气候变化领域集成服务门户(CCPortal): Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity

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DOI	10.1126/science.abf5972
	Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity
	Blum T.R.; Liu H.; Packer M.S.; Xiong X.; Lee P.-G.; Zhang S.; Richter M.; Minasov G.; Satchell K.J.F.; Dong M.; Liu D.R.
发表日期	2021
ISSN	0036-8075
起始页码	803
结束页码	810
卷号	371 期号:6531
英文摘要	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. © 2021 American Association for the Advancement of Science. All rights reserved.
英文关键词	botulinum toxin; botulinum toxin E; botulinum toxin F; botulinum toxin X; phosphatidylinositol 3,4,5 trisphosphate 3 phosphatase; proteinase; SNARE protein; synaptobrevin; unclassified drug; vesicle associated membrane protein 4; vesicle associated membrane protein 7; botulinum toxin; phosphatidylinositol 3,4,5 trisphosphate 3 phosphatase; synaptobrevin; synaptobrevin 2; VAMP2 protein, human; VAMP4 protein, human; VAMP7 protein, human; YKT6 protein, human; bacteriophage; biotechnology; enzyme activity; host specificity; membrane; protein; substrate; toxin; animal cell; Article; bacteriophage; controlled study; enzyme active site; enzyme activity; enzyme specificity; enzyme substrate; genetic selection; light chain; nerve cell culture; nonhuman; nuclear reprogramming; phage assisted continuous evolution; priority journal; protein cleavage; rat; viral evolution; animal; cell culture; cell line; chemistry; directed molecular evolution; Enterobacteria phage M13; enzyme specificity; genetics; human; metabolism; mutation; nerve cell; peptide library; protein domain; protein engineering; Animals; Bacteriophage M13; Botulinum Toxins; Catalytic Domain; Cell Line; Cells, Cultured; Directed Molecular Evolution; Humans; Mutation; Neurons; Peptide Library; Protein Domains; Protein Engineering; PTEN Phosphohydrolase; R-SNARE Proteins; Rats; Selection, Genetic; Substrate Specificity; Vesicle-Associated Membrane Protein 2
语种	英语
来源期刊	Science
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/243069
作者单位	Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, United States; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, United States; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, United States; Department of Urology, Boston Children’s Hospital, Boston, MA 02115, United States; Department of Microbiology, Department of Surgery, Harvard Medical School, Boston, MA 02115, United States; Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States
推荐引用方式 GB/T 7714	Blum T.R.,Liu H.,Packer M.S.,et al. Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity[J],2021,371(6531).
APA	Blum T.R..,Liu H..,Packer M.S..,Xiong X..,Lee P.-G..,...&Liu D.R..(2021).Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.Science,371(6531).
MLA	Blum T.R.,et al."Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity".Science 371.6531(2021).