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Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries

From National Research Council Canada

Linkhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC41833/
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Name affiliation
  1. National Research Council of Canada. NRC Institute for Biological Sciences
  2. National Research Council of Canada. NRC Biotechnology Research Institute
FormatText, Article
Subjectenvironmental
Abstract
A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementaritj-determining regions (CDRs) in a single-chain Fv specific for the 0 polysaccharide of SalmoneUla serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10%/1 while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.
Publication date
In
  • Proceedings of the National Academy of Sciences of the United States of America 92, no. 11 (23 May 1995): 4992–4996.
LanguageEnglish
NRC number34517
DENG1995A
NPARC number3539517
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