The PUREfrex^® reaction mixture has a relatively high salt concentration and may become turbid when heated after sample buffer containing SDS is directly added. To avoid turbidity, dilute the reaction mixture with at least equal volume of water before adding sample buffer. If this does not resolve the turbidity issue, heat the reaction mixture at a lower temperature (e.g., 37ºC) for a longer time (about 1 hour).

1) The synthesized protein may be insoluble. Check its solubility.

2) Factors required for proper activity (e.g., coenzymes, metal ions) may be missing. Since PUREfrex^® is a reconstituted system, it contains no small molecules not relating to transcription and translation. Add the factors required for activity.

3) If the protein of interest requires correct disulfide bond formation, synthesize it in the presence of an additive to promote this formation (e.g. GSSG, DsbC). In that case, use PUREfrex^® 2.1 in which reducing agent can be selected, because the efficiency of disulfide formation is affected by reducing agent.

1) Add molecular chaperones when synthesizing the protein. PUREfrex^® contains no molecular chaperones. When the protein of interest is synthesized in the presence of molecular chaperones, it may become soluble. Additionally, better results may be obtained with PUREfrex^® 1.0, in which the yield of the product is lower than PUREfrex^® 2.0.

2) Synthesize the protein at a lower translation speed. Proteins generally fold more slowly than they are translated. Reducing the translation speed by lowering the reaction temperature from 37°C to 30°C or 25°C may increase the proportion of the target protein that is soluble. This will reduce the yield, so be sure to select conditions to efficiently produce the soluble protein.

1)The template DNA may not contain the minimum required sequences. Template DNA for PUREfrex^® must contain T7 promoter, ribosome binding site (SD sequence), start codon, and stop codon.

2) Too much or little DNA may be added to the reaction mixture. Regardless of plasmid DNA or PCR product, the template DNA should be added at a concentration of 0.5 to 3 ng per 1 kbp for 1 µL of the reaction mixture. Adding too much may reduce the protein yield also. The concentration of the template DNA added to the reaction mixture of PUREfrex^® is defined as the number of DNA molecules (molar concentration) and it is added at a final concentration around 2 nM. About 2 nM is approximately 0.5 to 3 ng per 1 kbp for 1 µL of the reaction mixture. A 6 kbp plasmid DNA, for example, would be added at (0.5-3)×6 = 3-18 ng/µL, regardless of the length of the ORF.

3) The method for preparing the template DNA is inappropriate. Please see Tech Notes: “Preparation of the template DNA“.

4) The template DNA contains a difficult-to-translate sequence. Confirm that the template DNA does not contain the sequences described in Tech Notes: “6 Tips about the template DNA Design”.

1) Incubate the PUREfrex^® reaction mixture by directly heating the reaction tube with heat block or water bath. Gas-phase chamber (e.g., incubator) takes time to warm the reaction mixture and the yield may be reduced.

2) Some of the components of the kit may lose their activity. Store the kit at a proper temperature to prevent loss of activity. Divide the reagents into aliquots to avoid the repeated freeze-thaw cycles as much as possible.

3) Cysteine may not have been added. In PUREfrex^® 2.1, you can freely adjust the amount of reducing agents (Cysteine, DTT and GSH, etc.), but be sure to add Cysteine, which is a material of protein.

YES.
PUREfrex^® basically contains a final concentration of 1.5% glycerol, under which conditions protein is synthesized. When glycerol is added, if the final concentration in the reaction solution is 5% or less, there is almost no effect on protein synthesis, but high concentrations of glycerol inhibit protein synthesis.

NO.
If the final concentration of DMSO in the PUREfrex^® 1.0 reaction is only a few percent, there is no effect on protein synthesis.

YES.
The addition of a chelating agent to the PUREfrex^® reaction solution will greatly affect the amount of product, so avoid using it as much as possible.

YES.
Regarding magnesium ion, if the final concentration in the PUREfrex^® 1.0 reaction solution is only a few mM, there is little effect on protein synthesis. For other divalent cations, if the final concentration in the PUREfrex^® 1.0 reaction solution is 10 mM or more, the amount of product is often reduced, so avoid using them as much as possible.

NO.
If the final concentration of potassium ion in the PUREfrex^® 1.0 reaction is in the range of 10-20mM, there is no effect on protein synthesis.

YES.
The PUREfrex^® reaction solution is buffered so that the pH is around 7.5. Therefore, when adding an acid solution or alkaline solution, adjust the pH of the reaction solution so that it is near neutral.

YES.
Lowering the reaction temperature from 37°C to 30°C or 25℃ slows down the translation speed and generally reduces the amount of product. On the other hand, the folding is often slower than the translation, so slowing the translation may improve the solubility of the target protein.

Generally, we recommend reaction temperature of 37°C and a reaction time of 2 to 4 hours. However, the optimal conditions may differ depending on the proteins. For example, if synthesis at 37°C results in insolubilization or low activity, we recommend trying synthesis at other temperatures. For more information, Please see, “【Poster_MBSJ 2020】Investigation on how to synthesize active proteins by using a reconstituted cell-free protein synthesis system (PUREfrex^®)”

In case of using mRNA to synthesize the protein of interest, make sure that the mRNA contains SD sequence upstream of start codon. Add mRNA usually at a concentration of 0.1 to 1 µM to the reaction mixture. Since the optimal concentration depend on the sequence and purity of the mRNA, we recommend examining the optimal concentration in reference to this concentration range.

In preparing plasmid DNA, make sure that the RNase used for purification is not contaminated in the final purified DNA.

Using a filter-based purification kit such as QIAprep Spin Miniprep Kit (Qiagen) or Wizard Plus SV Minipreps DNA Purification System (Promega), for example, results in contamination of RNase A (contained in Lysis buffer) in to the final purified DNA solution. If you add this solution to the PUREfrex^® reaction mixture as template DNA without further treatment, transcription products and other RNAs in the reaction mixture are digested, which inhibit protein synthesis.

Therefore, treat the DNA with phenol/chloroform to denature RNase and then re-purify the DNA by ethanol precipitation to prepare it with free of RNase activity. Adding RNase inhibitor to the PUREfrex^® reaction mixture is also effective. Plasmid DNAs purified with Plasmid Mini Kit (Qiagen) has minimal contamination with RNase because the plasmid DNA eluted from resin is precipitated by adding isopropanol. Plasmid DNA purified with this kit are verified to use without any treatment.　
You can find the example with plasmid in the link here.

Vectors that contain T7 promoter, SD sequence, and T7 terminator can be used such as pET vectors (Merck) and pQE vectors (Qiagen). The presence of the lac operator sequence may decrease the protein yield. We recommend using vectors without the lac operator sequence (e.g., pET17).

YES.
Synthetic DNA fragments containing promoter sequences can be used as template DNA. In addition, template DNA can be prepared from synthetic DNA of ORFs by PCR using primers that include the promoter sequence and other necessary sequences.
However, depending on the manufacturer of the gene synthesis, the delivered product may contain RNase, which inhibits the protein synthesis reaction. If the protein of interest cannot be synthesized, try adding an RNase inhibitor or inactivating the RNase.
Also, if the product is delivered as a plasmid, please check the notes for using the plasmid as a template DNA.

PCR product should be a single band on an electrophoresis gel. If you found unexpected bands, modify the PCR condition to reduce byproducts. The purity of the PCR product affect the efficiency of protein synthesis because proteins can be also synthesized from PCR-byproducts.
If byproducts are unable to eliminate by changing the PCR conditions, excise the target band from the gel and purify the DNA. Excise the band from the gel without using ultraviolet light, which can damage DNA (i.e., impair transcription). Blue light may be used, but exposure time should be kept to a minimum.

Although unpurified PCR reaction mixture can be directly used with PUREfrex^®, do not add more than 10% (v/v) to the reaction mixture of PUREfrex^®. The efficiency of both transcription and translation reaction may decrease when the salt concentration is altered by carryover from the PCR mixture. If the concentration of the PCR product is low, prepare DNA solution with a sufficient concentration using a DNA purification kit instead of adding more unpurified PCR mixture to PUREfrex^®.

No.
Buffers containing EDTA should not be used for dissolving DNA, because EDTA inhibits transcription and translation reaction. We recommend dissolving the template DNA in EDTA-free buffer or Milli-Q water.

0.5-3 ng per 1 kbp DNA for 1 µL of the reaction mixture will be fine.
The amount of DNA should be 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 should be added for 1 µL of the reaction mixture.

“REV primer” is basically flexible, but there are some points to be considered.
・The “taatga” after the C-terminus of the ORF means two stop codons.
・The length is more important than the sequence. If the length is shorter than 10 bases, the efficiency of protein synthesis will be affected, but if it is longer than 10 bases(like 20 bases or more), it will be fine.
・Avoid sequences that form a rigid secondary structure (ex, high GC content, etc.) just after the stop codon as much as possible.
・For example, a restriction enzyme site can be inserted in this region.

When you use circular DNA, T7 terminator sequence to terminate transcription must be placed downstream of the gene encoding the protein of interest. When you use linear DNA, add at least 10 nucleotides downstream from stop codon. T7 terminator sequence is not necessary when linear DNA is used.

All of 3 stop codons (TAA, ochre; TAG, amber; TGA, opal) can be used with PUREfrex®, because it contains 2 release factors (translation termination factors) recognizing all 3 stop codons.

YES.
However, we know that the 5’UTR sequence affects the protein synthesis; see our poster on the effect of 5’UTR sequences on yield. So far, the 5’UTR sequence we have designed has shown the highest yield. Each sequences in 5’UTR have different effects on the yield, so please adjust the sequence according to the purpose of your experiment.

YES.
We recommend the template DNA with T7 promoter, because PUREfrex^® contains T7 RNA polymerase for transcription. However, if you use other polymerases, please generate template DNA with the suitable promoter for the selected polymerase.

The template DNA must contain T7 promoter sequence and ribosome binding site (SD sequence) upstream of the gene encoding the protein of interest.

Please refer to the following page on the points for template DNA preparation and optimization.
Tech Notes: “Preparation of the template DNA”
Tech Notes: “6 Tips about the template DNA Design”

YES.
The N-terminal methionine of the product synthesized by PUREfrex^® is formylated. And they are not deformylated during/after translation.

However, depending on the amount of synthesized protein, the formyl donor may be depleted and some non-formylated proteins may be present. PUREfrex^® proceeds with the translation reaction even if the N-terminal methionine is not formylated, but synthesis efficiency may be affected depending on the protein to be synthesized.

YES.
PUREfrex^® uses a tRNA mixture prepared from E. coli, so each tRNA concentration reflects the tRNA concentration in E. coli. Therefore, the amount of tRNA that is low in E. coli is also low in the PUREfrex^® reaction solution.

For the above reasons, we recommend basically that the template DNA for PUREfrex^® should use codons that match the codon frequency in E. coli. In addition to that, when you design your template DNA, there are some more points to be considered, so please see Question 1 of “FAQ about the template DNA”.

NO.
Signal peptide is not required for synthesis of secretory protein in PUREfrex^®, but removing a signal sequence may reduce the amount of the product. Therefore, we recommend optimizing the sequences (especially the N-terminus) excluding the signal sequence. For more information, please see Question 1 of “FAQ about the template DNA”.

YES.
A radiolabeled protein can be synthesized by adding radioisotope-labeled amino acids, such as [35S] methionine or [3H] leucine, into PUREfrex^®. PUREfrex^® 1.0 includes 0.5 mM of each amino acids. Please consider that point and optimize conditions for your labeling.

YES.
It is not possible with PUREfrex^® alone, as it does not contain post-translational modification enzymes.
However, synthesis is possible by adding post-translational modification enzymes and substrates to the reaction mixture.
For glycosylation, please see “【Poster_MBSJ 2021】 Synthesis of glycosylated proteins using PUREfrex^® with glycosyltransferase”.
For N-terminal modifications, please see the link: “https://pubs.acs.org/doi/10.1021/acssynbio.3c00191“.

YES.
In most 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 or nanodisc into PUREfrex^® when you synthesize the membrane protein.
Please see, “【Poster_MBSJ 2016】Improvement of translational efficiency by N-terminal codon optimization in the reconstituted cell-free protein synthesis system”

YES.
DsbC Set (#PF005-0.5-EX) or PDI Set (#PF006-05-EX) are supplements for PUREfrex^® to add for synthesizing proteins containing disulfide bonds in active form. PUREfrex^® 2.1 (#PF213-0.25-EX) is recommended to optimize the reaction conditions.
For antibodies (IgG, Fab, scFv) synthesis, please see the link: “https://www.nature.com/articles/s41598-018-36691-8“.

NO.
PUREfrex^® does not contain any chaperones. FYI, GeneFrontier provides molecular chaperones such as Hsp70 (DnaK Mix: #PF003-0.5-EX) and Hsp60 (GroE Mix: #PF004-0.5-EX) that can be added to PUREfrex^®.

YES.
We have synthesized the protein of 116 kDa using PUREfrex^®.

YES.
Using multiple template DNAs encoding different proteins, it is possible to synthesize multiple proteins simultaneously in one tube. If the amount of synthesized protein differs from each template DNA, the amount of each products can be controlled by adjusting the ratio of template DNA to add.
As an example, you can see the results of simultaneous synthesis of immunoglobulin (IgG) light chain (LC) and heavy chain (HC) on the link here: “https://www.nature.com/articles/s41598-018-36691-8“.

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 His-tag can be purified with metal-chelating resin by applying reaction mixture directly after the reaction. Please see Tech Notes: “Purification of His-tagged protein (DHFR-6xHis)”.

It depends on the target protein.
Dihydrofolate reductase (DHFR) from E.coli can be synthesized at approximately 160 µg per mL of PUREfrex^® 1.0 reaction and approximately 600 µg per mL of PUREfrex^® 2.0 reaction.

YES.
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. The nucleotide sequence and amino acid sequence of the template DNA, rather than the origin of the protein, may affect the amount of the product, and optimizing the causative sequences may improve the amount of the protein synthesized.

For more information, please see Question 1 of “FAQ about the template DNA”.