Contribution of antibody heavy chain CDR1 to digoxin binding analyzed by random mutagenesis of phage-displayed Fab 26-10 Journal Article


Authors: Short, M. K.; Jeffrey, P. D.; Kwong, R. F.; Margolies, M. N.
Article Title: Contribution of antibody heavy chain CDR1 to digoxin binding analyzed by random mutagenesis of phage-displayed Fab 26-10
Abstract: We constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 of the anti-digoxin antibody 26-10 Fab to investigate sequence constraints necessary for high affinity binding in an antibody of known crystal structure. Phage were selected by panning against digoxin and three C-16-substituted analogues. All antigen-positive mutants selected using other analogues also bound digoxin. Among 73 antigen-positive clones, 26 different nucleotide sequences were found. The majority of Fabs had high affinity for digoxin (K-alpha 3.4 x 10(9) M(-1)) despite wide sequence diversity, Two mutants displayed affinities 2- and 4-fold higher than the parental antibody. Analysis of the statistical distribution of sequences showed that highest affinity binding occurred with a restricted set of amino acid substitutions at positions H33-35. All clones save two retained the parental Asn-H35, which contacts hapten and hydrogen bonds to other binding site residues in the parental structure, Positions H30-32 display remarkable diversity, with 10-14 different substitutions for each residue, consistent with high affinity binding. Thus complementarity can be retained and even improved despite diversity in the conformation of the N-terminal portion of the H-CDR1 loop.
Keywords: recognition; mutations; specificity; escherichia-coli; affinity; site-directed mutagenesis; reshaping human-antibodies; antidigoxin antibodies; hypervariable regions; fv fragment
Journal Title: Journal of Biological Chemistry
Volume: 270
Issue: 48
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 1995-12-01
Start Page: 28541
End Page: 28550
Language: English
ACCESSION: WOS:A1995TH05600012
DOI: 10.1074/jbc.270.48.28541
PROVIDER: wos
PUBMED: 7499368
Notes: Article -- Source: Wos
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  1. Philip D Jeffrey
    30 Jeffrey