PF201-0.25-EX
PF201-2ML-EX
PF201-10ML-EX

Instruction ManualSep 2023

Table of Contents
Kit Information ............................................................................................................... 1
PUREfrex® 2.0 ...................................................................................................................... 1
Product information ............................................................................................................................. 1
Product contents .................................................................................................................................. 1

Caution about the use of the kit ........................................................................................... 2

About PUREfrex® ............................................................................................................ 3
What is PUREfrex®? ............................................................................................................. 3
About the PURE System ...................................................................................................... 3

Experiment workflow ...................................................................................................... 4
1. Preparation of the template DNA .................................................................................... 5
About the template DNA for PUREfrex® .............................................................................................. 5
In case using PCR product as the template DNA.................................................................................. 6
In case using plasmid DNA as the template DNA ................................................................................. 8
In case using RNA as a template ........................................................................................................... 8
Tips about the template DNA sequence ............................................................................................... 9

2. Synthesis of Protein using PUREfrex® 2.0 ..................................................................... 12
2-1 Synthesis of protein ...................................................................................................................... 12
2-2 Detection of the synthesized product ........................................................................................... 13
2-3 Evaluation of solubility ................................................................................................................. 14
2-4 Evaluation of disulfide bond formation ......................................................................................... 15

3. Addition of supplement .................................................................................................. 16

Examples of Protein Synthesis with PUREfrex® ........................................................... 17
1. Synthesis of DHFR ......................................................................................................... 17
What was used .................................................................................................................................... 17
DHFR gene and primer sequences ..................................................................................................... 18
Preparation of template DNA by 2-step PCR ..................................................................................... 18
Result of 2-step PCR .......................................................................................................................... 20
Synthesis with PUREfrex® and detection of synthesized DHFR .......................................................... 20
Result of synthesis of DHFR ............................................................................................................... 21

2. Purification of His-tagged protein (DHFR-6xHis)........................................................ 22
What was used .................................................................................................................................... 22
Synthesis with PUREfrex® and purification of synthesized DHFR-6xHis............................................ 22
Result of purification of DHFR-6xHis ................................................................................................ 23

3. Detection of the synthesized product with FluoroTect™ GreenLys (DHFR) ................. 24i

What was used .................................................................................................................................... 24
Protein Synthesis in the presence of FluoroTect™ GreenLys ............................................................... 24
Result of synthesis in the presence of FluoroTect ............................................................................... 25

4. Synthesis in the presence of molecular chaperones (luciferase) .................................... 26
What was used .................................................................................................................................... 26
Synthesis of luciferase in the presence of DnaK Mix and evaluation of its activity .............................. 26
Result of synthesis in the presence of DnaK Mix ................................................................................ 27

Troubleshooting ............................................................................................................ 28
The positive control (DHFR) is not synthesized. .............................................................. 28
The protein of interest is not synthesized. ......................................................................... 28
The template DNA does not contain the minimum required sequences. ............................................ 28
Too much or little DNA is added to the reaction mixture. .................................................................. 28
The method for preparing the template DNA is inappropriate. .......................................................... 28
The template DNA contains a difficult-to-translate sequence. ........................................................... 28

The synthesized protein is insoluble. ................................................................................. 29
Add molecular chaperones when synthesizing the protein. ................................................................. 29
Synthesize the protein at a lower translation speed. ............................................................................ 29

The synthesized protein has no activity. ............................................................................ 29
The reaction mixture becomes turbid when sample buffer for SDS-PAGE is added. ....... 29

Appendix ....................................................................................................................... 30
DHFR DNA sequence information .................................................................................... 30
Compositions of SDS-PAGE sample buffer....................................................................... 30
Publications ........................................................................................................................ 30

Related Products ........................................................................................................... 31
Kit for protein synthesis ..................................................................................................... 31
Supplement for protein synthesis....................................................................................... 31

Order support ............................................................................................................... 32
Technical support ......................................................................................................... 32ii

Kit Information
PUREfrex ® 2.0

Product information
Store the unopened kit at -80ºC.
Product no.

Product nameVolumePF201-0.25-EX

PUREfrex® 2.0

For 250 µL reactions

PF201-2ML-EX

PUREfrex® 2.0

For 2 mL reactions

PF201-10ML-EX

PUREfrex® 2.0

For 5x 2 mL reactions

Expiration date
See the attacheddocument
See the attacheddocument
See the attacheddocument

Product contents

VolumeVolume(250 µL rxn)

(2 mL rxn)

Solution I

125 µL

1000 µL

Amino acids, NTPs, tRNAs, enzyme substrates, etc.

≤ -20ºC

Solution II

12.5 µL

100 µL

Proteins (dissolved in 30% glycerol solution)≤ -20ºC*

Solution III

25 µL

200 µL20 µM Ribosomes-80ºC*

Reagent name

Description

StoragetemperatureTemplate DNA containing gene encoding E. coli
DHFR DNA

10 µL

10 µL

dihydrofolate reductase (DHFR) for positive control of≤ -20ºC

protein synthesis (20 ng/µL)*For storage at -80ºC: If storing the remaining Solution II and III at -80ºC after use, store after quickly freezing withliquid nitrogen or dry ice/ethanol. Divide into aliquots as necessary to avoid repeated freeze-thaw cycles wheneverpossible.

- 1 -Caution about the use of the kit
PUREfrex® is a kit for research purposes. Administration to human and other animals and use in the clinic, for diagnostic
purposes, and other uses are prohibited. Do not use in foods or for domestic purposes. It contains no compound regulated
under the relevant laws of Japan (Act on Confirmation of Release Amounts of Specific Chemical Substances in theEnvironment and Promotion of Improvements to the Management Thereof, Industrial Safety and Health Act, and Poisonousand Deleterious Substances Control Act).
PUREfrex® is a registered trademark of GeneFrontier Corporation. For any commercial use of PUREfrex®, please contact
GeneFrontier in advance.
e-mail : purefrex@genefrontier.com- 2 -About PUREfrex ®
What is PUREfrex ® ?
PUREfrex® is a product based on the PURE system technology that is a reconstituted cell-free protein synthesis systemdeveloped by a group led by Professor Takuya Ueda of the University of Tokyo (1). The kit mainly consists of 3 reagents.
Solution I contains amino acids, NTPs, other small-molecule compounds and tRNAs. Solution II contains RNA polymerase,translation factors and other proteins. Finally, Solution III contains purified ribosomes. The purified components are mixed
at a concentration to achieve the maximum protein synthesis activity. For the protein synthesis, DNA (or mRNA) encoding
the protein of interest is added to the mixture of Solutions I, II, and III and then the reaction mixture is incubated. The
preparation methods of the proteins, ribosomes, and tRNAs constituting the reagents of PUREfrex® are improved over thatof the original PURE system (1). The improved preparation method reduce contaminant substances unrelated to the protein
synthesis. Lipopolysaccharides from E. coli, for example, are reduced to around 0.1 EU per microliter of the reaction mixture.
Amounts of RNase, beta-galactosidase and other contaminating proteins are also reduced. All translation factors and otherproteins contained in the reagents of PUREfrex® do not contain any tag sequences. This enables the use of any tag sequencefor purification and detection of the synthesized protein.

About the PURE System
The PURE system is a reconstituted cell-free protein synthesis system developed by a group led by Professor Takuya
Ueda of the University of Tokyo (1, 2). The system contains 3 initiation factors (IF1, IF2, IF3), 3 elongation factors (EF-Tu,
EF-Ts, EF-G), 3 release factors (RF1, RF2, RF3), ribosome recycling factor, 20 aminoacyl-tRNA synthetases, andmethionyl-tRNA formyltransferase, that involved in translation in E. coli, as well as T7 RNA polymerase and 4 enzyme
proteins necessary for transcription and energy regeneration, respectively. These proteins are individually purified and thenmixed with amino acids, NTPs, other small-molecule compounds, tRNAs and purified ribosomes. In the PURE system
purified factors are only used rather than the cell extracts used in other cell-free protein synthesis systems. This means it isalmost free of proteins unrelated to the protein synthesis and allows its composition to be freely adjusted, which are thesuperior characteristics of the PURE system.
References:
1. Shimizu Y. et al. (2001) Nat. Biotechnol., vol. 19, p. 751.
2. Shimizu Y. et al. (2005) Methods, vol. 36, p. 299.

- 3 -Experiment workflow
General procedures for using PUREfrex® to synthesize functional proteins are shown in Figure 1.
Start by preparing the template DNA suitable to the protein synthesis with PUREfrex® and examine whether the protein
of interest is synthesized. Redesign the nucleotide sequence of the template DNA if too little protein is synthesized. Add
supplement such as molecular chaperones or changing the synthesis conditions if the solubility or activity of the synthesized
protein is low or absent. The kit contains enough reagents for examining whether the target protein can be synthesized.

1. Preparation of template DNA (page 5)x
No (no or too little product)2-1. Synthesis of protein (page 12)x2-2. Detection of synthesized product (page 13)xOK

2-3. Evaluation of solubility (page 14)
2-4. Evaluation of disulfide bond formation (page 15)x

No (insoluble, polymerization, etc.)

OK
3. Addition of supplement (page 16)

Analysis / Purification of synthesized productFigure 1. Workflow of protein synthesis with PUREfrex®

- 4 -1. Preparation of the template DNA
Transcription and translation take place sequentially in the same reaction mixture after template DNA is added to the
mixture by combining Solutions I, II, and III of the kit. Preparing the template DNA suited to the protein synthesis with
PUREfrex® is essential. Consider the following information when preparing the template DNA encoding the protein ofinterest to be synthesized with PUREfrex®. Please also use the support of DNA design for PUREfrex® (p. 11).

About the template DNA for PUREfrex ®
Both circular and linear DNA (including PCR product and circular DNA digested with a restriction enzyme) can beused as the template DNA for PUREfrex® in case it contains at least the sequence described in the following section.
Alternatively, mRNA can be also used to synthesize protein with PUREfrex®.
About the upstream region of the gene encoding the protein of interest
The template DNA must contain T7 promoter sequence and ribosome binding site (SD sequence) upstream of
the gene encoding the protein of interest (Figure 2).

Figure 2. Construct of the template DNA

About stop codon of the gene encoding the protein of interest
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) recognize all 3 stop codons.
About the downstream region of the gene encoding the protein of interest
When using circular DNA, T7 terminator sequence to terminate transcription must be placed downstream of thegene encoding the protein of interest. When using linear DNA, add at least 10 nucleotides downstream from stopcodon. See “Examples of primers used for 2-step PCR” (page 7) for an example of nucleotide sequence to add.
T7 terminator sequence is not necessary when linear DNA is used.

- 5 -Tips for dissolving the template DNA
EDTA contained in TE buffer may reduce the efficiency of protein synthesis because it chelates magnesium ion
necessary for transcription and translation reaction. We recommend dissolving the template DNA in EDTA-freebuffer or Milli-Q water. Since contaminating RNase digests RNA (transcription products and tRNAs) in thereaction mixture, we recommend using only nuclease-free water, reagents, and instruments and wearing gloves
and a mask.

In case using PCR product as the template DNA
PCR product purity
If bands other than that of the desired product are detected in electrophoresis after PCR, 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 andpurify the product. 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.
How much PCR product to add
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 isapproximately 0.5 to 3 ng per 1 kbp for 1 µL of the reaction mixture.
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 the reaction mixture of PUREfrex®.
Overview of preparing the template DNA by 2-step PCR
Two-step PCR for preparing the template DNA is summarized in Figure 3. Use a high fidelity PCR enzyme(e.g., KOD (Toyobo), PrimeSTAR (TakaraBio), Pfu (Promega)) to prepare the template DNA. At the first step,use FOR primer and REV primer to amplify fragments containing a sequence from the ribosome binding site (RBS
(SD sequence)) upstream of the gene encoding the protein of interest to a stop codon and at least 10 nucleotides.
Adjust the PCR condition so that even minimal byproducts are produced at 1st PCR. At the second step, use
T7PRO-SD primer and REV primer to amplify PCR fragments containing T7 promoter sequence.

- 6 -FOR, REV, and T7PRO-SD primers are not included in the kit and should be purchased separately.

Figure 3. Overview of 2-step PCR

Examples of primers used for 2-step PCR
Primer sequences used for 2-step PCR are shown in Table 1. Also refer to the following site:
https://purefrex.genefrontier.com/resources/tech_notes/sequence-of-dhfr-dna.html

Table 1. Primer sequences- 7 -In case using plasmid DNA as the template DNA
Usable vectors
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).
Plasmid DNA preparation
When preparing plasmid DNA, make sure that the activity of RNase used for purification is not contaminatedin the final purified product. 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 the final
purified DNA solution by the RNase A contained in Lysis buffer. If adding this solution without further treatment
to the PUREfrex® reaction mixture as template DNA, transcription products and other RNA in the reaction mixture
are digested, inhibiting protein synthesis. Therefore, treat with phenol/chloroform to denature RNase and then repurify by ethanol precipitation to prepare a DNA solution free of RNase activity. Adding RNase inhibitor to the
PUREfrex® reaction mixture is also effective.
Plasmid DNA purified with Plasmid Mini Kit (Qiagen) has minimal contamination with RNase because theplasmid DNA eluted from resin is precipitated by adding isopropanol. Plasmid DNA purified with this kit areverified to use without any treatment.
How much plasmid DNA to add
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 isapproximately 0.5 to 3 ng per 1 kbp for 1 µL of the reaction mixture. A 6 kbp plasmid DNA, for example, is beadded at (0.5-3)×6 = 3-18 ng/µL, regardless of the length of the ORF.

In case using RNA as a template
In case using mRNA to synthesize the protein of interest, make sure that the mRNA contains SD sequence upstreamof 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 concentrationin reference to this concentration range.

- 8 -Tips about the template DNA sequence
Certain nucleotide or amino acid sequence may reduce the yield of the protein of interest. In such cases, optimizing
the sequence may improve the yield. Ribosomes, tRNAs, and translation factors in PUREfrex® are derived from E. coli.
Therefore, in principle, it is recommended to design the gene encoding the protein of interest with codons optimized
for translation in E. coli. Consider the following points including the used codons when designing the template DNA
sequence for PUREfrex®.
Codon usage
Nucleotide sequences optimized for E. coli by available optimization tool may use single codon frequently usedin E. coli. Only CTG codon, for example, may be used for leucine. The yield of the synthesized protein may reduce
when using such DNA as a template DNA. If the codon usage is excessively imbalanced in the optimized sequence,reassign properly according to the codon usage in the E. coli genome.
The codon usage in the E. coli genome (W3110) is referred to the following web site:
https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=316407
AT content just after the start codon
Select codons to maximize AT content in the region immediately after the start codon (usually up to 6thcodon) (Figure 4 on page 10). In this region, use AT-rich codons over codons frequently used in E. coli.
Secondary structure of mRNA around the start codon
If mRNA can form a rigid secondary structure around the start codon, from SD sequence to the N-terminal of
the ORF (about 6-10 codons), and SD sequence is hidden, binding of mRNA to ribosome is impaired and then theyield may be decreased. Substitute N-terminal codons with synonymous codons if a rigid secondary structure toform in this region is predicted.
Example of secondary structure prediction is illustrated in Figure 5 (page 10). In this example, RNAfold(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) is used to analyze DHFR DNA included as the
positive control in the kit. Since the region around the start codon does not form rigid secondary structure, theyield is not reduced.
Amino acid sequence at N-terminus
A low yield may occur if the second and third amino acids right after the first methionine are proline or glycine.
Avoid using proline and glycine in this region if possible.

- 9 -Figure 4. Effects of codons near N-terminal region on protein yield
A. Codons of N-terminal region of heavy chain (HC) of trastuzumab (Herceptin)
B. Comparison of yields from template DNA with different N-terminal codons

Figure 5. Secondary structure prediction of the translation initiation region
A. Nucleotide sequence of 5'-terminal region of DHFR DNA. The region analyzed is highlighted in yellow.
B. Secondary structure prediction by RNAfold- 10 -

Sequence that cause frameshift
Nucleotide sequences that are likely to cause frameshift during elongation, such as X/XXY/YYZ, should be
replaced with other codons. A nucleotide sequence of “A/AAA/AAA” for 2 consecutive lysine residues, forexample, should be replaced with “A/AAG/AAA” to avoid frameshift.
Reference: Sharma V. et al. (2014) Nucleic Acids Res., vol.42, p.7210.
Sequences containing consecutive proline residues
Proteins with consecutive proline residues may be produced at a low yield. A translation factor called EF-P isknown to be involved in elongation at consecutive proline residues in E. coli, but the kit (PUREfrex® 2.0) contains
no EF-P and may therefore produce a lower-than-normal yield. EF-P is available as a supplement (#PFS052-0.5EX).
Examples of effect of EF-P on the increase of the yield are shown in the following poster.
https://www.genefrontier.com/files/sites/2/p21_MBSJ2018.pdf
Support of DNA design for PUREfrex ®
<Consultation is free of charge!> Send us the amino acid sequence of your target protein by the link below,
and we will return a suitable DNA sequence based on our algorism considering important points for protein
expression using PUREfrex®, such as codon optimization for whole ORF and N-terminus, UTRs, etc.

- 11 -

2. Synthesis of Protein using PUREfrex ® 2.0
Template DNA is added to the reaction mixture prepared by combining Solutions I, II, and III of the kit and then hereaction mixture is usually incubated for 2 to 6 hours at 37ºC.
Contamination of RNase digests the transcription products, tRNA, and other RNA, inhibiting synthesis or substantiallyreducing the yield. Be sure to use RNase-free tubes and tips and work with gloves.
For the first protein synthesis, we recommend concurrently performing the reaction without template DNA (negative
control) and the reaction with DHFR DNA included in the kit (positive control).

2-1 Synthesis of protein
What is needed
PUREfrex® 2.0 (GeneFrontier)Template DNA(containing gene encoding the protein of interest, and sequences necessary for transcription and translation)
Nuclease-free water
37ºC heat block or water bath
Protocol1.

Warm Solution I at a temperature between room temperature and 37ºC for approximately 1 minuteto completely thaw (so that any turbidity is removed) and then place on ice.
■ If the solution again becomes turbid after being placed on ice, keep at room temperature and use as soon
as possible, storing the remaining reagent at a temperature less than -20ºC.

2.

Thaw Solutions II and III on ice.

3.

Vortex or tap the thawed Solutions I, II, and III to achieve uniform and then centrifuge to collect thecontents at the bottom of the tubes.

4.

Prepare the following reaction mixture to synthesize protein in 20 µL:
Nuclease-free water

7-X µL

Solution I

10 µL

Solution II

1 µL

Solution III

2 µL

Template DNA

X µL

Total

20 µL

(0.5 to 3 ng/µL per 1 kbp)

■ Adjust the amount of template DNA added according to the type and concentration of the DNA used.
See “1. Preparation of the template DNA ” (page 5) for more information.

- 12 -

■ The volume of reaction mixture can be freely changed. Synthesis is possible with as little as several
microliters.
5.

Synthesize the protein of interest by allowing the tube to incubate for 2 to 6 hours on a 37ºC heat blockor water bath.
■ Gas-phase chambers (e.g., incubators) take time to warm the reaction mixture and produce a lower yield.
■ Adjust the temperature and incubation time of synthesis to best suit the protein synthesized.

6.

Detect the synthesized protein with SDS-PAGE.

2-2 Detection of the synthesized product
Apply the reaction mixture to SDS-PAGE to examine if the protein of interest is synthesized. Compare the lane of
the reaction mixture containing the template DNA to the lane of the reaction mixture without template DNA and see if
the band is present near the expected molecular weight of the synthesized protein. The maximum yield obtained by the
kit is almost the yield of DHFR synthesized from DHFR DNA included in the kit. Redesign the template DNA sequence
if the yield of the protein of interest is substantially lower than that of DHFR. Refer to “Tips about the template
DNA sequence ” (page 9) when designing the template DNA sequence.
The following method can be used for the detection of the synthesized product with SDS-PAGE.
• Stain the gel with fluorescent dye instead of CBB and then detect the protein bands by fluorescent imager.
• Detect the product by western blotting using an antibody against the protein of interest. Alternatively,
synthesize the target protein fused with tag sequence and detect with an antibody against the tag.
• Synthesize the protein in the presence of [35S] methionine and detect radioactive bands in the SDS-PAGE gel.
• Synthesize the protein in the presence of FluoroTect™ GreenLys (Promega) and detect fluorescent bands in the
SDS-PAGE gel.
An example of stain with Coomassie Brilliant Blue (CBB) following SDS-PAGE to detect the product is shown as
below. Change the reaction mixture volume and gel concentration for SDS-PAGE according to the molecular weightand yield of the protein of interest, the gel staining method, and other factors.
What is needed (in case of staining with CBB)
PUREfrex® reaction mixture after reaction
3x Sample Buffer for SDS-PAGE (see Appendix for composition on page 30)
Water
95ºC heat block or water bath
SDS-PAGE gel
Molecular weight marker- 13 -

CBB stain
Protocol1.

Add equal volume of water to the reaction mixture after protein synthesis.
■ Salt concentration of the reaction mixture is relatively high. Add 3x Sample Buffer after diluting withwater or buffer because addition of 3x Sample Buffer to the undiluted reaction mixture and heating may
cause a white precipitate to form.

2.

Add equal volume of 3x Sample Buffer to the diluted reaction mixture.

3.

Heat for 5 minutes at 95ºC.

4.

Apply 3 µL of the sample per lane and molecular weight marker to the SDS-PAGE gel and runelectrophoresis.
■ This is equivalent to 1 µL of reaction mixture per lane.
■ A 10-20% gradient gel is recommended for analyses over a wide range of molecular weight.

5.

After electrophoresis, stain the gel with CBB and detect the band of the protein of interest.
■ The band of the positive control (DHFR) is around 18 kDa.

2-3 Evaluation of solubility
Centrifuge the PUREfrex® reaction mixture containing the synthesized protein to separate it into supernatant andpellet fractions. Evaluate the solubility of the synthesized product by detecting the product in the supernatant with SDSPAGE.
What is needed
PUREfrex® reaction mixture after reaction
High-speed refrigerated microcentrifuge
3x Sample Buffer for SDS-PAGE (see Appendix for composition on page 30)
Water
95ºC heat block or water bath
SDS-PAGE gel
Molecular weight marker
Protocol1.

Add equal volume of water to the reaction mixture after protein synthesis.

2.

Withdraw an aliquot from the diluted reaction mixture. → “Total”- 14 -

3.

Centrifuge at 20,000 xg for 30 minutes at 4ºC.

4.

Withdraw an aliquot from the supernatant. → “Sup”

5.

Analyze the Total and Sup samples with SDS-PAGE.

2-4 Evaluation of disulfide bond formation
Evaluate disulfide bond formation by analyzing the synthesized protein with SDS-PAGE under non-reducing
condition. Proteins with disulfide bonds may exhibit altered mobility on SDS-PAGE under non-reducing conditions.
The product with disulfide bond may also be stuck in the sample well or detected as a smear band under non-reducing
condition. Confirm that disulfide bonds are correctly formed by assaying the activity of the synthesized product.
What is needed
PUREfrex® reaction mixture after reaction
3x Sample Buffer for SDS-PAGE (see Appendix for composition on page 30 without reducing agent)
Water
95ºC heat block or water bath
SDS-PAGE gel
Molecular weight marker
Protocol1.

Add equal volume of water to the reaction mixture after protein synthesis.

2.

Add equal volume of 3x Sample Buffer (without reducing agent) to the diluted reaction mixture.

3.

Heat for 5 minutes at 95ºC.

4.

Perform SDS-PAGE and analyze the bands of the synthesized product.

- 15 -

3. Addition of supplement
If the protein of interest aggregates or contains disulfide bond, we recommend addition of molecular chaperones oradditive to promote disulfide bond formation to PUREfrex® reaction mixture. See “Related Products” (page 31) for
information on related products.

- 16 -Examples of Protein Synthesis with PURE frex ®
Examples of the synthesis of various proteins are also shown in the following web site:
https://www.genefrontier.com/en/cases/purefrex/
Posters presented by GeneFrontier at conferences can be downloaded below:
https://purefrex.genefrontier.com/resources/posters/

1. Synthesis of DHFR
In this section, the preparation of template DNA for DHFR (the positive control in the kit) by PCR and synthesis of
DHFR with PUREfrex® 2.0 are described.

What was used
DNA containing ORF of DHFR
Primer (DHFR-F, DHFR-R3, T7PRO-SD Primer)
Water
PCR reagents (e.g., KOD-Plus-Neo (Toyobo, Japan))
Thermal cycler
PCR product purification kit (e.g., NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel))
6x Sample Buffer for agarose gel electrophoresis
Agarose gel (1%)
DNA size marker
UV gel imager
PUREfrex® 2.0 (#PF201-0.25-EX)
Nuclease-free water
Heat block or water bath (37°C and 95°C)
3x Sample Buffer for SDS-PAGE (see Appendix for composition on page 30)
SDS-PAGE gel (10-20%)
Molecular weight marker
CBB stain■ PCR enzymes and DNA purification kits from other manufacturers can be used. Be sure, however, that PCR
enzymes with high fidelity is used.

- 17 -

DHFR gene and primer sequences
DHFR gene
ATGATCAGTCTGATTGCGGCGTTAGCGGTAGATCGCGTTATCGGCATGGAAAACGCCATGCCGTGGAACCTGCCTGC
CGATCTCGCCTGGTTTAAACGCAACACCTTAAATAAACCCGTGATTATGGGCCGCCATACCTGGGAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATTATCCTCAGCAGTCAACCGGGTACGGACGATCGCGTAACGTGGGTGAAGTCGGTGGATGAAGCCATCGCGGCGTGTGGTGACGTACCAGAAATCATGGTGATTGGCGGCGGTCGCGTTTATGAACAGTT
CTTGCCAAAAGCGCAAAAACTGTATCTGACGCATATCGACGCAGAAGTGGAAGGCGACACCCATTTCCCGGATTACG
AGCCGGATGACTGGGAATCGGTATTCAGCGAATTCCACGATGCTGATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGGTAA
Primer
FOR Primer（DHFR-F）
AAGGAGATATACCAATGATCAGTCTGATTG
REV Primer（DHFR-R3）
GGATTAGTTATTCATTACCGCCGCTCCAGAAT
T7PRO-SD Primer
GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAG
ATATACCA

Preparation of template DNA by 2-step PCR
1.

The DHFR gene DNA and primer were diluted to 1 ng/µL and 2 µM, respectively, with water.

2.

The PCR reagents other than the enzymes were thawed at room temperature.

3.

The PCR reaction mixture was prepared as follows:water

3.5 µL10x buffer

1.0 µL

2 mM dNTPs

1.0 µL

25 mM MgSO4

0.6 µL2 µM FOR Primer (DHFR-F)

1.5 µL2 µM REV Primer (DHFR-R3)

1.5 µL

DMSO

0.2 µL

Enzyme (KOD-Plus- Neo)

0.2 µL

1 ng/µL DHFR gene DNA

0.5 µL

Total

10.0 µL- 18 -

4.

PCR was performed as below: (Step 1)
94ºC

2 min.

94ºC

15 sec.

58ºC

30 sec.

68ºC

1 min.

68ºC

2 min.

x 30 cycles

10ºC
5.

The PCR reaction mixture (1 µL) was analyzed with 1% agarose gel electrophoresis to confirm thepresence of the amplification products.

6.

The PCR products were purified with a PCR product purification kit (NucleoSpin® Gel and PCR Cleanup (Macherey-Nagel)). The bound DNA was eluted from the column with 20 µL of water.

7.

The PCR product purified in step 6 (PCR-1 Product) was diluted 50-fold with water.

8.

The PCR reaction mixture was prepared as follows:

9.

water

3.5 µL10x buffer

1.0 µL

2 mM dNTPs

1.0 µL

25 mM MgSO4

0.6 µL2 µM FOR Primer (T7PRO-SD)

1.5 µL2 µM REV Primer (DHFR-R3)

1.5 µL

DMSO

0.2 µL

Enzyme (KOD-Plus- Neo)

0.2 µL50-fold diluted PCR-1 product

0.5 µL

Total

10.0 µL

PCR was performed under the same condition used in step 4.

10. The PCR reaction mixture (1 µL) was analyzed with 1% agarose gel electrophoresis to confirm thepresence of the amplification products.
11. The PCR products were purified with a PCR product purification kit (NucleoSpin® Gel and PCR Cleanup (Macherey-Nagel)). The bound DNA was eluted from the column with 20 µL of water.
12. Absorbance at 260 nm was measured and the concentration of the purified PCR product was calculated.
13. The resulting DNA solution was adjusted to a concentration of 20 ng/µL.

- 19 -

Result of 2-step PCR

Figure 6. Preparation of the template DNA (DHFR DNA) by 2-step PCR

Synthesis with PUREfrex ® and detection of synthesized DHFR
1.

Solution I was warmed at room temperature for about 1 minute to thaw completely.

2.

Solutions II and III were thawed on ice.

3.

The thawed Solutions I, II, and III were vortexed to achieve uniform and then centrifuged to collect thecontents at the bottom of the tubes.

4.

The reaction mixture was prepared as follows:(+) DNA(-) DNA

Nuclease-free water

3.0 µL

3.5 µL

Solution I

5.0 µL5.0 µL

Solution II

0.5 µL0.5 µL

Solution III

1.0 µL1.0 µLDHFR DNA (20 ng/µL)

0.5 µL

0 µL

Total

10.0 µL

10.0 µL- 20 -

5.

The protein was synthesized by incubating the tubes for 2 hours on a 37ºC heat block.

6.

After incubation, 10 µL of water and 10 µL of 3x Sample Buffer were added to the reaction mixture.

7.

The samples were heated for 5 minutes at 95ºC.

8.

Three µL (equivalent to 1 µL of the reaction mixture) of samples were applied to 10-20% gradient gel,and electrophoresis was performed.

9.

After electrophoresis, the gel was stained with CBB to detect the synthesized product.

Result of synthesis of DHFR

Figure 7. Synthesis of DHFR with PUREfrex- 21 -

2. Purification of His-tagged protein (DHFR-6xHis)
Since all of proteins involved in protein synthesis in PUREfrex® reaction mixture are free of His-tag, the protein of interest
can be synthesized as His-tagged protein and purified with metal affinity resin. An example of purification of His-tagged
DHFR with a nickel affinity resin is shown below.

What was used
PUREfrex® 2.0 (#PF201-0.25-EX)
Template DNA (containing DHFR-6xHis gene)
Nuclease-free water
Heat block or water bath (37°C and 95°C)
Ni-Sepharose 6 FF (Cytiva)
Binding Buffer: 50 mM Tris-HCl pH 8, 500 mM NaCl, 20 mM imidazole, 20 mM Mg(OAc) 2
Wash Buffer: 50 mM Tris-HCl pH 8, 500 mM NaCl, 20 mM imidazole
Elution Buffer: 50 mM Tris-HCl pH 8, 500 mM NaCl, 100 mM imidazole
High-speed refrigerated microcentrifuge
3x Sample Buffer for SDS-PAGE (see Appendix for composition on page 30)
SDS-PAGE gel (10-20%)
Molecular weight marker
CBB stain

Synthesis with PUREfrex ® and purification of synthesized DHFR -6xHis
1.

The reaction mixture was prepared as in “1. Synthesis of DHFR” (page 20) (volume: 25 µL).

2.

The reaction mixture was incubated for 4 hours at 37ºC.

3.

Five µL was withdrawn from the reaction mixture. (→ “Syn” in Figure 8)

4.

The remaining reaction mixture (20 µL) was diluted with 80 µL of Binding Buffer.
■ Diluting with buffer without magnesium ion may destabilize ribosomes, introducing ribosomal proteins into
the eluted fraction. When purifying the synthesis product, dilute the reaction mixture with buffer containing
magnesium ion.

5.

Ten µL of Ni-Sepharose 6 FF equilibrated with Binding Buffer was added to the diluted reaction mixture,and the resulting mixture was incubated for 1 hour at 4ºC.
■ His-tagged proteins synthesized with PUREfrex® tend to bind poorly to nickel affinity resin. Be sure to use
more resin than recommended.

- 22 -

6.

The suspension was centrifuged briefly (about 5 seconds) to pellet the resin. The supernatant was isolated.
(→ FT in Figure 8)

7.

The resin was washed twice with 50 µL of Binding Buffer. (→ W1, W2 in Figure 8)

8.

The resin was washed 3 times with 50 µL of Wash Buffer. (→ W3, W4, W5 in Figure 8)

9.

Fifty µL of Elution Buffer was added to the resin to elute bound DHFR-6xHis. (→ E1, E2, E3 in Figure 8)

10. SDS-PAGE was performed with 10-20% gradient gel.
11. After electrophoresis, the gel was stained with CBB to detect the synthesized product.

Result of purification of DHFR -6xHis

Figure 8. Simple purification of DHFR-6xHis synthesized with PUREfrex®
An equivalent of 1 µL of the reaction mixture (Syn/FT/W1/W2) or 4 µL of the reaction mixture (W35/E1-3) was applied to SDS-PAGE.

- 23 -

3. Detection of the synthesized product with FluoroTect™ Green Ly s (DHFR)
FluoroTect™ GreenLys (Promega) is a reagent that introduce fluorescently (BODIPY®) labeled lysine at lysine residues
in the protein of interest. After SDS-PAGE, the synthesis of the target protein can be easily confirmed by detecting thefluorescently labeled band using a fluorescent imager.

What was used
DHFR DNA (positive control included in the kit)
PUREfrex® 2.0 (#PF201-0.25-EX)
Nuclease-free water
37ºC heat block or water bath
FluoroTect™ GreenLys in vitro Translation Labeling System (Promega)
1 mg/mL RNase A
SDS-PAGE gel (10-20%)
3x Sample Buffer for SDS-PAGE (see Appendix for composition on page 30)
Molecular weight marker
Fluorescent gel imager (LAS)
Oriole™ stain (Bio-Rad)Protein Synthesis in the presence of FluoroTect™ Green L ys
1.

The reaction mixture was prepared as in “1. Synthesis of DHFR” (page 20). At the end, 0.5 µL of
FluoroTect™ GreenLys was added per 10 µL of the reaction mixture.

2.

The reaction mixture was incubated for 1 hour at 37ºC.

3.

One µL of 1 mg/mL RNase A was added to the reaction mixture. The reaction mixture was additionally
incubated for 15 minutes at 37ºC to digest unreacted FluoroTect™ GreenLys.

4.

Equal volume of water and 3x Sample Buffer was added to the reaction mixture, which was heated for 5
minutes at 95ºC.

5.

Three µL (equivalent to 1 µL of the reaction mixture) of each sample was applied to SDS-PAGE gel, andelectrophoresis was performed.

6.

After electrophoresis, the band of the fluorescently labeled DHFR protein was detected by fluorescent gel
imager.

7.

The gel was stained with Oriole™ to detect all proteins in the reaction mixture.

- 24 -

Result of synthesis in the presence of FluoroTect

Figure 9. Synthesis of DHFR in the presence of FluoroTect™ GreenLys
A. Detection of the fluorescence of FluoroTect™ GreenLys (derived from BODIPY®)
B. Staining of all proteins in the reaction mixture with Oriole™

- 25 -

4. Synthesis in the presence of molecular chaperone s (luciferase)
Many proteins require molecular chaperones to achieve their proper conformation after synthesis. Since PUREfrex®
contains no molecular chaperones, add molecular chaperones to the reaction mixtures if the synthesized protein aggregate.
We provide DnaK Mix (#PF003-0.5-EX) and GroE Mix (#PF004-0.5-EX) as supplement for PUREfrex®. DnaK Mix is a
mixture containing E. coli Hsp70 DnaK, and DnaJ and GrpE which help the function of DnaK. GroE Mix is a mixture
containing E. coli Hsp60 GroEL and the helper protein GroES. Other molecular chaperones can be also used. Select theappropriate molecular chaperones because they depends on the synthesized protein.
Example of protein synthesis in the presence of DnaK Mix is shown below. Luciferase is an aggregate-prone protein andis tend to lose activity at 37ºC. When luciferase was synthesized in the presence of DnaK Mix, the activity was higher,
indicating that addition of DnaK Mix had an effect on the activity of luciferase synthesized with PUREfrex®.

What was usedTemplate DNA (Luciferase DNA)
PUREfrex® 2.0 (#PF201-0.25-EX)
DnaK Mix (#PF003-0.5-EX)
Nuclease-free water
30ºC heat block or water bath
Luciferase Assay System (Promega)
Luminometer

Synthesis of luciferase in the presence of DnaK Mix and evaluati on of its activity
1.

The reaction mixture was prepared as below.
(-) DnaK Mix(+) DnaK Mix

Nuclease-free water

3.0 µL

2.5 µL

Solution I

5.0 µL5.0 µL

Solution II

0.5 µL0.5 µL

Solution III

1.0 µL1.0 µLDnaK Mix0 µL

0.5 µL

Luciferase DNA (40 ng/µL)

0.5 µL0.5 µL

Total

10.0 µL

10.0 µL2.

The reaction mixture was incubated for 4 hours on 30ºC heat block.

3.

One µL of the reaction mixture was diluted with 29 µL of water.

- 26 -

4.

One µL of the diluted reaction mixture was added to 20 µL of Lumino-assay solution and mix by vortex.

5.

Luminescence was measured by luminometer.

Result of synthesis in the presence of DnaK Mix

Figure 10. Effect of DnaK Mix on the activity of the synthesized luciferase- 27 -

Troubleshooting
The positive control (DHFR) is not synthesized .
• Incubate the PUREfrex® reaction mixture by directly heating the reaction tube with heat block or water bath. Gas-phasechamber (e.g., incubator) takes time to warm the reaction mixture and the yield is reduced.
• 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 whenever possible.

The protein of interest is not synthesized .

The template DNA does not contain the minimum required sequences.
• Template DNA for PUREfrex® must contain T7 promoter, ribosome binding site (SD sequence), start codon, andstop codon.

Too much or little DNA is 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.
• 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 isapproximately 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.

The method for preparing the template DNA is inappropriate.
• See the tips described in “1. Preparation of the template DNA ” (page 5).

The template DNA contains a difficult -to-translate sequence.
• Confirm that the template DNA does not contain the sequences described in “Tips about the template DNA
sequence ” (page 9).

- 28 -

The synthesized protein is insoluble.

Add molecular chaperones when synthesizing the protein.
• PUREfrex® contains no molecular chaperones. When the protein of interest is synthesized in the presence of molecularchaperones, it may become soluble. Additionally, better results may be obtained with PUREfrex® 1.0, in which theyield of the product is lower than PUREfrex® 2.0. See “Related Products” (page 31) about molecular chaperones
and PUREfrex® 1.0.

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 37ºC to 30ºC or 25ºC 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.

The synthesized protein has no activity.
• The synthesized protein may be insoluble. Check its solubility.
• 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 related to transcription and translation. Add the factors required for activity.
• 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 theefficiency of disulfide formation is affected by reducing agent.

The reaction mixture becomes turbid when sample buffer for SDS-PAGE is added.
• 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).

- 29 -

Appendix
DHFR DNA sequence information
The sequence is available in text format here:
https://purefrex.genefrontier.com/resources/tech_notes/sequence-of-dhfr-dna.html

Compositions of SDS-PAGE sample buffer
3x Sample buffer (for 10 mL)
1 M Tris-HCl pH6.8

1.5 mL

Glycerol

3 mL

SDS

0.6 g

2-Mercaptoethanol

0.6 mL

Bromophenol BlueAs needed(Bring to a volume of 10 mL with H2O)

Publications
Publications on PUREfrex® and PURE system are available here:
https://purefrex.genefrontier.com/references.html- 30 -

Related Products
Kit for protein synthesis

Product no.

Product name

PF001-0. 25-EX
PF001-2ML-EX

VolumeFor 250 µL reactions

PUREfrex® 1.0

For 2 mL reactions

PF001-10ML-EX

For 5x 2 mL reactions

PF201-0.25-EX

For 250 µL reactions

PF201-2ML-EX

PUREfrex® 2.0

For 2 mL reactions

PF201-10ML-EX

For 5x 2 mL reactions

PF213-0.25-EX

For 250 µL reactions

PF213-2ML-EX

PUREfrex® 2.1

PF213-10ML-EX

For 2 mL reactions
For 5x 2 mL reactions

Supplement for protein synthesis

Product no.
PF003-0.5-EX
PF003-10ML-EX
PF004-0.5-EX
PF004-10ML-EX
PF005-0.5-EX
PF005-10ML-EX
PF006-0.5-EX
PF006-10ML-EX
PFS052-0.5-EX
PFS052-10ML-EX

Product nameDnaK Mix

VolumeFor 500 µL reactions
For 5x 2 mL reactions
For 500 µL reactions

GroE Mix

For 5x 2 mL reactions
For 500 µL reactions

DsbC Set

For 5x 2 mL reactions
For 500 µL reactions

PDI SetFor 5x 2 mL reactions
For 500 µL reactions

EF-P

For 5x 2 mL reactions- 31 -

Order support
International sales of GeneFrontier's products are carried out by Cosmo Bio USA.
Please contact here for requesting a quote and ordering information.
Cosmo Bio USA; https://www.cosmobiousa.com/

Technical support
Please contact us for a technical information about PUREfrex®.
https://purefrex.genefrontier.com/contact/

SHARP Kashiwa Building, 4F 273-1 Kashiwa, Kashiwa-shi, Chiba 277-0005, Japan
Phone: 04-7137-6301, fax: 04-7132-7530
URL: www.genefrontier.com- 32 -