Therapeutic antibodies are the standard of care for certain cancers, auto-immune and infectious diseases, but their efficacy decreases when the number of target molecules is too large. The engineering of proteases that specifically catalyze the cleavage of disease-promoting proteins would enable enzyme therapies for a wider range of diseases. HIV protease is a nearly ideal starting point for this purpose because three lines of evidence suggest that this enzyme is unusually evolvable. First, HIV protease evolves rapidly in nature in response to synthetic protease inhibitors. Second, this observed evolvability is consistent with evolutionary theory. Third, the investigator has already employed random mutagenesis and screening to create a variant that is specific for TNF-alpha (target protein implicated in rheumatoid arthritis). He now proposes studies with the following specific aims: 1. to improve the activity and TNF-alpha specificity of the HIV protease variant through a directed evolution process that mimics antibody affinity maturation. 2. to direct the evolution of HIV protease variants specific for the oncogenic protein HER2. 3. to discover the structural mechanisms of adaptive evolution by purifying the wild-type and variant HIV proteases and characterizing their biophysical and kinetic properties in vitro. These experiments are a first step toward a new approach to enzyme therapy. We will demonstrate the unusual evolvability of HIV protease, and better understand why some enzymes are more amenable to protein engineering than others. The high throughput screens developed for these studies could also be used to identify small molecule protease inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM074264-01
Application #
6908330
Study Section
Special Emphasis Panel (ZRG1-GDD (01))
Program Officer
Basavappa, Ravi
Project Start
2005-05-01
Project End
2010-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
1
Fiscal Year
2005
Total Cost
$272,829
Indirect Cost
Name
Emory University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
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Bryksin, Anton V; Matsumura, Ichiro (2010) Rational design of a plasmid origin that replicates efficiently in both gram-positive and gram-negative bacteria. PLoS One 5:e13244
Bryksin, Anton V; Matsumura, Ichiro (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 48:463-5
Patrick, Wayne M; Matsumura, Ichiro (2008) A study in molecular contingency: glutamine phosphoribosylpyrophosphate amidotransferase is a promiscuous and evolvable phosphoribosylanthranilate isomerase. J Mol Biol 377:323-36
Greene, Dina N; Whitney, Spencer M; Matsumura, Ichiro (2007) Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency. Biochem J 404:517-24
Patrick, Wayne M; Quandt, Erik M; Swartzlander, Dan B et al. (2007) Multicopy suppression underpins metabolic evolvability. Mol Biol Evol 24:2716-22
Geddie, Melissa L; Matsumura, Ichiro (2007) Antibody-induced oligomerization and activation of an engineered reporter enzyme. J Mol Biol 369:1052-9
O'Loughlin, Taryn L; Patrick, Wayne M; Matsumura, Ichiro (2006) Natural history as a predictor of protein evolvability. Protein Eng Des Sel 19:439-42
O'Loughlin, Taryn L; Greene, Dina N; Matsumura, Ichiro (2006) Diversification and specialization of HIV protease function during in vitro evolution. Mol Biol Evol 23:764-72
O'Loughlin, Taryn L; Matsumura, Ichiro (2006) HIV protease-activated molecular switches based on beta-glucuronidase and alkaline phosphatase. Comb Chem High Throughput Screen 9:313-20