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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_Contents

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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.

[Equipment and reagents to be supplied by others]

Protein Expression
Cell-free translation system (E.coli )
(e.g. RYTS Kit: E. coli Cell-free Protein Synthesis System,
PUREfrex®: Reconstituted Cell-free Protein Synthesis Kit)
Expression gene containing UAG or CGGG codon (plasmid DNA, linear DNA, or messenger RNA).
(Suitable expression vector should be used for the cell-tree translation system.)

Purification, buffer exchange, and concentration
Affinity column and buffer for purification
(e.g. His SpinTrap kit; GE Healthcare Science, #28-9321-71)
Ultrafiltration membrane and buffer (e.g. ULTRAFREE-0.5 Centrifugal Filter Devices 10k; Millipore , UFV5BC00)

Brief protocol

Step 1. Protein Expression
Dissolve unnatural aminoacyl-tRNA with tRNA buffer, and mix with template DNA and cell-free translation system. The mixture is incubated for 1 hour to synthesize protein containing unnatural amino acid.

Step 2. Purification
Reaction mixture includes several proteins derived from cell-free translation system and unnatural amino acid which is not incorporated into protein. Full-length protein containing unnatural amino acid can be isolated by purification for C-terminal tag such as His tag.

Step 3. Buffer exchange and concentration (optional)
Buffer exchange and concentration can be done by ultrafiltration membrane. Purified protein containing unnatural amino acid can be directly used for the downstream experiment.

Points to note

1. Protein expression
Confirm your protein can be expressed in E.coli cell-free translation system. In case of very low expression of a wild-type gene that does not contain UAG codon or CGGG codon, optimization of nucleotide sequence (codon usage, addition of N-terminal tags, etc.) is required to improve the expression. ProteinExpress provides custom service for protein expression using CloverDirect™ including gene construction for efficient expression.

2. Incorporation site-dependency
Some unnatural amino acids are allowed to be incorporated only at the N-terminal region (within 20 amino acid residues from the N-terminus). Please check the product list [PDF] for details.

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.

3. Custom Services ProteinExpress provides custom services for the synthesis of unnatural aminoacyl-tRNAs (fluorescent-labeled, functional amino acids, etc.), which allows the expression of proteins with your requested unnatural amino acids.
Custom Services for Unnatural Aminoacyl-tRNA (contact us)

ProteinExpress also provides custom services for the expression of proteins with unnatural amino acids including construction of recombinant DNA, cell-free translation, and purification of proteins.
Custom Service for Protein Expression using CloverDirect™ (contact us)

Product List

Site-Directed Fluorescence Labeling
Site-Directed Biotin Labeling
Site-Directed Post-Translational Modification
Site-Directed Unnatural Mutagenesis
Product List [PDF]

*Please note that the delivery lead time may be 2 - 3 months if the product is out of stock. Please inquire about the stock us

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.