UNNATURAL AMINO ACID INCORPORATION INTO PROTEINS AND QUANTIFICATION THEROF

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 由Peter Schultz教授（Scripps）开创的一种方法允许在蛋白质序列内的任何地方位点特异性地掺入非天然氨基酸。 该技术已被证明可用于标记蛋白质与荧光，13 C和15 N，和化学交联的非天然氨基酸在体内和体外的结构-功能研究。 通过使用正交M.詹氏tRNA/tRNA-合成酶对，即，当tRNA和合成酶都不与内源性大肠杆菌tRNA或氨酰tRNA合成酶交叉反应时，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蒙泰拉诺实验室从Schultz实验室获得了这种标记技术，并正在使用它来标记各种含血红素的蛋白质，用于生物物理和生物化学研究。关于非天然氨基酸的掺入程度和蛋白质的相对表达水平的信息可以严重限制该技术的应用。出于这个原因，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，而且将使这项强大的技术得到更广泛的采用。

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.