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Protein Engineering Design and Selection 2008 21(5):343-351; doi:10.1093/protein/gzn013
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants

Matthieu Chodorge1, Laurent Fourage2,3, Gilles Ravot2, Lutz Jermutus1 and Ralph Minter1,3

1Cambridge Antibody Technology, Milstein Building, Granta Park, Cambridge CB21 6GH, UK 2 Proteus S.A., 70 allee Graham Bell, Parc Georges Besse, 30000 Nimes, France

3 To whom correspondence should be addressed. E-mail: ralph.minter{at}cambridgeantibody.com (R.M.); lfourage{at}proteus.fr (L.F.)

The use of random mutagenesis in concert with protein display technologies to rapidly select high affinity antibody variants is an established methodology. In some cases, DNA recombination has been included in the strategy to enable selection of mutations which act cooperatively to improve antibody function. In this study, the impact of L-Shuffling DNA recombination on the eventual outcome of an in vitro affinity maturation has been experimentally determined. Parallel evolution strategies, with and without a recombination step, were carried out and both methods improved the affinity of an anti-Fas single chain variable fragment (scFv). The recombination step resulted in an increased population of affinity-improved variants. Moreover, the most improved variant, with a 22-fold affinity gain, emerged only from the recombination-based approach. An analysis of mutations preferentially selected in the recombined population demonstrated strong cooperative effects when tested in combination with other mutations but small, or even negative, effects on affinity when tested in isolation. These results underline the ability of combinatorial library approaches to explore very large regions of sequence space to find optimal solutions in antibody evolution studies.

Keywords: affinity maturation/directed evolution/Fas/in vitro recombination/ribosome display

Received November 7, 2007; revised March 5, 2008; accepted March 7, 2008.


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