Protein Studies with an Expanded Genetic Code

描述(申请人提供)：蛋白质几乎处于每个生物过程的十字路口。这项研究的目标是利用体内特定部位掺入的非天然氨基酸来探索蛋白质的结构和功能，这是舒尔茨和他的同事最近开创的一项技术。这项研究将促进生物医学所有领域的研究，这些领域最终与蛋白质有关。特别是，在大肠杆菌系统中，将发现更多的正交tRNA-合成酶对将更多有趣的非天然氨基酸如3-乙酰苯丙氨酸结合到感兴趣的蛋白质中。掺入非天然3-乙酰苯丙氨酸作为“酮柄”的应用将包括：1)用荧光分子对蛋白质进行定点修饰，以实时显示目标蛋白质在体内的动态；2)生物素，从细胞裂解液中检测和纯化感兴趣的蛋白质。此外，还将开发一种基因选择，将非天然氨基酸整合到哺乳动物细胞的蛋白质中。这项研究的成功可能会通过直接适应蛋白质中的定制功能基团来彻底改变哺乳动物细胞的研究。这项技术将非常有用：1)产生特定位点的蛋白质修饰(磷酸化、糖基化等)。通过随意传递翻译后修改。2)从二十种常见的氨基酸中产生自然没有创造的新的蛋白质功能。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.