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Author Notes:

E-mail: imatsum@emory.edu.

D.N.G. first expressed and purified the hexa-histidine tagged HIV PR, although she did not refold it. We are grateful to Lauren Ancel-Meyers, Stefan Lutz, Wayne Patrick, and Jennifer Stratton for their critical readings of the manuscript.

Claudia Schmidt-Dannert, Associate Editor


Research Funding:

We thank the National Institutes of Health/National Institute of Allergy and Infectious Diseases (1 R21AI054602-01) and National Institute of General Medical Services (1 R01 GM074264-01) for support.


  • directed evolution
  • random mutagenesis
  • high-throughput screening
  • aspartic protease

Diversification and Specialization of HIV Protease Function During In Vitro Evolution


Journal Title:

Molecular Biology and Evolution


Volume 23, Number 4


, Pages 764-772

Type of Work:

Article | Post-print: After Peer Review


Our goal 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. Broad-specificity enzymes are generally deleterious to living cells, so this 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 human immunodeficiency virus type I protease (HIV PR) is cytotoxic and exhibits no detectable activity in reactions with beta-galactosidase (BGAL). In contrast, the selected variants were nontoxic and exhibited greater activity and specificity against BGAL 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 engenders more rapid adaptation than positive selection alone.

Copyright information:

©The Author 2006. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

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