Site-specific incorporation of 4-Iodo-l-phenylalanine through opal suppression

被引:3
作者
Kodama, Koichiro [1 ]
Nakayama, Hiroshi [2 ]
Sakamoto, Kensaku [1 ,3 ]
Fukuzawa, Seketsu [4 ]
Kigawa, Takanori [3 ]
Yabuki, Takashi [1 ]
Kitabatake, Makoto [5 ]
Takio, Koji [6 ]
Yokoyama, Shigeyuki [1 ,3 ]
机构
[1] Univ Tokyo, Dept Biophys & Biochem, Sch Sci, Bunkyo Ku, Tokyo 1130033, Japan
[2] RIKEN, Wako Inst, Biomol Characterizat Team, Adv Dev Supporting Ctr, Wako, Saitama 3510033, Japan
[3] RIKEN, Syst & Struct Biol Ctr, Tsurumi Ku, Yokohama, Kanagawa 2300045, Japan
[4] Univ Tokyo, Dept Chem, Sch Sci, Bunkyo Ku, Tokyo 1130033, Japan
[5] Kyoto Univ, Inst Virus Res, Kyoto 6068507, Japan
[6] RIKEN, Harima Inst, Sayo, Hyogo 6795148, Japan
关键词
cell-free protein synthesis; indolmycin; non-natural amino acid; opal codon; Transfer RNA; TRANSFER-RNA-SYNTHETASE; NONNATURAL AMINO-ACIDS; CELL-FREE SYNTHESIS; ESCHERICHIA-COLI; IN-VITRO; GENETIC-CODE; SUBSTRATE-SPECIFICITY; PROTEIN-BIOSYNTHESIS; MAMMALIAN-CELLS; SINGLE PROTEIN;
D O I
10.1093/jb/mvq051
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A variety of unique codons have been employed to expand the genetic code. The use of the opal (UGA) codon is promising, but insufficient information is available about the UGA suppression approach, which facilitates the incorporation of non-natural amino acids through suppression of the UGA codon. In this study, the UGA codon was used to incorporate 4-iodo-l-phenylalanine into position 32 of the Ras protein in an Escherichia coli cell-free translation system. The undesired incorporation of tryptophan in response to the UGA codon was completely repressed by the addition of indolmycin. The minor amount (3%) of contaminating 4-bromo-l-phenylalanine in the building block 4-iodo-l-phenylalanine led to the significant incorporation of 4-bromo-l-phenylalanine (21%), and this problem was solved by using a purified 4-iodo-l-phenylalanine sample. Optimization of the incubation time was also important, since the undesired incorporation of free phenylalanine increased during the cell-free translation reaction. The 4-iodo-l-phenylalanine residue can be used for the chemoselective modification of proteins. This method will contribute to advancements in protein engineering studies with non-natural amino acid substitutions.
引用
收藏
页码:179 / 187
页数:9
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