Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity
Year: 2021
Type: article
Abstract: Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.
Source: Science
Language: English
Subfield: Clinical Neurology
Field: Medicine
Domain: Health Sciences
Open Access status: green
Awards: REPROGRAMMING BOTULINUM NEUROTOXIN PROTEASES THROUGH CONTINUOUSEVOLUTION AS A STRATEGY FOR DEVELOPING SELECTIVE INTRACELLULAR THERAPIES, G533379926, Molecular mechanism for retrograde transport of tetanus neurotoxin, Structure and Function of C. Difficile Toxins, Continuous Evolution of Proteins with Novel Therapeutic Potential, Continuous Evolution of Proteins with Novel Therapeutic Potential, Molecular basis of action and pathogenesis of Clostridium difficile toxin B, ePACE: automation platforms for adaptable and scalable continuous evolution of biomolecules with therapeutic potential, Integrating Chemistry and Evolution to Illuminate Biology and Enable Novel Therapeutics, Biology and Engineering of Botulinum Neurotoxins.
OpenAlex ID: W3129903208