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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. RTS100 E.coli HY kit; Roche Applied Science, #3186156)
· 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 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)

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