Open AccessPhage display of ricin B chain and its single binding domains: system for screening galactose-binding mutants.
Author(s) -
Candace Swimmer,
Sophie M. Lehar,
John McCafferty,
David J. Chiswell,
Walter A. Blättler,
Braydon C. Guild
Publication year - 1992
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.89.9.3756
Subject(s) - ricin , mutant , asparagine , lectin , fusion protein , biochemistry , phage display , binding domain , binding site , biology , plasma protein binding , amino acid , microbiology and biotechnology , chemistry , toxin , recombinant dna , peptide , gene
We demonstrate that the B chain of ricin toxin preserves its lectin activity when expressed as a fusion protein on the surface of fd phage. Moreover, B chain, which folds into two topologically similar globular domains, can be dissected into amino-terminal and carboxyl-terminal domains to form single binding domains (SBDs) of B chain, each of which displays specificity for complex galactosides. The specific binding exhibited by the fusion protein of these SBDs was eliminated when amino acid substitutions Gly-46 in SBD1 or Gly-255 in SBD2 for native asparagine were introduced to alter key residues implicated in hydrogen bonding with substrate. These data demonstrate that it is possible to use a prokaryotic expression system to stably express and screen ricin B chain and its SBDs for sugar-binding mutants. Expression of ricin B chain on the surface of fd phage provides a method that can be used to efficiently select mutants with altered binding activities from a randomly generated library.