Site-specific incorporation of 4-iodo-L-phenylalanine through opal suppression.
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.
Kodama
Koichiro
K
Department of Biophysics and Biochemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo. k.kodama@kurenai.waseda.jp
Nakayama
Hiroshi
H
Sakamoto
Kensaku
K
Fukuzawa
Seketsu
S
Kigawa
Takanori
T
Yabuki
Takashi
T
Kitabatake
Makoto
M
Takio
Koji
K
Yokoyama
Shigeyuki
S
D001483:Base Sequence; D002474:Cell-Free System; D018388:Codon, Terminator / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D018631:ras Proteins / Q000096:biosynthesis*
D018388:Codon, Terminator; D007211:Indoles; D010649:Phenylalanine; D012343:RNA, Transfer; D018631:ras Proteins; C049181:4-iodophenylalanine; C001447:indolmycin