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

Hughes, Marcus D., Zhang, Zhan-Ren, Sutherland, Andrew, Santos, Albert F. and Hine, Anna V. (2005). Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing:identification of novel zinc finger proteins. Nucleic Acids Research, 33 (3),

Abstract

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.

Publication DOI: https://doi.org/10.1093/nar/gni031
Divisions: Life & Health Sciences > Biosciences
Life & Health Sciences
Additional Information: © 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@oupjournals.org
Uncontrolled Keywords: MAX randomisation,saturation mutagenesis,combinatorial biology,protein engineering,phage display,Genetics
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Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
http://nar.oxfo ... stract/33/3/e32 (Publisher URL)
Published Date: 2005-02-18
Authors: Hughes, Marcus D.
Zhang, Zhan-Ren
Sutherland, Andrew ( 0000-0003-4065-831X)
Santos, Albert F.
Hine, Anna V. ( 0000-0003-4065-831X)

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