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Technology - CellFree Sciences Co., Ltd.|The natural power of wheat driving science -wheat germ cell-free protein expression system-

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Technology

What is a cell-free expression system ?

Various protein expression systems

 

Living Cells

Cell-free

prokaryotes

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e.g. E.coli
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E.coli cell-free

eukaryotes

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e.g. 
Yeast 
Insect cell
Mammalian cell
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e.g.
Wheat germ cell-free

Differences between living cells and cell-free methods

Protein expression using living cells

This method incorporates the genes for the target protein into a living cell to produce the protein inside the living cell.

 

宿主細胞を用いたタンパク質発現

Protein expression using a cell-free

This method produces proteins inside a test tube by using the genes for the target protein and a cell extract or the various factors included in cell extracts that are necessary to synthesize protein.

 

無細胞系を用いたタンパク質発現

What is the Wheat germ cell-free protein expression system(ENDEXT Technology)

This technology synthesizes proteins from genes inside a test tube 

using an extract from wheat germ.

 

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A wheat kernel is comprised of the bran, endosperm, and germ, and the germ contains an abundance of the translation factors that are required for protein synthesis.

 

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The effectiveness of a wheat germ cell-free protein synthesis system was first reported in the world by Marcus et al in Russia in 1964.

However, its translation activity was extremely low, and at the time, the technology was far from being used to produce proteins.

 

Years later, Yaeta Endo (Professor Emeritus at Ehime University) discovered that the reason for the extremely low translation activity to date was due to the translation inhibitors present in the reaction solution, and in 2000, the wheat germ cell-free protein expression system (ENDEXT Technology), which improved synthesized volumes dramatically by removing the translation inhibitors originating in the germ, was born.

This made the production of proteins using a cell-free protein synthesis system a reality.

Now, our company has an even more improved wheat extract with high synthetic activity: the “WEPRO7240 Series”.

 

 

Various methods of synthesis

The method of synthesis is also important in order to maximize the benefits of the high synthetic activity of wheat germ extracts.

We have developed proprietary technology that solves the problems of conventional methods such as the batch method and dialysis method.

 

Batch

method

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Method of reaction where the reagents are all mixed together in the reaction solution

Operability:Easy and perfect for synthesizing multiple samples

Yield:Since the substrates are completely consumed within a few hours, synthetic efficiency is low, as are yields

Dialysis 

method

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Method of reaction using a dialysis membrane

Operability:Not suited to synthesizing multiple samples

Yield:Since the dialysis membrane can remove by-products while supplying substrates, synthetic efficiency and yields are high

Our Technology

Bilayer

method

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Method of reaction where two layers are formed within the reaction solution: the extract and the substrate solution

Operability:Easy and also perfect for synthesizing multiple samples

Yield:Since the substrates are supplied slowly from the two contact layers, synthetic efficiency and yields are high

About the Bilayer method

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By forming two layers in the form of the wheat germ extract and the substrate solution, the substrates are supplied slowly and dispersed through the contact surface, providing a high yield of proteins. 

This method has a higher synthetic efficiency than the batch method, where the substrates are completely consumed within a few hours and the reactions stop.

 

*Our products can also be used to synthesize target proteins using the dialysis method.

Flow of operations

1 Template DNA construction

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Work time Vector:1 week  /  PCR products: 1 day

2 Transcription reaction

Reaction Temperature:37 ℃

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Work time  1-6 hour

3 Translation reaction

Reaction Temperature:15 ℃

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Work time  20 hour

Performs purification and determines expression, solubility, activity, etc. of the target protein according to the customer’s needs.

 

The target protein can be confirmed in a minimum of four days when using a PCR product as the Template DNA, and a minimum of 10 days when using a vector.

Comparison of work involved against other representative protein expression systems

Wheat germ
cell-free

Work time

4-10 days*2

E.coli

Work time

2+ weeks

Insect cells

Work time  

5+ weeks

CHO cells*1

Work time  

2+ weeks

コムギ胚芽無細胞 大腸菌 昆虫細胞 CHO細胞

※※Smartphone users can view the chart in horizontal scrolling mode.

*1:CEO stands for Chinese Hamster Ovary. 

*2:The target protein can be confirmed in four days when using a PCR product as the Template DNA, and a 10 days when using a vector.

Features of the wheat germ cell-free expression system

  • Can manufacture proteins derived from a variety of species.
  • Easy to obtain the proper folding.
  • Easy to obtain as soluble proteins.
  • Limited degradation of the synthesized proteins.
  • Unlike other expression systems, there is no glycosylation, so there is no attachment of foreign sugar moieties.

Comparison against other representative protein expression systems

 

Wheat germ

cell-free

E.coli

cell-free

E.coli

Insect cell

Yeast

CHO cells

Scope of species that can be created

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Folding

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Solubility

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Reproducibility

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Ease of operation

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Simultaneous throughput

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Total work time

3~10 days

9 days

2 week~

5 week~

3 week~

2 week~

※Smartphone users can view the chart in horizontal scrolling mode.

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