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PERRC:保护酶工程与反应剂停留时间控制

Sage Nelson¹, Jokent Gaza², Seyednima Ajayebi¹

¹Department of Chemical Engineering, University of Florida, Gainesville, 32611, USA.

bioRxiv : the preprint server for biology

|March 17, 2025

概括

研究人员使用Protease Engineering与反应物停留时间控制 (PERRC) 设计了一种具有增强特异性的蛋白酶. 这种方法精确地控制了进化的严格性,创造了一个新的蛋白酶变体 (TEVESNp),对其基质的偏好是65倍.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
蛋白质工程是指蛋白质工程.

背景情况:

具有工程特异性的蛋白酶对治疗和生物技术有价值.
自然蛋白酶的广泛基质特异性限制了它们的应用.
工程蛋白酶特异性是具有挑战性的,因为其在本地基质上的残留活性.

研究的目的:

开发一种用于工程蛋白酶特异性的新平台.
创建一个直角蛋白质酶变体,具有高基质选择性.
为了证明平台在构建蛋白质电路中的实用性.

主要方法:

开发了使用反应物停留时间控制 (PERRC) 平台的蛋白酶工程.
PERRC利用内等质网膜 (ER) 保留序列强度和停留时间.
为精确的进化控制,调整了对抗选择与选择基质比率.

主要成果:

进化了一个正交的烟草蚀刻病毒蛋白酶变体 (TEVESNp).
TEVESNp显示对单氨基酸改变基质 (ENLYFES) 的偏好是其母基质 (ENLYFQS) 的 65 倍.
证明了TEVESNp在细菌直角蛋白质电路结构中的功能.

结论:

PERRC使得蛋白酶特异性的精确工程成为可能.
TEVESNp是第一个基于单个氨基酸差异的高选择性工程直角蛋白酶.
分子动力学模拟显示了功能性活性部位的重新排列.

关键词:

高通量选高通量选保护酶工程是保护酶的工程.烟草蚀刻病毒 (TEV) 蛋白酶酵母表面显示显示器

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