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Exploiting recombination in single bacteria to make large phage antibody libraries

Nature Biotechnology volume 18, pages 75–80 (2000)Cite this article

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 3×1011. A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigens.

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Figure 1: (A) Map of the display vector pDAN5 with an scFv cloned.
Figure 2: scheme of D1.3 recombination experiment.
Figure 3: V gene use and CDR3 length in primary, recombined, and selected scFvs.

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Acknowledgements

We are grateful to Roberto Marzari, Jianlong Lou, and Chonglin Yang for helpful discussions; to Francesco Tedesco for RNA from human lymphocytes; to Gabriella Rossi and Jessica Franzot for excellent technical help; and to the following for the proteins they kindly provided for selection: Min Park (Rad52), Scott Peterson (cyclin D, cdk2, cdc25A), Tom Peat (phosphoglycerate dehydrogenase). We would also like to thank Brian Sauer for providing BS1365 as well as many useful discussions on Cre recombinase. This work was partly financed by Fondo Trieste.

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  1. Andrew Bradbury

    Present address: Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM, 87545

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  1. International School for Advanced Studies (SISSA), Biophysics Sector, Via Beirut 2-4, Trieste, 34014 , Italy

    Daniele Sblattero & Andrew Bradbury

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Correspondence to Andrew Bradbury.

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Sblattero, D., Bradbury, A. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnol 18, 75–80 (2000). https://doi.org/10.1038/71958

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