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- W2034405192 abstract "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 beta-lactamase enzyme, which acts as a reporter. Linear dodecapeptide and cysteine-constrained decapeptide libraries were created at the amino-terminus of the Enterobacter cloacae P99 cephalosporinase molecule (P99 beta-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 (ErbB2(ECD)). The target-selected clones were already conjugated to an enzyme reporter, therefore, did not require subcloning or any other post-panning modifications. We used beta-lactamase enzyme activity-based assays for sample normalizations and clone binding evaluation. Clones were identified that bound to purified ErbB2(ECD) 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 ErbB2(ECD). beta-Lactamase fusion to peptides saved time and resources otherwise required by the phage-ELISA of a typical phage display screening protocol. The beta-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 beta-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." @default.
- W2034405192 created "2016-06-24" @default.
- W2034405192 creator A5041123859 @default.
- W2034405192 creator A5081120943 @default.
- W2034405192 date "2010-01-01" @default.
- W2034405192 modified "2023-09-27" @default.
- W2034405192 title "Phage-Displayed Combinatorial Peptide Libraries in Fusion to β-Lactamase as Reporter for an Accelerated Clone Screening: Potential Uses of Selected Enzyme-Linked Affinity Reagents in Downstream Applications" @default.
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- W2034405192 doi "https://doi.org/10.2174/138620710790218258" @default.
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