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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 tRNA Phe
Author(s) -
Kodama Koichiro,
Fukuzawa Seketsu,
Sakamoto Kensaku,
Nakayama Hiroshi,
Kigawa Takanori,
Yabuki Takashi,
Matsuda Natsuko,
Shirouzu Mikako,
Takio Koji,
Tachibana Kazuo,
Yokoyama Shigeyuki
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200600137
Subject(s) - transfer rna , phenylalanine , escherichia coli , biochemistry , aminoacyl trna synthetase , protein biosynthesis , chemistry , protein engineering , cell free protein synthesis , aminoacyl trna , stereochemistry , biology , enzyme , amino acid , rna , gene
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.