Protein Studies with an Expanded Genetic Code

扩展遗传密码的蛋白质研究

• 批准号: 6630422
• 负责人: ZHIWEN Jonathan ZHANG
• 金额: $ 4.3万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6630422
• 关键词: Escherichia coli; aminoacid; aminoacid tRNA ligase; biotechnology; cell line; peptide library; phenylalanine; postdoctoral investigator; protein biosynthesis; protein engineering; protein purification; protein structure function; transfection

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.

描述（由申请人提供）：蛋白质几乎处于每一个生物过程的十字路口。这项研究的目标是利用体内特异性结合的非天然氨基酸来探测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。这项研究将促进最终与蛋白质相关的生物医学所有领域的研究。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。加入的非天然3-乙酰基苯丙氨酸作为“酮处理”的应用将包括位点特异性蛋白质修饰，1)荧光分子允许实时可视化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，将开发一种遗传选择，将非天然氨基酸纳入哺乳动物细胞中的蛋白质中。这项研究的成功可能会彻底改变哺乳动物细胞的研究，直接调节蛋白质中定制的功能基团。这项技术将非常有用：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)从20种常见的氨基酸中产生自然界没有创造出来的新的蛋白质功能。这些蛋白质的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。