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对于HRV 3C蛋白酶工程的一种组合策略,以实现N端自由裂变

Meng Mei¹, Xian Fan¹, Yu Zhou¹

¹State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China.

International journal of biological macromolecules

|March 23, 2024

概括

研究人员设计了人类犀牛病毒3C蛋白酶 (HRV 3CP),以提高其分裂特异性. 新的C3变种在P1上识别了更广泛的氨基酸范围.

科学领域:

生物化学生化学
酶工程是什么? 酶工程是什么?
结构生物学结构生物学

背景情况:

人类犀病毒3C蛋白酶 (HRV 3CP) 是生物技术和学术界广泛使用的融合标签去除工具.
HRV 3CP的一个关键限制是其严格的P1'基质特异性,导致裂变后不需要的N端甘氨酸残留物.
这种残留的甘氨酸可能会对重组蛋白的功能和免疫性产生负面影响.

研究的目的:

设计HRV 3CP以扩大其P1'基质特异性.
为了克服蛋白质加工中残留的N-终端甘氨酸的限制.
为蛋白质工程和生物技术开发改进的酶工具.

主要方法:

采用了结合策略,整合了结构导向的图书馆设计.
用于HRV 3CP工程的 eYESS 方法利用高通量选.
进行生物化学测定和下一代测序 (NGS) 介导的基质概况.
进行结构模拟来分析酶基质相互作用.

主要成果:

开发了HRV 3CP的C3变体,具有显著扩展的P1'特异性,识别了20种不同的氨基酸.
这种C3变种对LEVLFQ↓M的活性最高 (kcat/KM = 3.72 ± 0.04 mM−1s−1).
在P1位点保持基质强度,在P2'位点保持残留耐受性.
结构分析显示,在C3变体中重建了S1'结合口袋.

结论:

工程 C3 变体为精确的蛋白质加工提供了一个有价值的工具,克服了 HRV 3CP 的局限性.
扩大的P1'特异性提高了其在各种生物技术应用中的实用性.
提供了对酶基质识别机制的见解,并为进一步的酶工程提供了一个平台.

关键词:

定向进化是指导进化的.在 HRV 3C 蛋白酶中,基质特异性基质特异性

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