genetic incorporation of unnatural amino acids into proteins in mycobacterium tuberculosis基因的非自然氨基酸成蛋白质在结核分枝杆菌.pdfVIP

Genetic Incorporation of Unnatural Amino Acids into Proteins in Mycobacterium tuberculosis 1 2 1 1 1,2 Feng Wang , Scott Robbins , Jiantao Guo , Weijun Shen , Peter G. Schultz * 1 Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America, 2 Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America Abstract 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 ami