M13 bacteriophage displaying disulfide-constrained microproteins.

A display-phage library (TN2), displaying an 18-residue peptide fused to coat protein III, represents a collection of up to 8.55 x 10(6) peptides encoded by only 1.68 x 10(7) DNA sequences. Each displayed peptide has two fixed cysteine residues (allowing disulfide formation) and six variegated residues, four between the cysteines and one either side of the cysteines. Screening this library against streptavidin (Sv) and the anti-beta-endorphin monoclonal antibody, 3-E7, yielded phage displaying disulfide-constrained microproteins with sequences similar to those published for the linear-peptide display phage. Analysis of selected clones indicated that a disulfide bond is required for high-affinity binding to each of the target proteins. The microproteins selected for binding to Sv and 3-E7 show more stringent sequence specificity than do linear peptides selected for binding to the same targets.