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

Analytical Biochemistry
M BalassE Katchalski-Katzir

Abstract

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...Continue Reading

Citations

Jun 10, 1997·Proceedings of the National Academy of Sciences of the United States of America·T ScherfJ Anglister
May 12, 2005·Biomolecular Engineering·Giang H NguyenCassandra L Smith
Feb 27, 2001·Toxicon : Official Journal of the International Society on Toxinology·M BalassE Katchalski-Katzir
Aug 6, 2004·Protein Expression and Purification·Michael D ScholleBrian K Kay
Sep 22, 2010·BioTechniques·William D Thomas, George P Smith
Apr 21, 2005·The Journal of Biological Chemistry·Sau-Ching Wu, Sui-Lam Wong

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