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含有光氧醇氨基酸的蛋白质的细胞质合成

Shengxi Chen¹, Xun Ji¹, Mingxuan Gao¹

¹Biodesign Center for BioEnergetics, and School of Molecular Sciences , Arizona State University , Tempe , Arizona 85287 , United States.

Journal of the American Chemical Society

|March 21, 2019

概括

研究人员扩展了基因编码, 将光氧醇氨基酸纳入活体大肠杆菌细胞中的蛋白质. 这种新型的氨基酸扩大了蛋白质的工程能力,

科学领域:

生物化学
分子生物学
合成生物学

背景情况:

基因代码扩展允许蛋白质中的非正规氨基酸 (ncAAs),主要是α-l-氨基酸.
已使用修改后的核糖体来结合二和二二胺剂.
自然系统有利于特定的氨基酸结构,限制了纳入的ncAAs的多样性.

研究的目的:

在细胞质中实现非正规的,缺乏α-氨基基组的光氧醇氨基酸的结合.
为了证明这种酸氨基酸作为光蛋白标签的实用性.
扩大用于蛋白质工程的基因编码氨基酸库.

主要方法:

使用修饰的大肠杆菌核糖体和pyrrolysyl-tRNA合成酶 (PylRS).
编码目标蛋白质 (hnRNP LL RRM1 域) 和 PylRS 系统的同转化质粒.
在含有氧醇氨基酸 (氧醇2) 的介质中化细胞,并通过质谱检测验证其结合.
表达和分析的绿色光蛋白和MreB蛋白.

主要成果:

在大肠杆菌细胞中成功地将oxazol 2 纳入 hnRNP LL RRM1 域,GFP 和 MreB.
氧化2的加入显著增强了蛋白质的光.
修改后的MreB保留了野生型蛋白质功能和细胞形态.
通过质谱检测验证了成功的结合.

结论:

在使用修改后的核糖体和PylRS的非正规性素氨基酸的细胞体内证明.
加入的oxazole 2作为一种强烈的光标签,增强蛋白质的光.
这种方法显著扩大了可用于体内蛋白质合成的氨基酸范围.

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