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-p eptide Libraries is described. The methodology employs a chemically mo dified form of streptavidin, termed nitrostreptavidin, which exhibits a reversible attraction for biotin. The new approach emulates conventi onal procedures in that a biotinylated probe, in this case biotinylate d alpha-bungarotoxin, is attached to an immobilized streptavidin matri x 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 immobili zed toxin. These can be released by virtue of the reversible biotin-bi nding properties of nitrostreptavidin. For this purpose, alkaline solu tions (pH 10) or free biotin can be used. Using this approach, phage p articles that recognize alpha-bungarotoxin were isolated; their peptid e sequences were found to be similar to, but clearly distinct from, th ose collected by conventional acid elution. The affinity of the isolat ed phage was dramatically higher than that of phage obtained by the co nventional methodology. In contrast, their synthetically prepared 15-m er peptides actually exhibited a lower affinity for the toxin than tha t shown by peptides prepared on the basis of the sequence obtained fro m conventional acid-eluted phage, This apparent discrepancy can be exp lained by an altered conformational state of the peptides in solution, compared to the epitopes expressed in situ on the phage surface. (C) 1996 Academic Press, Inc