Q1：How do I place an order?
HuCAL® Custom Monoclonal Antibody Generation service is provided only in Japan.
If you live in outside of Japan, please visit their site.
Q1: Can I synthesize eukaryotic proteins using the PUREfrex® kit?
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?
It depends on the target protein.
Dihydrofolate reductase from E.coli can be synthesized with about 600 μg per mL of the reaction.
Q3: Can I synthesize proteins of more than 100 kDa?
We have synthesized the protein of 116 kDa using PUREfrex®.
Q4: What do you recommend the reaction condition for PUREfrex®?
We recommend that the protein synthesis using PUREfrex® is carried out at 37℃ for 2-4 hours.
Q5: Can I synthesize and purify a tagged protein?
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?
There is no post-translational modification, because PUREfrex® is constituted by translation factors only.
Q7: Does PUREfrex® contain any molecular chaperones?
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®?
DsbC Set (#PF005-0.5-EX) and PDI Set (#PF006-05-EX) are supplements by adding to PUREfrex® for synthesizing proteins containing a disulfide bond in an active form. PUREfrex® 2.1 (#PF213-0.25-EX) is recommended to optimize the reaction conditions.
Q9: Can I synthesize a membrane protein with PUREfrex®?
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 radiolabeled protein can be synthesized by adding radioisotope-labeled amino acids, such as [35S] methionine or [3H] leucine, to PUREfrex®. The amino acid content is 0.5 mM for PUREfrex®1.0 and 2 mM PUREfrex®2.0. Please consider that point and optimize conditions for your labeling.
Q11: Can I use any promoter besides T7 promoter?
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.
- 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.
- 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.
- 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®.
- Secondary structure of transcripts may prevent the translation reaction. In this case, please optimize the template sequence to solve the problem of secondary structure.
Q1: What amount of the template DNA is added to the reaction mixture?
0.5-3 ng per 1 kbp DNA for 1 µL of the reaction mixture.
The amount of DNA is calculated based on number of DNA molecules. For example, in case of 6 kbp of plasmid, regardless of length of ORF, 3 to 18 ng of DNA is added for 1 µL of the reaction mixture.
Q2: Can TE buffer be used for dissolving the template DNA?
Buffers containing EDTA should not be used for dissolving DNA, because EDTA inhibits transcription and translation reaction.
Q3: What are key points to use PCR product as the template DNA?
We recommend that the template DNA is prepared by two-step PCR.
Overview of two-step PCR is illustrated in Fig. 5 and primer sequence for two-step PCR is shown in Table 2.
Text sequence is also available.
Purity and Amount of PCR product
PCR product should be a single band on an electrophoresis gel. Otherwise unexpected polypeptides may be synthesized. If there are still extra bands in spite of optimizing PCR condition, please purify the desired band by gel extraction.
We recommend blue light for gel extraction, not UV light. UV light causes DNA damage, which can lead to an unexpected termination of transcription. You should cut the gel quickly even using blue light.
PCR reaction mixture containing the amplified template DNA can be directly added to PUREfrex® reaction mixture. However, some components in the PCR reaction mixture inhibit transcription and/or translation reaction.
To prevent carrying inhibitory components from the PCR reaction mixture, we recommend adding less than 0.1 volume of the PCR reaction mixture to PUREfrex® reaction mixture. If the concentration of the template DNA is not enough for protein synthesis, you may concentrate DNA solution with a purification kit or by gel extraction.
Q4: What are key points to use plasmid DNA as the template DNA?
To include essential elements
T7 promotor, SD sequence, and T7 terminator are essential elements. We don’t recommend using vectors including Lac operator because it causes decrease on productivity depending on the target protein.
RNase digests the transcribed RNA in the reaction mixture, resulting reduction of the productivity of the target protein. Many commercial kits can be used for purifying plasmid DNA. However, in some cases, RNase in the lysis buffer is brought over to the purified DNA fraction.
If there is a possibility that RNase is contained in the purified plasmid DNA fraction, we recommend Phenol/Chloroform extraction followed by ethanol or isopropanol precipitation for removing RNase.
Addition of RNase inhibitor to the PUREfrex® reaction mixture is also effective.
Q5: Do I need optimization of the sequence of template DNA for PUREfrex®?
We recommend that you use the optimal codons for E. coli because PUREfrex® utilizes translation system derived from E. coli. Moreover, N-terminal codons (2nd-6th codon following 1st ATG) should be changed to AT-rich codon (not rather than the most frequently used codon) to increase the productivity.
Please refer the results in our posters, which are partially described in Japanese but all results in English.
“Improvement of translational efficiency by N-terminal codon optimization in the reconstituted cell-free protein synthesis system” (2016)
»Poster1; Fab, His tag, etc. (0.7 MB)
»Poster2; various proteins, membrane protein, etc. (2.5 MB)
Q1: Do you sell kits for ribosome display with PUREfrex? Or are you planning to sell it?
Kit for ribosome display is not on sale.
There will be no sales schedule in the future.
Q2 Is it possible to perform ribosome display using a commercially available PUREfrex® kit?
We introduce the paper published by Takuya Ueda of the University of Tokyo who developed the PURE system which is the basic technology of PUREfrex®.
In this paper, a reaction solution contains Release factors same as the commercially available PUREfrex® kit. Therefore the stop codon is removed from the library.
J Biochem(2009)145(5):693-700 Osada
In addition, we recommend that you refer to the above mentioned paper especially the composition of buffers for ribosome display selection even when using a commercially available PUREfrex® kit.
Sometimes the ribosome display may not work as expected since the commercially available PUREfrex® kit is optimized for protein synthesis.
Please contact us for any questions or concerns.
Q3 Is it possible to optimize the PUREfrex reaction solution for our owned protein / peptide libraries?
Yes. It is possible.
The optimization condition also depends on your library and target.
Please note that before using the reaction solution optimized for ribosome display, there is a limitation and a license agreement may be required depending on its purpose of use.
Q4: Do I need a license when I perform ribosome display using PUREfrex®?
If you perform ribosome display using a commercially available PUREfrex® kit, you do not need a license.
However, when using a customized PUREfrex® reaction solution, you need a license according to the purpose, so please contact us in advance.