一个合理设计的pyrrolysyl-tRNA合成酶突变体,具有广泛的基质谱
Yane-Shih Wang1, Xinqiang Fang, Ashley L Wallace
1Department of Chemistry, Texas A&M University, College Station, Texas 77843, USA.
Journal of the American Chemical Society
|February 1, 2012
概括
研究人员使用设计的酶和tRNA将新型氨衍生物遗传纳入蛋白质中. 这扩大了创建蛋白质的工具包,在大肠杆菌中增加了新的氨基酸.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 合成生物学 合成生物学
背景情况:
- 遗传密码的扩展使非正规氨基酸 (ncAAs) 可以被纳入蛋白质中.
- 解-tRNA合成酶 (PylRS) 和tRNA (CUA) (Pyl) 是将非自然氨基酸解结合的关键组成部分.
- 扩大PylRS的范围以接受多种类型的氨衍生物对于蛋白质工程至关重要.
研究的目的:
- 将氨衍生物与大型替代物遗传地纳入超级绿色光蛋白 (sfGFP).
- 为了设计一种能够接受这些修饰后的 fenylalanine 基质的 pyrrolysyl-tRNA 合成酶 (PylRS) 突变体.
- 为了证明工程PylRS-tRNA(CUA) ((Pyl) 对在扩大基因编码的氨基酸谱的实用性.
主要方法:
- 皮洛西尔-tRNA合成酶 (PylRS) 的理性设计和突变发生,特别针对N346A和C348A突变.
- 使用工程PylRS突变体与tRNA(CUA) ((Pyl) 结合使用以抑制大肠杆菌中的珀.
- 表达超级绿色光蛋白 (sfGFP) 含有特定地点的氨酸衍生物.
主要成果:
- 理性设计的PylRS突变体 (N346A/C348A) 成功地促进了各种氨酸衍生物的基因结合,并使用了大量的副替代剂.
- 这些经过修改的氨基酸在珀色的停止符号上被特定地插入 sfGFP 中.
- 经过工程设计的PylRS-tRNA (CUA) (Pyl) 系统证明了对多种类型的氨酸类似物具有广泛的基质接受性.
结论:
- 一种合理设计的PylRS突变使得可以将氨衍生物与重的替代物进行基因结合.
- 这一进步显著扩大了基因编码的非正规氨基酸的库存.
- 经过工程设计的PylRS-tRNA (CUA) (Pyl) 对为未来的遗传密码扩展工作提供了一个强大的平台.
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