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在大肠杆菌的遗传密码中添加L-3-(2-Naphthyl) alanine

Lei Wang¹, Ansgar Brock, Peter G Schultz

¹Department of Chemistry, University of California at Berkeley, Berkeley, California 94720, USA.

Journal of the American Chemical Society

|February 28, 2002

概括

研究人员在特定地点将一种非自然的氨基酸纳入大肠杆菌中的蛋白质中,使用一种进化的酶和tRNA. 这种遗传密码扩展方法实现了高保真性,使蛋白质工程的新可能性成为可能.

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:

遗传密码包括20种标准氨基酸,限制了蛋白质的多样性和功能.
对非自然氨基酸 (UAA) 的特定位点内置为蛋白质提供了一种扩展该代码的途径.
以前的UAA整合方法在效率和忠实性方面遇到了挑战.

研究的目的:

开发一种可靠的方法,用于将UAA具体纳入大肠杆菌中的蛋白质.
为了证明一个进化的正交氨基酸-tRNA合成酶 (aaRS) 系统在UAA传递中的实用性.
评估这种方法对于遗传密码扩展的忠实性和适用性.

主要方法:

一个对立的氨基-tRNA合成酶 (aaRS) 的演变,它是特定于L-3-(2-naphthyl) alanine的.
一个直角珀抑制器tRNA的设计和使用.
在E. coli中通过珀无意义的codon抑制将L-3-(2-naphthyl) alanine纳入E. coli中的蛋白质.
使用工程系统评估翻译忠实性.

主要成果:

成功地将非自然氨基酸L-3-(2-naphthyl) alanine局部特异性纳入了大肠杆菌中的蛋白质.
开发了一种正交的aaRS,可以选择性地将UAA氨基化到正交tRNA上,效率高.
在响应珀色无意义代码的过程中,UAA结合的翻译保真度超过99%.
证明了这种方法对广泛的UAA的潜在适用性.

结论:

开发的方法使UAA能够高效,高保真地将UAA纳入蛋白质.
这种方法显著扩大了蛋白质工程和功能研究的可能性.
用UAA扩展遗传密码有望增强蛋白质和潜在的生物体功能.

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