Incorporation of fluorescent non-natural amino acids into N-terminal tag of proteins in cell-free translation and its dependence on position and neighboring codons.
Fluorescence labeling is a useful technique for structural and functional analyses of proteins. In a previous study, we developed position-specific incorporation of visible wavelength fluorescent non-natural amino acids carrying relatively small BODIPY fluorophores into proteins, in response to a four-base codon CGGG. Here, we have expanded this position-specific fluorescence labeling method to include relatively large non-natural amino acids carrying photostable rhodamine dyes. TAMRA-linked aminophenylalanine was synthesized and attached to a tRNA having a four-base anticodon, and its incorporation into proteins was examined in an Escherichia coli cell-free translation system. TAMRA-labeled amino acids were successfully incorporated into proteins, although incorporation was allowed only at the N-terminal region. Insertion of two codons encoding a stop codon in the +1 frame before four-base codon suppressed the expression of non-labeled proteins that may have been produced by spontaneous +1 frameshift upstream of the four-base codon. Alternation of the incorporation position affected the expression level of the TAMRA-labeled protein. In addition, alternation of upstream and downstream codons affected the efficiency and accuracy of TAMRA-labeled amino acid incorporation. Based on these results, a novel tag peptide was developed; it contained the four-base codon at the 9th position with optimized upstream and downstream codons. This tag peptide was effective for producing proteins with various fluorescent labels at the N-terminal region.
Abe
Ryoji
R
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Ishikawa 923-1292, Japan.
Shiraga
Kaori
K
Ebisu
Shogo
S
Takagi
Hiroaki
H
Hohsaka
Takahiro
T
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods*
D000596:Amino Acids; D003062:Codon; D005456:Fluorescent Dyes; D009842:Oligopeptides; D011506:Proteins; C037841:tyrosyl-alanyl-glycine