Recovery of high-affinity phage from a nitrostreptavidin matrix in phage-display technology.

A novel approach for the selection of high-affinity phage from phage-peptide libraries is described. The methodology employs a chemically modified form of streptavidin, termed nitrostreptavidin, which exhibits a reversible attraction for biotin. The new approach emulates conventional procedures in that a biotinylated probe, in this case biotinylated alpha-bungarotoxin, is attached to an immobilized streptavidin matrix. The phage library is introduced, and interacting phage particles are released under conventional acidic conditions (pH 2.2). At this stage, the primary peptide sequences characterizing the released phage are found to be identical with those previously known to interact with the toxin. However, other phage particles, which presumably interact more strongly than those released by acid, remain attached to the immobilized toxin. These can be released by virtue of the reversible biotin-binding properties of nitrostreptavidin. For this purpose, alkaline solutions (pH 10) or free biotin can be used. Using this approach, phage particles that recognize alpha-bungarotoxin were isolated; their peptide sequences were found to be similar to, but clearly distinct from, those collected by conventional acid elution. The affinity of the isolated phage was dramatically higher than that of phage obtained by the conventional methodology. In contrast, their synthetically prepared 15-mer peptides actually exhibited a lower affinity for the toxin than that shown by peptides prepared on the basis of the sequence obtained from conventional acid-eluted phage. This apparent discrepancy can be explained by an altered conformational state of the peptides in solution, compared to the epitopes expressed in situ on the phage surface.