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.
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.
Kodama
Koichiro
K
Department of Biophysics and Biochemistry, School of Science The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
Fukuzawa
Seketsu
S
Sakamoto
Kensaku
K
Nakayama
Hiroshi
H
Kigawa
Takanori
T
Yabuki
Takashi
T
Matsuda
Natsuko
N
Shirouzu
Mikako
M
Takio
Koji
K
Tachibana
Kazuo
K
Yokoyama
Shigeyuki
S
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
D012360:RNA, Transfer, Phe; D010649:Phenylalanine; D012343:RNA, Transfer; D018631:ras Proteins; C049181:4-iodophenylalanine