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Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.09.29.319145v1?rss=1
Authors: Dyer, R. P., Isoda, H. M., Salcedo, G. S., Speciale, G., Fletcher, M. H., Le, L. Q., Liu, Y., Malik, S. Z., Vazquez-Cintron, E. J., Chu, A. C., Rupp, D. C., Jacky, B., Nguyen, T. T., Steward, L. E., Majumdar, S., Brideau-Andersen, A. D., Weiss, G. A.
Abstract:
The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (approx. 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A's substrate and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A infiltrates neurons and retains its SNAP23 activity. The identification of substrate control loops outside BoNT/A's active site could guide the design of improved BoNT proteases and inhibitors.
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By Multimodal LLCLink to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.09.29.319145v1?rss=1
Authors: Dyer, R. P., Isoda, H. M., Salcedo, G. S., Speciale, G., Fletcher, M. H., Le, L. Q., Liu, Y., Malik, S. Z., Vazquez-Cintron, E. J., Chu, A. C., Rupp, D. C., Jacky, B., Nguyen, T. T., Steward, L. E., Majumdar, S., Brideau-Andersen, A. D., Weiss, G. A.
Abstract:
The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (approx. 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A's substrate and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A infiltrates neurons and retains its SNAP23 activity. The identification of substrate control loops outside BoNT/A's active site could guide the design of improved BoNT proteases and inhibitors.
Copy rights belong to original authors. Visit the link for more info