Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America.
PLoS One. 2010 Feb 22;5(2):e9354. doi: 10.1371/journal.pone.0009354.
New tools are needed to study the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), to facilitate new drug discovery and vaccine development. We have developed methodology to genetically incorporate unnatural amino acids into proteins in Mycobacterium smegmatis, BCG and Mtb, grown both extracellularly in culture and inside host cells. Orthogonal mutant tRNATyr/tyrosyl-tRNA synthetase pairs derived from Methanococcus jannaschii and evolved in Escherichia coli incorporate a variety of unnatural amino acids (including photocrosslinking, chemically reactive, heavy atom containing, and immunogenic amino acids) into proteins in response to the amber nonsense codon. By taking advantage of the fidelity and suppression efficiency of the MjtRNA/pIpaRS pair in mycobacteria, we are also able to use p-iodophenylalanine to induce the expression of proteins in mycobacteria both extracellularly in culture and inside of mammalian host cells. This provides a new approach to regulate the expression of reporter genes or mycobacteria endogenous genes of interest. The establishment of the unnatural amino acid expression system in Mtb, an intracellular pathogen, should facilitate studies of TB biology and vaccine development.
需要新的工具来研究细胞内病原体结核分枝杆菌（Mtb），这是结核病（TB）的病原体，以促进新药发现和疫苗开发。我们已经开发了一种方法，将非天然氨基酸基因整合到分枝杆菌、卡介苗和结核分枝杆菌的蛋白质中，这些蛋白质在体外培养和宿主细胞内生长。来自产甲烷球菌和在大肠杆菌中进化的正交突变 tRNATyr/tyrosyl-tRNA 合成酶对可以将各种非天然氨基酸（包括光交联、反应性化学、含重原子和免疫原性氨基酸）掺入到蛋白质中，以响应琥珀终止密码子。利用分枝杆菌中 MjtRNA/pIpaRS 对的保真度和抑制效率，我们还能够使用对碘苯丙氨酸在体外培养的和哺乳动物宿主细胞内诱导蛋白质的表达。这为调节报告基因或分枝杆菌感兴趣的内源基因的表达提供了一种新方法。在细胞内病原体结核分枝杆菌中建立非天然氨基酸表达系统，应该有助于研究结核病生物学和疫苗开发。
