title: Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis.
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 amino acid expression system in Mtb, an intracellular pathogen, should facilitate studies of TB biology and vaccine development.
authors.affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Scott
authors.initials: S
authors.lastname: Robbins
authors.forename: Jiantao
authors.initials: J
authors.lastname: Guo
authors.forename: Weijun
authors.initials: W
authors.lastname: Shen
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D000818:Animals; D001386:Azides; D001483:Base Sequence; D001577:Benzophenones; D001896:Boron Compounds; D002460:Cell Line; D018389:Codon, Nonsense / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000382:microbiology; D017017:Methanococcus / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012365:RNA, Transfer, Tyr / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism
chemical_list: C488060:4-benzoylphenylalanine; D001386:Azides; D001577:Benzophenones; D001896:Boron Compounds; D018389:Codon, Nonsense; D012365:RNA, Transfer, Tyr; D049452:Green Fluorescent Proteins; C040239:4-nitrophenylalanine; C027162:4-azidophenylalanine; D010649:Phenylalanine; D014448:Tyrosine-tRNA Ligase; C033685:4-boronophenylalanine