Protein Studies with an Expanded Genetic Code

描述（由申请人提供）：蛋白质几乎处于每个生物过程的十字路口。这项研究的目标是使用体内位点特异性掺入的非天然氨基酸来探测蛋白质的结构和功能，这是Schultz及其同事最近开创的一项技术。这项研究将促进最终与蛋白质相关的生物医学所有领域的研究。特别是在E. coli系统中，将发现更多的正交tRNA-合成酶对将更多感兴趣的非天然氨基酸例如3-乙酰基苯丙氨酸掺入感兴趣的蛋白质中。掺入的非天然3-乙酰基苯丙氨酸作为“酮柄”的应用将包括用1）荧光分子进行位点特异性蛋白质修饰以允许靶蛋白在体内的动态实时可视化，2）生物素以从细胞裂解物中检测和纯化目的蛋白质。此外，将开发一种遗传选择，将非天然氨基酸掺入哺乳动物细胞的蛋白质中。这项研究的成功可能会彻底改变哺乳动物细胞的研究，直接适应蛋白质中定制的功能基团。该技术将非常有用：1）产生位点特异性蛋白质修饰（磷酸化、糖基化等）。随意-绕过翻译后修饰。2)产生新的蛋白质功能，自然界没有从20种常见氨基酸中创造出来。这些蛋白质功能将有助于理解细胞生物学和创造用于治疗干预的试剂。

DESCRIPTION (provided by applicant): Proteins are at the crossroads of virtually every biological process. The goal of this research is to probe protein structure and function using in vivo site-specifically incorporated unnatural amino acids, a technology recently pioneered by Schultz and coworkers. This study will facilitate research in all areas of biomedicine which is ultimately protein-related. In particular, in the E. coli system, more orthogonal tRNA-synthetase pairs will be found to incorporate more interesting unnatural amino acids e.g. 3-acetyl phenylalanine into proteins of interest. Applications of incorporated unnatural 3-acetyl phenylalanine as a "ketone handle" will include site-specifically protein-modification with 1)fluorescent molecules to allow the real-time visualization of dynamics of target protein in vivo, 2) biotin to detect and purify the protein of interest from cell lysate. Furthermore, a genetic selection will be developed to incorporate unnatural amino acids into proteins in mammalian cells. The success of this research may revolutionize the study of mammalian cells by directly accommodating tailored functional groups in proteins. This technology would be very useful for: 1) generating site-specific protein modification (phosphorylation, glycosylation etc.) at will - by-passing post-translational modifications. 2) generating new protein functions that nature has not created from the twenty common amino acids. These protein functions would be useful for understanding cell biology and creating reagents for therapeutic intervention.