UNNATURAL AMINO ACID INCORPORATION INTO PROTEINS AND QUANTIFICATION THEROF

非天然氨基酸掺入蛋白质及其定量

• 批准号: 8169801
• 负责人: Paul R Ortiz De Montellano
• 金额: $ 0.18万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-12 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169801
• 关键词: Adoption; Amino Acid Sequence; Amino Acids; Amino Acyl-tRNA Synthetases; Biochemical; Chemicals; Codon Nucleotides; Computer Retrieval of Information on Scientific Projects Database; Funding; Grant; Heme; In Vitro; Institution; Knowledge; Label; Ligase; Mass Spectrum Analysis; Peptide Sequence Determination; Positioning Attribute; Proteins; Relative (related person); Research; Research Personnel; Resources; Site; Source; Structure; Technology; Transfer RNA; United States National Institutes of Health; Work; crosslink; in vivo

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. An approach pioneered by Prof. Peter Schultz (Scripps) allows for the site-specific incorporation of an unnatural amino acid anywhere within a protein sequence. This technology has proven useful for labeling proteins with fluorescent, 13C and 15N, and chemical-crosslinking unnatural amino acids for in vivo and in vitro structure-function studies. By using an orthogonal M. jannaschii tRNA/tRNA-synthetase pair, i.e., one where neither the tRNA nor the synthetase cross-reacts with the endogenous E.coli tRNA's or amino acyl tRNA synthetases, it is possible to introduce an unnatural amino acid residue at any position in the protein using a codon that is only recognized by the orthogonal tRNA. The Ortiz de Montellano lab acquired this labeling technology courtesy of the Schultz lab and is using it to label diverse heme containing proteins for biophysical and biochemical studies. Information on the extent of incorporation of the unnatural amino acid and the relative expression level of the protein can severely limit the application of this technology. For that reason, we utilize the UCSF Mass Spectrometry Facility to determine the extent and site of incorporation of the unnatural amino acids into the proteins, and to determine their relative expression levels. This knowledge is not only critical for our work, but will make possible the more widespread adoption of this powerful technology.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Peter Schultz 教授（斯克里普斯）首创的一种方法允许在蛋白质序列中的任何位置进行非天然氨基酸的位点特异性掺入。 事实证明，该技术可用于用荧光、13C 和 15N 以及化学交联非天然氨基酸标记蛋白质，以进行体内和体外结构功能研究。 通过使用正交 M. jannaschii tRNA/tRNA 合成酶对，即 t​​RNA 和合成酶都不与内源性大肠杆菌 tRNA 或氨酰 tRNA 合成酶发生交叉反应的一对，可以使用仅被正交 tRNA 识别的密码子在蛋白质的任何位置引入非天然氨基酸残基。 Ortiz de Montellano 实验室从 Schultz 实验室获得了这项标记技术，并使用它来标记多种含有血红素的蛋白质，用于生物物理和生化研究。有关非天然氨基酸掺入程度和蛋白质相对表达水平的信息可能会严重限制该技术的应用。因此，我们利用 UCSF 质谱设备来确定非天然氨基酸掺入蛋白质的程度和位点，并确定它们的相对表达水平。这些知识不仅对我们的工作至关重要，而且将使这种强大技术得到更广泛的采用成为可能。