Expression vectors for the introduction of highly diverged sequences into the six complementarity-determining regions of an antibody

Yoshitaka Iba, Wataru Ito, Yoshikazu Kurosawa

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)

Abstract

We prepared three kinds of phagemid vector that permit the simultaneous introduction of highly diverged sequences into six complementarity-determining regions (CDRs) of an antibody (Ab) by the polymerase chain reaction (PCR) with degenerate oligodeoxynucleotide (oligo) primers. The phages expressed either the Fv, single-chain Fv (sc Fv) or Fab form of an Ab fused with a half-molecule of cpIII on the surface of M13 phage. A phage-display library, composed of 2 x 108 independent clones, was constructed; the phages that were specific for hen egg-white lysozyme (HEL) were selected by three rounds of panning; and 20 clones were isolated. The isolated clones consisted of 17 different clones. Among them, 16 clones expressed proteins that were able to bind to HEL. The association constants for binding of the encoded proteins to HEL, ranged from 1.48 x 106 to 7.71 x 106/M. These vectors allowed us to prepare many libraries of artificial Ab in which the sequences of six CDRs were very different and reflected the artificial sequences that had been designed for the degenerate oligo that we used as primers for PCR. The libraries should be also useful for the analysis of relationships between the sequences of the CDRs and antigen (Ag) specificity.

Original languageEnglish
Pages (from-to)35-46
Number of pages12
JournalGene
Volume194
Issue number1
DOIs
Publication statusPublished - 18-07-1997

All Science Journal Classification (ASJC) codes

  • Genetics

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