Q1: Can I synthesize eukaryotic proteins using PUREfrex® kit?

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".

Q2: How much can I synthesize the target protein using PUREfrex® kit?

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.

Q3: Can I synthesize and purify a tagged protein?

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)".

Q4: Can multiple proteins be synthesized simultaneously?

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".

Q5: Can I synthesize proteins of more than 100 kDa?

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

Q6: Does PUREfrex® contain any molecular chaperones?

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®.

Q7: Can I synthesize proteins containing disulfide bonds (such as antibodies) with PUREfrex®?

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".

Q8: Can I synthesize a membrane protein with PUREfrex®?

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”.

Q9: Are post-translational modifications such as glycosylation and N-terminal modifications possible with PUREfrex®?

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 "【Cases】 N-terminal modification of cell-free synthesized proteins (acetylation and myristoylation)".

Q10: Can I synthesize a radiolabeled protein with [35S] methionine or [3H] leucine?

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.

Q11: Is a signal sequence required for secretory protein synthesis?

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".

Q12: Should the codon usage of PUREfrex® be the same as that of E. coli?

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".

Q13: Are the N-terminal methionines of proteins/peptides expressed in PUREfrex® formylated? If so, are they deformylated during/after translation?

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.

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