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Pin-point Protein Labeling An amber stop codon (UAG) is used for pin-point fluorescence or biotin labeling.

CloverDirect™ tRNA Reagents for Site-Directed Protein Functionalization_Principle


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Principle

[Principle of incorporation of unnatural amino acids]
Incorporation position of unnatural amino acids is defined by a UAG amber codon or CGGG four-base codon. An unnatural aminoacyl-tRNA recognizes the UAG amber codon or the CGGG codon during translation. Consequently, the unnatural amino acid is incorporated at the directed site of the protein. By using two tRNAs for amber and four-base codons, dual-labeled proteins can be obtained which are available for fluorescence resonance energy transfer (FRET).

[UAG amber codon]
If the UAG codon is recognized by the amber suppressor tRNA, full-length protein containing the unnatural amino acid is successfully synthesized. On the contrary, if the UAG codon is recognized by release factor 1 (RF1) which is one of the termination factors, the protein synthesis is terminated. Therefore, the translation product obtained as a full-length protein contains the unnatural amino acid at 100% efficiency.

[CGGG four-base codon]
If the CGGG codon is recognized by the four-base anticodon tRNA, full-length protein containing the unnatural amino acid is successfully synthesized. On the contrary, if the CGG is recognized as a triplet codon by Arg-tRNA, the reading frame shifts to +1 frame and a downstream stop codon terminates the protein synthesis. Therefore, the translation product obtained as a full-length protein contains the unnatural amino acid at 100% efficiency.

Incorporation at N-terminal regions (within 20 amino acid residues from the N-terminus) in response to CGGG codon sometimes results in the production of full-length proteins without unnatural amino acids, possibly because of spontaneous +1 frameshifting. In such case, ProteinExpress recommend the use of ProXTM tag, which is original peptide tag developed for the CGGG codon-mediated incorporation of unnatural amino acids.

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