title: A new protein engineering approach combining chemistry and biology, part I; site-specific incorporation of 4-iodo-L-phenylalanine in vitro by using misacylated suppressor tRNAPhe.
abstract: An Escherichia coli suppressor tRNA(Phe) (tRNA(Phe) (CUA)) was misacylated with 4-iodo-L-phenylalanine by using the A294G phenylalanyl-tRNA synthetase mutant (G294-PheRS) from E. coli at a high magnesium-ion concentration. The preacylated tRNA was added to an E. coli cell-free system and a Ras protein that contained the 4-iodo-L-phenylalanine residue at a specific target position was synthesized. Site-specific incorporation of 4-iodo-L-phenylalanine was confirmed by using LC-MS/MS. Free tRNA(Phe) (CUA) was not aminoacylated by aminoacyl-tRNA synthetases (aaRSs) present in the E. coli cell-free system. Our approach will find wide application in protein engineering since an aryl iodide tag on proteins can be used for site-specific functionalization of proteins.
authors.affiliation: Department of Biophysics and Biochemistry, School of Science The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kodama
authors.forename: Seketsu
authors.initials: S
authors.lastname: Fukuzawa
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakayama
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuki
authors.forename: Natsuko
authors.initials: N
authors.lastname: Matsuda
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Koji
authors.initials: K
authors.lastname: Takio
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tachibana
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000215:Acylation; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D012343:RNA, Transfer / Q000737:chemistry*; D012360:RNA, Transfer, Phe / Q000737:chemistry*; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D012360:RNA, Transfer, Phe; D010649:Phenylalanine; D012343:RNA, Transfer; D018631:ras Proteins; C049181:4-iodophenylalanine