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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 彼得·舒尔茨（Peter Schultz）教授（Scripps）率先提出的一种方法允许在蛋白质序列中任何地方的非天然氨基酸特异性掺入。 这项技术已被证明可用于将蛋白质用荧光，13C和15N标记，以及用于体内和体外结构功能研究的化学连接非天然氨基酸。 通过使用正交M. jannaschii tRNA/tRNA-合成酶对，即，tRNA和合成酶与内源性大肠杆菌tRNA或氨基酰基TRNA合成酶的交叉反应都不是在任何一个protna中以任何位置识别的，只能引入任何位置。 Ortiz de Montellano实验室获得了Schultz Lab提供的这种标签技术，并将其用于将多种血红素标记为含有生物物理和生化研究的蛋白质。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，可以严重限制该技术的应用。因此，我们利用UCSF质谱设施来确定非天然氨基酸掺入蛋白质的程度和部位，并确定其相对表达水平。这些知识不仅对我们的工作至关重要，而且将使这种强大的技术更广泛地采用。