Exploiting recombination in single bacteria to make large phage antibody libraries

Nat Biotechnol. 2000 Jan;18(1):75-80. doi: 10.1038/71958.

Abstract

The creation of large phage antibody libraries has become an important goal in selecting antibodies against any antigen. Here we describe a method for making libraries so large that the complete diversity cannot be accessed using traditional phage technology. This involves the creation of a primary phage scFv library in a phagemid vector containing two nonhomologous lox sites. Contrary to the current dogma, we found that infecting Cre recombinase-expressing bacteria by such a primary library at a high multiplicity of infection results in the entry of many different phagemid into the cell. Exchange of Vh and Vl genes between such phagemids creates many new V h/Vl combinations, all of which are functional. On the basis of the observed recombination, the library is calculated to have a diversity of 3x1011. A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigens.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / genetics*
  • Antibodies / immunology*
  • Antibody Diversity* / genetics
  • Antibody Diversity* / immunology
  • Antibody Specificity / genetics
  • Antibody Specificity / immunology
  • Antigens / immunology
  • Attachment Sites, Microbiological / genetics
  • Bacteriophages / enzymology
  • Bacteriophages / genetics
  • Binding Sites, Antibody / genetics
  • Binding Sites, Antibody / immunology
  • Cloning, Molecular
  • Escherichia coli / genetics*
  • Escherichia coli / virology
  • Gene Rearrangement / genetics
  • Genetic Vectors / genetics
  • Humans
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin Fragments / immunology
  • Integrases / genetics
  • Integrases / metabolism
  • Peptide Library*
  • Recombination, Genetic / genetics*
  • Reproducibility of Results
  • Sequence Analysis, DNA
  • Viral Proteins*

Substances

  • Antibodies
  • Antigens
  • Immunoglobulin Fragments
  • Peptide Library
  • Viral Proteins
  • Cre recombinase
  • Integrases