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Nucleic Acids Research 2005 33(3):e32; doi:10.1093/nar/gni031
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Published online 18 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

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Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

Marcus D. Hughes1,3, Zhan-Ren Zhang1, Andrew J. Sutherland2, Albert F. Santos4 and Anna V. Hine1,3,*

1 School of Life and Health Sciences, Aston University Aston Triangle, Birmingham B4 7ET, UK 2 Chemical Engineering and Applied Chemistry, School of Engineering & Applied Science, Aston University Aston Triangle, Birmingham B4 7ET, UK 3 ProtaMAX Ltd 55 Colmore Row, Birmingham B3 2AS, UK 4 GE Healthcare, Cardiff Laboratories Forest Farm, Whitchurch, Cardiff CF14 7YT, UK

*To whom correspondence should be addressed. Tel: +44 121 204 3961; Fax: +44 121 359 0733; Email: a.v.hine{at}aston.ac.uk

Received December 7, 2004. Revised January 27, 2005. Accepted January 27, 2005.

We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using ‘MAX’ randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40 000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants.

Present address: Zhan-Ren Zhang, Millipore Bioprocessing Ltd, No. 1 Industrial Estate, Medomsley Road, Consett, Co. Durham DH8 6SZ, UK


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