具有重编程特异性的植物毒素蛋白酶的菌体辅助进化
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
概括
科学家们使用菌体辅助进化系统设计了具有新功能的定制蛋白质酶. 这一突破使得特定蛋白质的精确向成为可能,
科学领域:
- 生物化学
- 分子生物学
- 生物技术
背景情况:
- 开发具有定制裂变特性的蛋白酶对于生物技术和医学至关重要.
- 现有的蛋白酶工程方法具有挑战性和局限性.
- 肉毒神经毒素 (BoNT) 轻链蛋白酶为工程新特异性提供了一个起点.
研究的目的:
- 开发一个用于重新编程蛋白酶特异性的多功能平台.
- 设计肉毒神经毒素 (BoNT) 轻链蛋白酶,使其能够切割新的非原生基质.
- 为了证明治疗应用的蛋白酶特异性的显著变化.
主要方法:
- 使用菌体辅助蛋白酶进化系统,同时进行正负选择.
- 将该系统应用于三个不同的BoNT轻链蛋白酶 (BoNT/X,BoNT/F和BoNT/E).
- 对已演化的蛋白酶进行了针对特定标蛋白和自然基质的裂变活性测试.
主要成果:
- 设计了BoNT/X变种, 专门切割VAMP4和Ykt6.
- 开发了BoNT/F蛋白酶变体,可以选择性地切割非原生基质VAMP7.
- 创建了能够在不影响自然神经元基质的情况下切割PTEN的BoNT/E蛋白酶变体.
- 观察到蛋白酶特异性的显著增加,从218倍到1100万倍以上.
- 进化的蛋白酶保持了它们在原始神经元中形成自我递送的全毒素的能力.
结论:
- 为工程蛋白酶特异性建立了一个强大的多功能平台.
- 证明了BoNT蛋白酶的成功重新编程以准具有治疗意义的新基质.
- 开发的系统具有促进基于蛋白酶的生物技术和治疗的巨大潜力.
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