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PEDS Advance Access published online on June 27, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi042
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received May 25, 2005
Accepted May 26, 2005

Article

Generating molecular diversity by homologous recombination in Escherichia coli

Peter L. Wang 1*, Benny K. C. Lo 2, and Greg Winter 3

1 Centre for Protein Engineering, University of Cambridge, Hills Road, Cambridge CB2 2QH, UK; Present address: Department of Biochemistry, Stanford University Medical School, Stanford, CA 94305, USA
2 Laboratory of Molecular Biology, University of Cambridge, Hills Road, Cambridge CB2 2QH, UK; Present address: St Edmund's College, University of Cambridge, Cambridge CB3 0BN, UK
3 Centre for Protein Engineering, University of Cambridge, Hills Road, Cambridge CB2 2QH, UK; Laboratory of Molecular Biology, University of Cambridge, Hills Road, Cambridge CB2 2QH, UK

* To whom correspondence should be addressed.
Peter L. Wang, E-mail: plwang{at}stanford.edu


  Abstract

We explored the use of recE-mediated homologous recombination to generate molecular diversity in Escherichia coli. Two homologous genes were placed on different phagemid vectors each comprising multiple EcoRI restriction sites and overlapping N- and C-terminal portions of {beta}-lactamase. By co-infection of these phage into RecE+ EcoRI+ E.coli, we were able to introduce double-strand breaks into these vectors, allowing efficient homologous recombination (in up to 10% of bacteria) by the recE pathway and selection of the recombinants by resistance to ampicillin. Recombination gave single crossovers; these were more frequent near the EcoRI sites and the recombination frequency increased with the target length and degree of homology. The system was used to create a large combinatorial chicken antibody library (1010) for display on filamentous phage and to isolate several antibody fragments with binding affinities in the 10-100 nM range.

Keywords: antibody phage display; chicken bursa; single-chain Fv library.
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