Custom synthesis service for non-natural amino acyl-tRNA

Non-natural amino acids (nnAAs) are used for several purposes: creating proteins with a novel function, studying the protein function and so on. They are incorporated into proteins just add them to the in vitro translation system like PUREfrex® from GeneFrontier.
ProteinExpress offers the custom nnAA-charged tRNA synthesis and following custom nnAA-charged protein production service. You could get a custom tRNA with an amino acid and with a tRNA sequence of your interest.

Schematic procedure
Fig.1 nnAA and its incorporation into proteins

Advantages

You could choose any codons of your interest (UAG or CGGG are generally used.)
Many nnAAs or labeled tRNAs are available (Fig.2)

target molecule binding peptide
Fig.2 Off-the-shelf tRNA products and its usage

TAMRA-X-AF
CR110-X-AF
HiLyte Fluor 488-AF
ATTO633-AF
ATTO655-X-AF

Biotin-AF
Biotin-X-AF
Biotin-XX-AF

Lys (Me)
Lys (Me2)
Lys (Ac)

PEG4-AF
PEG8-AF
PEG12-AF
BPA
AcPhe
AzoAla
Phe(4-F)
Phe(4-I)

※Above tRNAs are available for both UAG and CGGG codon.

※Please inquire if the tRNA of your interest is not listed!

Application

target molecule binding peptide
Fig.3 Comparison of the incorporation rates among 5 nnAAs

5 nnAAs (lane 3-7) were incorporated into the amber codon (UAG) on the same protein. All the tRNA had the same sequence, but the incorporation rate widely varied depending on the charged nnAAs.

HiLyte Flour 488 target molecule binding peptide

TAMRA target molecule binding peptide

ATTO633 target molecule binding peptide

Biotin target molecule binding peptide

Fig.4 Pin-point protein labeling in the E.coli based cell-free expression system

2UAG: Proteins with UAG codon right after the start codon (AUG)

ProX tag: Proteins with ProX tag (UAG) This makes it possible to increase the productivity of the protein itself and improve the efficiency of introducing non-natural amino acids.

Reference paper related to the service or product

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Development of a novel PPARγ ligand screening system using pinpoint fluorescence-probed protein.
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Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence.
Abe R, Jeong HJ, Arakawa D, Dong J, Ohashi H, Kaigome R, Saiki F, Yamane K, Takagi H, Ueda H.
Sci Rep. 2014 Apr 11;4:4640.
Strategy for making a superior Quenchbody to proteins: effect of the fluorophore position.
Jeong HJ, Ueda H.
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In vitro selection of a photoresponsive peptide aptamer to glutathione-immobilized microbeads.
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A fluorogenic peptide probe developed by in vitro selection using tRNA carrying a fluorogenic amino acid.
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In vitro selection of a photo-responsive peptide aptamer using ribosome display.
Liu M, Tada S, Ito M, Abe H, Ito Y.
Chem Commun (Camb). 2012 Dec 18;48(97):11871-3.
A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.
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PLoS One. 2014 Dec 8;9(12):e112874.
Farnesyl pyrophosphate regulates adipocyte functions as an endogenous PPARγ agonist.
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Detection method for quantifying global DNA methylation by fluorescence correlation spectroscopy
Tomohiro Umezu, Kazuma Ohyashiki, Junko H Ohyashiki
Anal Biochem. 2011 Aug 15;415(2):145-50.
Autonomous synthesis and assembly of a ribosomal subunit on a chip
Michael Levy, Reuven Falkovich, Shirley S Daube, Roy H Bar-Ziv
Sci Adv. 2020 Apr 15;6(16):eaaz6020.
Reconstitution of Bam Complex-Mediated Assembly of a Trimeric Porin into Proteoliposomes
Sunyia Hussain, Janine H Peterson, Harris D Bernstein
mBio. 2021 Aug 31;12(4):e0169621.

This product is "Research reagent". It is prohibited to use against humans or animals in medical care, clinical diagnoses, or as food.