TITLE:
Binding specificity and affinity analysis of an anti-protective antigen peptide reagent using capillary electrophoresis
AUTHORS:
Joshua M. Kogot, Deborah A. Sarkes, Joseph M. Pennington, Paul M. Pellegrino, Dimitra Stratis-Cullum
KEYWORDS:
Affinity Capillary Electrophoresis; Peptide Biosensor; Peptide-Protein; Display Library
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.5 No.1,
January
10,
2014
ABSTRACT: Peptide biosensor reagents are emerging as an alternative
to typical antibody-based detection methods. Peptides can be rapidly isolated
using bacterial display methods for new and emerging biothreats and can be
chemically synthesized for rapid, large-scale production. With the emergence of
peptide biosensor reagents, there is a growing need to develop methods for characterizing binding interactions. Capillary
electrophoresis (CE) is a free-solution separation method that is able to determine target and analyte binding
association (Kb) and dissociation constants (Kd). In this
study, the Kb, Kd, and peptide specificity of an isolated
peptide binding reagent to protective antigen (PA) of Bacillus anthracis were evaluated using capillary electrophoresis
at 10 and 20 kV. The relative binding specificity was rapidly assessed by
measuring the peptide relative mobility shift at 20 kV at nonequilibrium using
bovine serum albumin (BSA), horseradish peroxidase (HRP), and an anti-PA monoclonal
antibody (mAb). The αPA peptide was
shown to be highly specific for PA, with a Kd = 177 nM measured at
20 kV and Kd = 312 nM measured at 10 kV. These results show that
peptides from bacterial display libraries can be rapidly tested for specificity
and binding affinity, in solution, for use as a potential biosensor reagent
against new and emerging biothreats.