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PEDS Advance Access originally published online on September 26, 2006
Protein Engineering Design and Selection 2006 19(11):517-524; doi:10.1093/protein/gzl039
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Bioinformatics-driven, rational engineering of protein thermostability

Mary Kate DiTursi, Seok-Joon Kwon, Philippa J. Reeder and Jonathan S. Dordick1

Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute Troy, NY 12180-3590, USA

1To whom correspondence should be addressed. E-mail: dordick{at}rpi.edu

A longstanding goal in protein engineering is to identify specific sequence changes that endow proteins with desired functional properties. As opposed to traditional rational and random protein engineering techniques, we have employed a bioinformatic approach to identify specific sequence changes that influence key functional properties of a protein within a defined superfamily. Specifically, we have used the Bayesian sequence-based algorithms PROBE and Classifier to identify a strand–turn–strand motif that contributes to thermophilicity among members of the serine protease subtilase superfamily. By replacing a 16 amino acid sequence in the mesophilic subtilisin E (from Bacillus subtilis) with a bioinformatics-generated thermophilic model sequence, the melting temperature of subtilisin E was increased by 13°C. While wild-type subtilisin E was inactive at 90°C, the mutant retained a substantial fraction of its function, with ca. one-third of the activity that it has at 45°C.

Keywords: PROBE and Classifier/strand–turn–strand motif/subtilisin/thermostability

Received April 28, 2006; revised July 24, 2006; accepted August 18, 2006.


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