Selection of phage-display library peptides recognizing ethanol targets on proteins

Abstract 

There is a forthcoming link between chronic alcohol consumption and proteins covalently modified by ethanol metabolites and their antibodies. To identify sensitive probes of protein–ethanol conjugates, we screened for the ethanol-altered protein domains with a phage-display combinatorial peptide library. In principle, recognition of the epitopes by the library peptides occurs through protein–protein interactions. A general screening, M13-based library with 10⁹ random sequences of linear heptameric peptides was used. The peptides were displayed in five copies each, as fusion proteins with phage’s minor coat protein III. They were located on one end of the surface of the phage particles. The targets were a model protein, streptavidin, and protein–ethanol conjugates (hydroxyethyl radical- or acetaldehyde-modified bovine serum albumin). They were either immobilized on a surface by direct coating or affinity captured on floating beads. An enriched library of phages with the tightest peptide binders for each target was selected and amplified in a multiple-cycle biopanning in vitro procedure. Binders were characterized by DNA sequencing of the corresponding phages and by counter-screening with positive and negative targets in either an enzyme-linked immunosorbent assay or plaque assay. We obtained the HPQ motif for streptavidin and two unique subsets of peptides that recognized each ethanol target with a selectivity of two orders of magnitude above the carrier protein and controls. The application of biopanning processes, coupled with phage-display peptide libraries on biological fluids and tissues, could provide a systematic mapping of protein–ethanol conjugates and supply a means for early diagnosis and prognosis of chronic alcohol consumption in human beings.

Keywords: Ethanol, Hydroxyethyl radicals, Acetaldehyde, Bovine serum albumin, Protein conjugates, Phage-display peptide libraries, Heptameric peptides, Biopanning, Screening, Disease-specific epitopes, Biomarkers, Alcoholism