Fluorescence binding assay for a small peptide based on a GFP fusion protein

Abstract

A fluorescence binding assay was developed for a small peptide based on a fusion protein between the peptide and the green fluorescent protein, GFP. The assay employs genetic engineering methods to prepare the analyte-label (peptide-GFP) conjugate as a fusion protein in order to produce a one-to-one, homogenous population of labeled-peptide. Specifically, a plasmid was constructed in which the C-terminus of a model octapeptide was fused to the N-terminus of GFP. Following expression of the octapeptide-GFP fusion protein in Escherichia coli, an immunoassay was developed based on sequential binding of the free octapeptide and labeled-octapeptide to an anti-octapeptide antibody immobilized on a solid surface. The naturally fluorescent protein acts as a label to provide sensitive detection for peptides. To our knowledge, this is the first time that GFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for a peptide analyte. Copyright (C) 1999 Elsevier Science B.V.