Protein Studies with an Expanded Genetic Code

扩展遗传密码的蛋白质研究

• 批准号: 6551488
• 负责人: ZHIWEN Jonathan ZHANG
• 金额: $ 3.83万
• 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
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6551488
• 关键词: Escherichia coli; aminoacid; aminoacid analog; 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）：蛋白质几乎处于每个生物过程的十字路口。这项研究的目标是利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，这是舒尔茨和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，将发现更多正交 tRNA 合成酶对掺入更多有趣的非天然氨基酸，例如3-乙酰基苯丙氨酸转化为感兴趣的蛋白质。掺入的非天然 3-乙酰基苯丙氨酸作为“酮手柄”的应用将包括使用 1) 荧光分子进行位点特异性蛋白质修饰，以实现体内目标蛋白质动态的实时可视化，2) 生物素以检测和纯化细胞裂解物中的目标蛋白质。此外，还将开发遗传选择，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通过直接在蛋白质中容纳定制的功能基团来彻底改变哺乳动物细胞的研究。该技术对于以下方面非常有用：1) 随意生成位点特异性蛋白质修饰（磷酸化、糖基化等）——绕过翻译后修饰。 2）从二十种常见氨基酸中产生自然界尚未创造的新蛋白质功能。这些蛋白质功能将有助于理解细胞生物学和创建用于治疗干预的试剂。