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


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.

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Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
Additional Information: © The Author 2005. Published by Oxford University Press. All rights reserved
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Uncontrolled Keywords: MAX randomisation,saturation mutagenesis,combinatorial biology,protein engineering,phage display,Genetics
Publication ISSN: 1362-4962
Last Modified: 09 Jan 2024 08:05
Date Deposited: 14 Apr 2010 13:00
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Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
http://nar.oxfo ... stract/33/3/e32 (Publisher URL)
PURE Output Type: Article
Published Date: 2005-02-18
Authors: Hughes, Marcus D.
Zhang, Zhan-Ren
Sutherland, Andrew (ORCID Profile 0000-0003-4065-831X)
Santos, Albert F.
Hine, Anna V. (ORCID Profile 0000-0003-4065-831X)



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