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Author Notes:

Address correspondence to this author at the 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, USA; Tel: (404) 727-5625; Fax: (404) 727-3452; E-mail: imatsum@emory.edu

IM constructed PBAD-HIV PR-pCDF and the Plac*-6his-gusA-HA-(NNN)12-pET20 library; he isolated GUS (clone 461) and fused the gene (and the selected peptide) to lacZ.


Research Funding:

National Institutes of Health

This work was supported by the NIH/NIAID (1 R21AI054602-01).


  • Molecular switch
  • biosensor
  • reporter
  • random elongation mutagenesis
  • directed evolution

HIV Protease-Activated Molecular Switches Based on Beta-Glucuronidase and Alkaline Phosphatase


Journal Title:

Combinatorial Chemistry and High Throughput Screening


Volume 9, Number 4


, Pages 313-320

Type of Work:

Article | Post-print: After Peer Review


Our long-term goal is to direct the evolution of novel protease variants. To this end we have engineered a new type of protease-activated reporter enzyme. Many protease-activated enzymes evolved in nature, but the introduction of novel regulatory mechanisms into normally unregulated enzymes poses a difficult design challenge. Random Elongation Mutagenesis [1] was used to fuse the p6 peptide, which is recognized and cleaved by HIV protease, and twelve random sequence amino acids to the C-termini of beta-glucuronidase (GUS) and alkaline phosphatase (AP). The resulting GUS- p6-(NNN)12 and AP-p6-(NNN)12 libraries were expressed in E. coli and screened for clones that were inactivated by the C-terminal extension (tail). The inactivated clones were co-expressed with HIV protease, and those that were re-activated were isolated. The AP and GUS activities of the most responsive clones were each >3.5-fold higher when co-expressed with HIV protease, and this activation is correlated with in vivo proteolysis. It should be possible to generalize this strategy to different reporter enzymes, different target proteases, and perhaps to other types of protein-modifying enzymes.
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