throughput platform. Target-specific ??-lactamase-linked affinity reagents selected by this procedure can be produced in bulk, purified, and used, without any modification, for a variety of downstream applications, including targeted prodrug therapy.
Peptide libraries, phage-display, β-lactamase reporter, enzyme-linked peptides, accelerated screening, `
Department of Surgery, E309 Given Building, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405, USA.