FAQs for Next Generation Cell Free Protein Expression Kit (Wheat Germ) - CFPS700

Easily scalable protein production using Next Generation Cell Free Protein Expression Kit (Wheat Germ).

1) Post-translational modification or glycosilation modification

In this kit, Glycosilation modification or plant glycosilation is not generated In other word, the uniform protein can be expected. Phosphorylation modification may be generated.

2) Folding and S-S Bond

Synthesized proteins including membrane proteins have activity and proper folding.

Intramolecular SS binding can be induced by using this cell-free protein synthesis kit

It is reported that if the refolding reagent BMC (trans-1,2-bis (2-mercaptoacetamido) cyclohexane) is given to the amino acid solution at 0.1 mg / mL, SS binding is well induced. It can be used as an additive reagent when it is not expressed or when the activity is low, etc. Also, if you can use BMC with dialysis method with amino acid solution it is expected to increase the yield of active protein further.

BMC Cas-No: 257641-01-3

3) Which Tag?

We suggest FLAG tag, given that it confers very high specificity to purify the expressed target protein in the wheat germ extract. His-tag is not recommended, as it cross-reacts with few of proteins in the wheat germ extract.

4) Translation by dialysis method

  • The yield tends to increase about 1.5 times by the simple batch method with a scale larger than one reaction (110 µL) in our standard protocol (5 reactions for 550 µL). If the protein yield is low with 110 µL of sample, please consider first trying with sample size about 550 µL. Wheat Germ Extract, Amino Acid Mix will be “n” times as much as preparation of translation reagent for n times sample.
  • A general dialysis method can be used, in which a translation solution is placed inside the dialysis cup (sample solution) and an amino acid mix is used as a dialysis external solution. Ordinary dialysis external fluid is generally 20 times to 100 times more than internal solution.
  • An example of composition of translation reaction solution by dialysis method for one sample is shown.

Sample solution (translation solution) composition

Reagents
Wheat Germ Extract 10 μL
Amino Acid Mix 20 μL
mRNA 80 μL
Total 110 μL

Dialysis cup used in the following example: Slide-A-Lyzer ™ Mini dialysis tool (sample 0.1 mL, molecular weight cutoff 3.5 K) manufactured by Thermo Fisher Scientific Inc.

  1. In the above composition, first prepare a mixture excluding the mRNA solution, in the room temperature, and then add it to the mRNA solution and gently pump to avoid foam formation.
  2. Place 2 mL of Amino Acid Mix into 1 well of a 24-well titer plate, diluted 4 times with Milli-Q® into, and cover the plate.
    * Since 2 mL Eppendorf tube matches the dialysis cup, the dialysis method can be done with 2 mL Eppendorf tube
  3. Take the total volume of 110 μL of the prepared translation reaction solution and transfer to dialysis cup gently so bubbles do not enter on to the dialysis cup, two-step pushing of the pipette can causes foaming. Then close the lid of the dialysis cup. At this time, please set the level of the sample solution (reaction layer) and the level of the external dialysate (Amino Acid Mix) are at the same level or the level of the sample solution is slightly lower.
  4. Incubate at 16 °C and let it react overnight (over 10 hours)
  5. Sample is mixed by pipet gently, collected in an Eppendorf tube, centrifuged at 15,000 rpm for 10 minutes at 4 ° C, and the supernatant is stored at -80 ° C.

5) Tips for Transcription

2nd PCR product is not necessary to be purified for transcription

Please do not mix 2nd PCR product and Transcription buffer directory

Using 4M ammonium acetate is essential for purifying mRNA.

6) Silent mutation, Codon Transformation

Basically no codon transformation is necessary. But if ORF is optimized for E-coli, codon transformation may be suggested

7) Scale-up

In this protocol, 1 translation reaction is 110 µL, but scale-up is possible. In that case, please increase the ratio of translation reaction system as it is.

Example: When translation reaction 110 µL is scaled up to 220 µL, the composition is
Wheat germ extract 10µL >> 20µL
Amino acid mix 20µL >> 40µL
mRNA 80µL >> 160µL

Once scaled up, the yield of the target protein may be improved. Example: In the case one translation reaction of 110 µL have a yield 10 µg, one translation reaction of 550 µL improves the yield per reaction solution with the target yield of about 70 µg.

Therefore, if it is desired to further improve the protein yield after adjusting the amount of mRNA of one reaction in the standard protocol, you can try the following:

  • Increase the translation reaction time from 10 hours to 20 hours
  • Scale-up
  • Dialysis method

8) Yield

In the standard protocol, from 110 μL of the translation solution in the simple batch method, the average yeild is about 10 μg. It is expected to be several times more by dialysis method.

The yield tends to increase about 1.5 times by the simple batch method with a scale 55 µL, larger than one reaction (110 µL) If the protein yield is low with 110 µL of sample, please consider first trying with sample size about 550 µL.

9) Preparation of primer

Primers must be prepared beforehand in the preparation of templates.

Multiple primers are required to perform two steps of PCR. The primer list provided in the protocol shows five different tag sequences and shows the forward primer and the reverse primer when each tag is attached to the N-terminus or the C-terminus, respectively. For the primer, the primer described as 1st_gene-specific-XX is an ORF-specific primer, and the one described as 2nd-XXX is a universal primer. The universal primer is designed so that it can be used with other ORFs, which makes it possible to efficiently create transcription templates containing multiple tag sequences.

Please refer to the primer list for specific primer to be prepared.

10) Required reagents

In addition to our Next Generation Cell Free Protein Expression Kit (Wheat Germ) (CFPS700), necessary reagents are shown for each steps, template preparation, transcription and translation. M shows a must , and B shows better to have.

Reagent Name Spec Template preparation Transcription Translation
Ultra pure water   M B M
RNAse free water   B M B
DNA Template 1 μg/μL M    
PCR Enzyme   M    
PCR Primer   M    
TAE Agarose gel   M M  
Ammonium Acetate 4 M Stock   M  
Ethanol 99.5%   M  

11) Required Equipments

The equipment necessary for each step of template preparation, mRNA synthesis (transcription), protein synthesis (translation) is shown below. Protein synthesis is possible with equipment in common bio laboratories.

Equipment Spec Template preparation transcription translation Always required
RCR device   M      
high speed cooling centrifuge 15000rpm/4 ° C M M    
Tabeletop Centrifuge   M M M  
DNA/RNA Electrophoresis 120V M M    
Transilluminator   M M    
Nucleic acid densiometer 1μL If there is M    
Incubator / waterbath 37° C   M    
Vortex mixer     M    
Incubator 16-25° C     M  
Ultrapure water system 18.3MΩ       M
Clean bench         M
Freezer -20 ° C       M
Deep freezer -80 ° C       M
Consumables         M

Materials