CloverDirect™ tRNA Reagents for Site-Directed Protein Functionalization

Background:

CloverDirect™ tRNA Reagents for Site-Directed Protein Functionalization allow the incorporation of unnatural amino acids at defined positions of proteins using in vitro translation. Unnatural amino acids containing fluorescent groups, biotin, PEG, photo-crosslink, etc are available. Proteins with unnatural amino acids will be obtained within a few hours just by adding CloverDirect™ reagents and DNA template having an amber stop codon (UAG) or a four-base codon (CGGG) to an in vitro translation system. CloverDirect™ covers the following four applications. In addition, we provide custom services for the expression of proteins with unnatural amino acids (contact us).

It is not easy to incorporate fluorescent groups into proteins in a site-direct and quantitative fashion by chemical modification. CloverDirect™ tRNA Reagents for Site-Directed Fluorescence Labeling allow the incorporation of fluorescent unnatural amino acids into proteins in a site-direct and quantitative fashion. Various fluorescent dyes are available including those for 488 nm, 543 nm and 633 nm excitation.

Site-Directed Biotin Labeling:

CloverDirect™ tRNA Reagents for Site-Directed Biotin Labeling allow the incorporation of biotinylated unnatural amino acids into proteins in a site-direct and quantitative fashion. Labeling proteins are available for the oriented immobilization onto avidin-coated plates and beads. The biotinylated amino acids have one or two aminohexyl liners between amino acid and biotin.

Site-Directed Post-Translational Modification:

It is not easy to prepare post-translationally modified proteins (phosphorylation, methylation, etc.). CloverDirect™ tRNA Reagents for Site-Directed Post-Translational Modification allow the incorporation of modified amino acids into proteins to obtain post-translationally modified proteins in a site-direct and quantitative fashion.

Site-Directed Unnatural Mutagenesis:

By incorporation of unnatural amino acids containing functional groups, novel functional proteins can be designed and synthesized. CloverDirect™ tRNA Reagents for Site-Directed Unnatural Mutagenesis allow the incorporation of unnatural amino acids with PEG, photo-crosslinking, photo-isomerizable groups, etc.

Principe:

Principe 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.

Kit component:

Unnatural aminoacyl-tRNA X 1
tRNA dissolving buffer X 1

Note 1 : One tube contains unnatural aminoacyl-tRNA sufficient for 300 µL of in vitro translation reaction. Once thawed, unnatural aminoacyl-tRNA can be stored at -70 ?C for 2 months.