title: A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli.
abstract: We developed an efficient method for introduction of 3-azidotyrosine (N(3)-Y) into proteins in Escherichia coli cells. We constructed a plasmid that is adaptable for the constitutive expression of both Methanosarcina acetivorans tyrosyl-tRNA synthetase (TyrRS) and tRNA(()(CUA)), and made an orthogonal tRNA((CUA)) that is recognized as a substrate only by the archaeal TyrRS. Random mutations were introduced into M. acetivorans TyrRS around the tyrosine binding pocket, and a TyrRS mutant recognizing N(3)-Y was selected. We then expressed rat calmodulin (CaM) containing N(3)-Y, using the CaM gene with an amber codon at position 80. Mass analyses confirmed production of CaM containing N(3)-Y, but a significant amount of CaM containing 3-aminotyrosine was also detected. To more efficiently express CaM containing N(3)-Y, we added an arabinose-inducible gene for the mutant TyrRS to the plasmid carrying the mutant TyrRS/tRNA(()(CUA)) gene. Although the yields of full-length CaM increased ~3-fold, the ratio of N(3)-Y introduction was not significantly improved. Following screening for a suitable host cell, we found that CaM expressed in E. coli SHuffle (K-12) had 97% N(3)-Y at the pre-determined site. Finally, we obtained up to 2 mg of CaM containing N(3)-Y per 100 ml of culture media, sufficient for use in various proteomics experiments, including photo-crosslinking.
authors: Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors: Akiyoshi
authors: A
authors: Ikeda-Boku
authors: Satoshi
authors: S
authors: Ohno
authors: Yuuka
authors: Y
authors: Hibino
authors: Takashi
authors: T
authors: Yokogawa
authors: Nobuhiro
authors: N
authors: Hayashi
authors: Kazuya
authors: K
authors: Nishikawa
mesh_terms: D000818:Animals; D001089:Arabinose / Q000494:pharmacology; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001386:Azides / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D018388:Codon, Terminator / Q000235:genetics; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D017020:Methanosarcina / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D001386:Azides; D002147:Calmodulin; D018388:Codon, Terminator; D012365:RNA, Transfer, Tyr; D011994:Recombinant Proteins; D014443:Tyrosine; D001089:Arabinose; D014448:Tyrosine-tRNA Ligase