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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity
Travis R Blum1,2,3, Hao Liu4,5, Michael S Packer1,2,3
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.
Science (New York, N.Y.)
|February 19, 2021
Summary
Scientists engineered custom proteases with new functions using a phage-assisted evolution system. This breakthrough allows for precise targeting of specific proteins, advancing biotechnology and therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Developing proteases with custom cleavage specificities is crucial for biotechnology and medicine.
- Existing methods for protease engineering are challenging and limited.
- Botulinum neurotoxin (BoNT) light-chain proteases offer a starting point for engineering novel specificities.
Purpose of the Study:
- To develop a versatile platform for reprogramming protease specificity.
- To engineer botulinum neurotoxin (BoNT) light-chain proteases to cleave novel, non-native substrates.
- To demonstrate significant changes in protease specificity for therapeutic applications.
Main Methods:
- Utilized a phage-assisted protease evolution system incorporating simultaneous positive and negative selection.
- Applied the system to three distinct BoNT light-chain proteases (BoNT/X, BoNT/F, and BoNT/E).
- Evolved proteases were tested for cleavage activity against specific target proteins and natural substrates.
Main Results:
- Engineered BoNT/X variants to specifically cleave VAMP4 and Ykt6.
- Developed BoNT/F protease variants that selectively cleave the non-native substrate VAMP7.
- Created BoNT/E protease variants capable of cleaving PTEN without affecting natural neuronal substrates.
- Observed substantial increases in protease specificity, ranging from 218-fold to over 11,000,000-fold.
- Evolved proteases maintained their ability to form self-delivering holotoxins in primary neurons.
Conclusions:
- Established a robust and versatile platform for engineering protease specificity.
- Demonstrated the successful reprogramming of BoNT proteases to target new substrates of therapeutic interest.
- The developed system holds significant potential for advancing protease-based biotechnologies and therapeutics.
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