Skip Navigation



MBE Advance Access published online on January 19, 2006
Molecular Biology and Evolution, doi:10.1093/molbev/msj098
This Article
Right arrow Advance Access manuscript (PDF) Freely available
Right arrow All Versions of this Article:
23/4/764    most recent
msj098v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by O'Loughlin, T. L.
Right arrow Articles by Matsumura, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by O'Loughlin, T. L.
Right arrow Articles by Matsumura, I.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org
Accepted January 17, 2006

Research Article

Diversification and Specialization of HIV Protease Function During in vitro Evolution

Taryn L. O'Loughlin 1, Dina N. Greene 1, and Ichiro Matsumura 1 *

1 Department of Biochemistry, Center for Fundamental and Molecular Evolution, Emory University School of Medicine, Rollins Research Center, Room 4119, 1510 Clifton Road, Atlanta, GA 30322

* To whom correspondence should be addressed.
Ichiro Matsumura, E-mail: imatsum{at}emory.edu


  Abstract

Our goal endpoint is to understand how enzymes adapt to utilize novel substrates. We, and others have shown that directed evolution tends to generate enzyme variants with broadened substrate specificity. Most broad-specificity enzymes would be deleterious to contemporary cells so the observed trend might be an artifact of the most commonly employed high throughput screens. Here we demonstrate a more natural and effective screening strategy for directed evolution. The gene encoding model enzyme HIV protease was randomly mutated, and the resulting library was expressed in Escherichia coli cells to eliminate cytotoxic broad-specificity variants. The surviving variants were screened for clones with activity against a reporter enzyme. The wild-type HIV PR is cytotoxic, and exhibits no detectable activity in reactions with beta-galactosidase. In contrast, the selected variants were non-toxic and exhibited greater activity and specificity against beta-galactosidase than did the wild-type HIV PR in reactions with any substrate. A single round of whole gene random mutagenesis and conventional high throughput screening does not usually effect complete inversions of substrate specificity. This suggests that a combination of positive and purifying selection leads to more rapid adaptation than positive selection alone.

Keywords: directed evolution; random mutagenesis; high throughput screening; aspartic protease.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
T. L. O'Loughlin, W. M. Patrick, and I. Matsumura
Natural history as a predictor of protein evolvability
Protein Eng. Des. Sel., October 1, 2006; 19(10): 439 - 442.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.