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Contribution of Antibody Heavy Chain CDR1 to Digoxin Binding Analyzed by Random Mutagenesis of Phage-displayed Fab 26-10
- Source :
- Journal of Biological Chemistry. 270:28541-28550
- Publication Year :
- 1995
- Publisher :
- Elsevier BV, 1995.
-
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 (Ka 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.
- Subjects :
- Digoxin
Stereochemistry
Molecular Sequence Data
Mutant
Immunoglobulin Variable Region
Biochemistry
Antibodies
Cell Line
Immunoglobulin Fab Fragments
Mice
Animals
Bacteriophages
Nucleotide
Amino Acid Sequence
Cloning, Molecular
Binding site
Molecular Biology
DNA Primers
chemistry.chemical_classification
Binding Sites
Base Sequence
biology
Chemistry
Hydrogen bond
Cell Biology
Affinities
Amino acid
Mutagenesis
biology.protein
Antibody
Hapten
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 270
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....84fe76cdb31038038b7a116bde481d17