Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.
Science
; 371(6531): 803-810, 2021 02 19.
Article
em En
| MEDLINE
| ID: mdl-33602850
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Toxinas Botulínicas
/
Engenharia de Proteínas
/
Evolução Molecular Direcionada
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Science
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos