CloverDirect, PEG4-AF

CloverDirect, PEG4-AF


Product ID: FNK-CLD2301
Quantity: 5x300 μL


About CloverDirect

CloverDirect^TM 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^TM reagents and DNA template having an amber stop codon (UAG) or a four-base codon (CGGG) to an in vitro translation system. CloverDirect^TM covers the following four applications. In addition, we provide custom services for the expression of proteins with unnatural amino acids.

 

Site-Directed Unnatural Mutagenesis (PEGylated amino acids)

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

 

Site-Directed Unnatural Mutagenesis	Product No	Site-dependency (*1)		codon	Q’ty
		N-terminal	Internal, C-terminal
PEG4-AF [Methyl-PEG4]	FNK-CLD2301	O	O	Amber	5 X 300 μL

*1 : Incorporation site-dependency
Some unnatural amino acids are incorporated in a incorporation site-dependent manner.
(N-terminal region = within 20 amino acid residues from the N-terminus.)
O : Available
△ : Available in some sites
X : Unavailable


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 Cterminal 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 TM 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.




Applications for site-directly fluorescent labeled proteins

The site-directly fluorescent-labeled proteins are available to the following measurements.

• Interaction analysis using single molecule fluorescence analysis: Single Molecule fluorescence detection system (MF20 / Fluor Point-Light MF20; OLYMPUS)
• Conformation analysis of protein by inter- or intra-molecular fluorescence resonance energy transfer (FRET)
• Functional analysis in cell imaging
• Interaction analysis in protein array
• Expression analysis by in-gel fluorescent detection of SDS-PAGE

References

1. Daisuke Kajihara, Ryoji Abe, Issei Iijima, Chie Komiyama, Masahiko Sisido, Takahiro Hohsaka. FRET analysis of protein
    conformational change through position-specific incorporation of fluorescent amino acids. 
    Nature Methods., 3, 923-929 (2006).

2. Takayoshi Watanabe, Norihito Muranaka, Issei Iijima, Takahiro Hohsaka. Position-specific incorporation of biotinylated
    non-natural amino acids into a protein in a cell-free translation system. 
    Biochem. Biophys. Res. Commun., 361, 794-799 (2007)

3. Hikaru Taira, Yosuke Matsushita, Kenji Kojima, Kaori Shiraga, Takahiro Hohsaka. Comprehensive screening of amber
    suppressor tRNAs suitable for incorporation of non-natural amino acids in a cellfree translation system. 
    Biochem. Biophys. Res. Commun., 374, 304-308 (2008).

4. Takahiro Hohsaka, Daisuke Kajihara, Yuki Ashizuka, Hiroshi Murakami, Masahiko Sisido. Efficient Incorporation of Nonnatural
    Amino Acids with Large Aromatic Groups into Streptavidin in In Vitro Protein Synthesizing Systems. 
    J. Am. Chem. Soc., 121, 34-40 (1999).