PUREfrex™ FAQ


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Q1 Can I synthesize eukaryotic proteins using the PUREfrex™ kit?
Q2 How much can I synthesize the target protein using the PUREfrex™ kit?
Q3 Can I synthesize proteins of more than 100 kDa?
Q4 What do you recommend the reaction condition for PUREfrex™?
Q5 Can I synthesize and purify a tagged protein?
Q6 Is synthesized protein modified by glycosylation or phosphorylation?
Q7 Does PUREfrex™ contain any molecular chaperones?
Q8 Can I synthesize a protein containing disulfide bonds with PUREfrex™?
Q9 Can I synthesize a membrane protein with PUREfrex™?
Q10 Can I synthesize a radiolabeled protein with [35S] methionine or [3H] leucine?
Q11 Can I use any promoter besides T7 promoter?
Q12 I can’t get DHFR using DHFR DNA in the kit as positive control.
Q13 I can get DHFR using DHFR DNA in the kit.
But I can’t get my protein of interest or can get it with very low amount.

 

 

Q1 Can I synthesize eukaryotic proteins using the PUREfrex™ kit?
A YES.
The PUREfrex™ is a reconstituted cell-free protein synthesis kit composed of E. coli ribosomes and translation factors, but eukaryotic proteins from such as mammalian and plant can also be synthesized. The productivity of the target protein may depend on the property of nucleotide sequence encoding the target protein, such as GC contents or frequency of rare codon.

 

Q2 How much can I synthesize the target protein using the PUREfrex™ kit?
A It depends on the target protein.
Dihydrofolate reductase from E.coli can be synthesized with about 150 μg per mL of the reaction.

 

Q3 Can I synthesize proteins of more than 100 kDa?
A YES.
We have synthesized the protein of 116 kDa using PUREfrex™.

 

Q4 What do you recommend the reaction condition for PUREfrex™?
A We recommend that the protein synthesis using PUREfrex™ is carried out at 37Ž for 2-4 hours.

 

Q5 Can I synthesize and purify a tagged protein?
A YES.
You can use any tag, because all of protein components of PUREfrex™ have no tag for purification or detection. For example, the target protein with histidine tag can be purified with metal-chelating resin by applying reaction mixture directly, after synthesizing.

 

Q6 Is synthesized protein modified by glycosylation or phosphorylation?
A NO.
There is no post-translational modification, because PUREfrex™ is constituted by translation factors only.

 

Q7 Does PUREfrex™ contain any molecular chaperones?
A NO.
PUREfrex™ does not contain any chaperones, but you can add chaperones, such as Hsp70. You may prepare a chaperone by yourself or buy it from another company.
GeneFrontier has been developing molecular chaperones suitable for PUREfrex™ kit. For more information about products under developing, please contact us.

 

Q8 Can I synthesize a protein containing disulfide bonds with PUREfrex™?
A NO.
The target protein is synthesized without forming disulfide bonds, because translation is carried out in the presence of a reducing agent, DTT. Most of the proteins, which require the disulfide bond for its activity, may have no activity.
GeneFrontier has been developing the improved PUREfrex™ kit for synthesizing the proteins containing disulfide bonds. For more information about products under developing, please contact us.

 

Q9 Can I synthesize a membrane protein with PUREfrex™?
A YES.
In the most of the cases, a synthesized membrane protein may form aggregates. To obtain a membrane protein inserted into a lipid bilayer, you may add a lipid bilayer such as liposome to PUREfrex™ when you synthesize the membrane protein.

 

Q10 Can I synthesize a radiolabeled protein with [35S] methionine or [3H] leucine?
A YES.
A radiolabeled protein can be synthesized by adding radioisotope-labeled amino acids, such as [35S] methionine or [3H] leucine, to PUREfrex™. PUREfrex™ contains 20 of natural amino acids at 0.5 mM each. So, please consider that point and optimize conditions for your labeling.

 

Q11 Can I use any promoter besides T7 promoter?
A We recommend the template DNA with T7 promoter, because PUREfrex™ contains T7 RNA polymerase for transcription. When you use other polymerases, please generate template DNA with the suitable promoter for the selected polymerase.

 

Q12 I can’t get DHFR using DHFR DNA in the kit as positive control.
A1 The kit may be inactivated for some reasons. To avoid the inactivation, please store the kit at suitable temperature and divide into aliquots to reduce freeze-thaw cycles as much as possible.
A2 The kit may be contaminated by nuclease. To avoid a nuclease contamination, please use nuclease-free water, reagents and materials.

 

Q13 I can get DHFR using DHFR DNA in the kit.
But I can’t get my protein of interest or can get it with very low amount.
A1 The kit may be inactivated for some reasons. To avoid the inactivation, please store the kit at suitable temperature and divide into aliquots to reduce freeze-thaw cycles as much as possible.
A2 The kit may be contaminated by nuclease. To avoid a nuclease contamination, please use nuclease-free water, reagents and materials.
Sometimes the plasmid is contaminated with RNase through a plasmid preparation process. So, please confirm that RNase is removed from the plasmid.
A3 The construct of template DNA may be incorrect. It is necessary to contain the sequence of T7 promoter, ribosome binding site, initiation codon, and stop codon for the template DNA for PUREfrex™.
A4 Secondary structure of transcripts may prevent the translation reaction. In this case, please optimize the template sequence to solve the problem of secondary structure.

 

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