Engineering Environmentally-Stable Proteases to Specifically Neutralize Protein Toxins
Final rept. 15 Sep 2010-14 Sep 2013
POTOMAC AFFINITY PROTEINS LLC NORTH POTOMAC MD
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This project is intended to develop the tools and principles necessary to engineer subtilisin proteases which specifically target and deactivate biological warfare agent BWA toxins. We are engineering and evolving subtilisin proteases that specifically target and deactivate BoNT, SEB, ricin, and B. anthracis lethal factor LF, representing four functionally distinct families of toxins. The centerpiece of our design effort is a phage-display selection method for creating tightly-regulated proteases of high specificity. In this system the protease, substrate sequence, and regulatory co-factor are co-evolved. The key accomplishments this past year were 1. Determined the structure of an evolved variant pT2077 in complex with the substrate sequence used to select it. 2. Designevolution of a highly active enzyme that can cut P4 I pT2050. 3. Computational design of specificity for an ionic P4 amino acid P4 E, pT2121 and P4 K, pT2114 4. Engineered protease chain reactions that can reliably measure concentrations of 250 fM range in a 20 hour assay.
- Medicine and Medical Research