The principle of Pin-point labeling technology by CloverDirect TM
Labeling position of a protein is decided by an UAG amber codon which is
one of the stop codons.
In preparation of the DNA template, the coding triplet
corresponding to the selected labeling position should be replaced or inserted
by the UAG amber codon. The labeling position is strongly recommended from
2nd to 20th residues of N-terminus, because the incorporation efficiency is high at
N-terminal region and tend to decrease dramatically after 20th residue of a protein.
When the labeled aminoacyl-tRNA (Figure 1) is added to an E.coli cell-free translation system with the DNA template coding amber codon,
the labeled aminoacyl-tRNA recognizes the UAG amber codon during translation.
Consequently, labeled amino acid is incorporated at defined position of
a protein (Figure 2). As competitive reaction, when the amber codon is recognized by release
factor 1 (RF1) which is one of the termination factors, then protein synthesis
is terminated. But, our tRNAs are designed to be more highly effective
in incorporating than RF1. As a result, synthesized full-length protein
is labeled at 100% efficiency.
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