WO2006051908A1

WO2006051908A1 - Cell-free protein synthesizing process

Info

Publication number: WO2006051908A1
Application number: PCT/JP2005/020727
Authority: WO
Inventors: Yaeta Endo; Tatsuya Sawasaki; Tomio Ogasawara
Original assignee: Cellfree Sciences Co., Ltd.
Priority date: 2004-11-12
Filing date: 2005-11-11
Publication date: 2006-05-18
Also published as: JP2008029204A

Abstract

A protein synthesizing process in which simple and easy mass synthesis of proteins can be carried out by cell-free protein synthesizing means; and a cell-free protein synthesizing apparatus utilizing the process. To realize these, study has been made on means for feeding a supply solution and on a protein synthesizing process in which a synthetic reaction utilizing an overlay technique is repeated. As a result, a cell-free protein synthesizing process in which an overlay technique and a supply batch technique are repeated has been found, leading to completion of this invention.

Description

Specification

Cell-free protein synthesis method

Technical field

[0001] This application, which is incorporated herein by reference, claims priority from Japanese Patent Application No. 2004-329798.

The present invention relates to a method for repeated protein synthesis using a cell-free protein synthesis system.

Background art

[0002] With the completion of the genome project coming soon, the focus of research is rapidly expanding from gene structure analysis to gene function analysis. Proteins in cells are not functioning alone. It functions by interacting with a wide variety of protein factors, nucleic acids, low molecular weight biologically active substances, cell membrane components, etc., and the biological function is performed as the sum of these interactions. It is thought that One of the main tasks of the post-genome project is to analyze the relationship between the structure and function of various protein factor complexes. The results obtained here provide extremely important knowledge in a wide range of research fields such as structural biology and basic biology including biochemistry, elucidation of the relationship between gene translation products and pathogenesis in the medical field, and drug development. It is expected to be.

As a method for conducting protein synthesis reactions in vitro, protein is synthesized in vitro by adding translational trapezoids, amino acids serving as substrates, energy sources, various ions, buffers, and other effective factors to cell extracts. Researches such as so-called cell-free protein synthesis methods have been actively conducted (Patent Documents:! To 5).

The cell extract or biological tissue extract for protein synthesis used in this cell-free protein synthesis system is prepared using Escherichia coli, wheat germ, rabbit reticulocyte or the like as a raw material. The cell-free protein synthesis system maintains performance comparable to that of living cells in two respects: the peptide synthesis rate and the accuracy of the translation reaction. This system does not require complicated chemical reaction steps or complicated cell culture steps. Because of this advantage, this system The practical system has been developed. However, in general, cell extracts extracted from biological cells have low protein synthesis efficiency due to their extremely unstable protein synthesis ability. In addition, the quality of the cell extract during storage was significantly reduced. For this reason, the amount of the compound obtained by the cell-free protein synthesis system was small enough to be detectable by radioisotope labeling or the like. As a result, this system could not be used as a practical protein production tool.

[0003] The present inventors previously provided the following as a method for solving the drawbacks of the conventional cell-free protein synthesis system.

(1) Cell extract preparation for cell-free protein synthesis and cell-free protein synthesis method (Patent Document 6), (2) Spider-type molecule with versatility and high-efficiency function, and cell-free protein synthesis method using the same (Patent Document 7), (3) Diffusion continuous batch method (sometimes called multi-layer method) (Patent Document 8).

In addition, a device that continuously performs cell-free protein synthesis to increase the efficiency of protein synthesis has been reported. Conventional continuous cell-free protein synthesizers include those using an ultrafiltration membrane method, a dialysis membrane method, and a column chromatography method in which a translation cage is immobilized on a resin (Non-patent Document 1). . In particular, the ultrafiltration membrane method and the dialysis membrane method are widely used because they are easy to handle. The continuous process using these membranes still has the following problems to be solved: (1) The material strength of the membrane used is low; (2) Deterioration of membrane function due to clogging (3) The skill is required because the operation is complicated.

[0004] The inventors have previously invented a diffusion continuous batch cell-free protein synthesis method (multilayer method) and a repetitive batch method as novel cell-free protein synthesis reaction methods. Even using these methods that surpassed conventional synthesis efficiency, there was a limit to the amount of protein synthesis. Therefore, there has been a need to develop elemental technologies for establishing a high-throughput protein synthesis method that enables highly efficient protein production from a large number of genes with simpler operations.

Non-Patent Document 1: Spirin, A., et al., (1993) Methods in Enzymology, 217, 123-142

Patent Document 1: JP-A-6-98790 Patent Document 2: JP-A-6-225783

Patent Document 3: JP-A-7-194

Patent Document 4: JP-A-9 291

Patent Document 5: JP-A-7_147992

Patent Document 6: WO00 / 68412

Patent Document 7: WOOlZ27260

Patent Document 8: WO02Z24939

Disclosure of the invention

Problems to be solved by the invention

[0005] An object of the present invention is to provide a protein synthesis method that enables simple and large-scale synthesis in a cell-free protein synthesis means and a cell-free protein synthesis apparatus using the method.

Means for solving the problem

[0006] As an important factor for automation, the present inventor has studied a means for supplying a supply liquid and a method for repeatedly synthesizing a protein using a multilayer method. As a result, the inventors have found a cell-free protein synthesis method in which the layering method and the supply batch method are repeated, thereby completing the present invention.

[0007] That is, the present invention comprises:

1. a cell-free protein synthesis method by an iterative method including at least the following steps;

1) The process of supplying the supply liquid from the supply phase to the reaction phase, which is the reaction liquid, leading to the synthesis reaction

2) A process of mixing the supply phase and the reaction phase, or stopping the supply of the supply liquid, before or after substantially decreasing the synthesis rate, before or after substantially stopping the synthesis reaction

3) Concentration process

4) The process of restarting the synthesis reaction by supplying the supply liquid from the supply phase to the reaction phase

2. A synthesis method consisting of upper and lower two layers via an interface, with the upper layer as the supply phase and the lower layer as the reaction phase, comprising the following steps and a cell-free protein synthesis method by the repeated layer method;

1) The process of superposing the supply liquid that is the supply phase on the reaction phase that is the reaction liquid to lead to the synthesis reaction

2) Supply before or after substantially lowering the synthesis rate, before or after the stop of the synthesis reaction, or on the way Process of mixing the reaction phase and the reaction phase

3) Concentration process

4) The process of resuming the synthesis reaction by overlaying the supply liquid as the supply phase on the reaction phase

3. Cell-free protein synthesis method by repeated feeding batch method including the following steps;

1) Process of supplying the supply liquid continuously or discontinuously to the reaction phase, which is the reaction liquid, leading to the synthesis reaction

2) A process of stopping the supply of the feed liquid before or after substantially lowering the synthesis rate, before or after substantially stopping the synthesis reaction

3) Concentration process

4) Step of restarting the synthesis reaction by supplying the reaction solution to the reaction phase continuously or discontinuously

4. The synthesis method according to item 3 above, wherein the synthesis reaction is controlled at the feed addition rate (feed solution amount Z seconds).

5. The method according to item 4 above, wherein the same amount of the supply liquid as the reaction phase at the start of the reaction is continuously or discontinuously supplied to the reaction phase in 10 minutes to 10 hours.

6. The synthesis method according to any one of 1 to 15 above, wherein the step 2) —4) is repeated a plurality of times.

7. The synthesis method according to any one of 1 to 16, wherein the reaction phase reaction solution comprises a transcription solution containing unpurified mRNA after the transcription reaction and a wheat seed embryo-derived extract.

8. The synthesis method according to 1 or 7 of item 17 above, wherein a transcription solution containing purified mRNA after the transcription reaction or purified mRNA is supplemented and added to the supply phase at the time of use.

9. The synthesis method according to any one of 1 to 8, wherein the by-product of the reaction phase is removed by concentration treatment.

10. The synthesis method according to any one of 4 above, wherein magnesium ions and Z or nucleotides derived from the transcription solution are removed by concentration treatment.

11. The wheat seed germ extract is a wheat seed germ extract from which the wheat endosperm component and the low-molecular protein synthesis inhibitor are substantially removed. Synthesis method.

12. From an extract derived from wheat seed germ, via one of the following ATP The synthesis method according to item 11 above, wherein the sugar phosphorylation system is controlled;

1) removal of monosaccharides,

2) removal of phosphorylated sugar,

3) control of the production of monosaccharides from polysaccharides,

4) Control of production of phosphorylated saccharide from monosaccharides.

13. A synthesis kit comprising at least 1 of the reagents used in the synthesis method according to any one of 4 to 12 in the preceding paragraph.

14. An apparatus for carrying out the cell-free protein synthesis method according to the repeated layering method according to any one of the preceding paragraphs 2, 6-12, and a cell-free protein synthesis apparatus comprising at least the following control means;

(1) Means to overlay the supply phase on the reaction phase

(2) Means for mixing

(3) Means for concentration

15. A device for cell-free protein synthesis by the repetitive supply batch method according to any one of the preceding paragraphs 3-12, wherein the cell-free protein synthesis device comprises at least the following control means;

(1) Means for continuously or discontinuously supplying the supply liquid to the reaction phase

(2) Means for concentration

The invention's effect

[0008] The cell-free protein synthesis means of the present invention has achieved an unprecedented simplicity and mass synthesis of cell-free protein synthesis means, and has cleared the most important issue in the automated protein synthesis method.

BEST MODE FOR CARRYING OUT THE INVENTION

[0009] The present invention is a cell-free protein synthesis method by an iterative method including at least the following steps in a cell-free protein synthesis system using mRNA as a raw material.

2) A process of mixing the supply phase and the reaction phase, or stopping the supply of the supply liquid, before or after substantially decreasing the synthesis rate, before or after substantially stopping the synthesis reaction 3) Concentration process

4) Step of restarting the synthesis reaction by supplying the supply liquid from the supply phase to the reaction phase In other words, the present invention provides characteristics of the initial phase of the synthesis reaction having a high reaction rate by supplying the supply liquid continuously or discontinuously. It is a synthesis method characterized by maximum utilization. The means is to repeatedly supply the supply liquid before or after substantially decreasing the synthesis rate, before or after substantially stopping the synthesis reaction, or in the middle thereof. This method can be applied by either a batch method or a multilayer method.

[0010] In the repetitive supply batch method, a supply solution is supplied continuously or discontinuously to a reaction phase that is a reaction solution, leading to a synthesis reaction. On the way, the supply of supply liquid is stopped. Subsequently, the reaction solution (reaction phase) is concentrated. The concentrated reaction solution (reaction phase) is diluted by continuous or discontinuous supply of the supply solution. This dilution process replenishes components such as substrates, energy sources, ions, buffers, and saddle-type substances required for synthesis using mRNA as a raw material. By this treatment, the reaction solution (reaction phase) is returned to the original optimal concentration, and protein synthesis is reactivated.

This dilution and concentration treatment can be repeated discontinuously to synthesize a large amount of protein.

[0011] The present invention also relates to a synthesis method characterized by making the best use of the characteristics of the initial phase of the synthesis reaction having a high reaction rate of the multilayer method in a cell-free protein synthesis system using mRNA as a raw material (hereinafter, repeated Sometimes called the multi-layer method).

That means is that the supply phase and the reaction phase are mixed before and after the synthesis rate is substantially reduced or before or after the synthesis reaction is substantially stopped. Subsequently, a concentration process is performed. After concentration, the feed phase is subsequently layered.

This multi-layer treatment replenishes protein synthesis by replenishing components such as substrates, energy sources, ions, and buffers required for synthesis using mRNA as a raw material.

This mixing, concentration, and multi-layer treatment is repeated discontinuously, so that a large amount of protein can be synthesized.

[0012] In order to increase the efficiency of the synthesis reaction, the reaction solution or the supply phase and the reaction phase A dilution operation can be added to the mixed solution as necessary. The dilution operation can be performed before or after the concentration treatment.

[0013] Further, the present invention is a cell-free protein synthesizer characterized in that a synthesis method that makes the best use of the characteristics of the above two synthesis methods can be automatically made.

[0014] (1) Production process of transfer mold

In the present specification, the “transcription type” refers to a DNA that can be used as a type molecule in an in vitro transcription reaction, and has at least a base sequence encoding a target protein downstream of an appropriate promoter sequence. Suitable promoter sequences include promoter sequences that can be recognized by RNA polymerase used in the transcription reaction, and examples thereof include SP6 promoter and T7 promoter. Any DNA encoding the target protein may be used.

It is preferable that the transcription type has a base sequence having an activity to control translation efficiency between the promoter sequence and the base sequence encoding the target protein. For example, derived from RNA virus such as Ω sequence derived from tobacco mosaic virus 5 'untranslated region and / or Kozak sequence can be used. Furthermore, it is preferable that the transcription type includes a 3 ′ untranslated region including a transcription termination region and the like downstream of the base sequence encoding the target protein. As the 3 ′ untranslated region, about 1.0 to about 3.0 kilobases downstream from the stop codon is preferably used. These 3 'untranslated regions are not necessarily those of the gene encoding the target protein.

One of the preferred embodiments of the method of the present invention is to amplify and synthesize a transcription cage that encodes the target protein by a PCR method, and directly convert it into a transcription cage without purification.

In order to prevent the production of short-chain DNA (resulting in a decrease in yield of the target product and resulting in low-molecular translation product noise) generated by non-specific amplification, a promoter-splitting primer described in International Publication No. WO02Z18586 may be used. it can.

The transcription type DNA obtained as described above may be subjected to transcription reaction after purification by chloroform extraction or alcohol precipitation, but in the protein synthesis method of the present invention, the reaction solution after the PCR reaction is used as it is. It can be used as a transfer mold solution. Transcription In the production of a saddle type, since the type DNA is not introduced into the plasmid, the steps can be greatly omitted compared to a method in which a large amount of plasmid is prepared and treated with a restriction enzyme to obtain a transcription type. This makes it possible to synthesize a large number of transfer molds in a short time with a small number of steps. That is, since a step of preparing a plasmid incorporating a DNA encoding the target protein is not required, the time required for ultracentrifugation for plasmid purification can be shortened. In addition, restriction enzyme treatment to cut out the transcript from the plasmid, phenol treatment to remove the restriction enzyme, etc., chloroform treatment, alcohol precipitation for purification of the transcript, DNA that is a transcription template The step of dissolving the precipitate can be omitted, so that there is no inhibition of the transcription reaction due to residual phenol / chloroform, and there is no loss of transcription type due to the multi-step purification operation. In addition, since the number of steps required for the reaction can be reduced, there is an additional advantage that the number of chips used can be reduced.

(2) Transcription reaction process

Transcription mold is a solution (also called transcription reaction solution) that contains components necessary for transcription reaction, such as RNA polymerase compatible with the promoter in the transcription mold (4 types of ribonucleoside triphosphates). ) And incubating at about 20 to about 60 ° C., preferably about 30 to about 42 ° C., for about 30 minutes to about 16 hours, more preferably about 2 to 5 hours to initiate the transcription reaction. Nau.

Further, as one of the preferred embodiments, the transcription type DNA encoding the target protein prepared by a method known per se or the DNA type amplified and synthesized by the PCR method described in (1) is purified. Thus, mRNA that is a translation type is generated by in vitro transcription reaction from transcription type DNA encoding the target protein prepared as it is as a transcription type. The transcription reaction is performed using a solution containing a transcription template (transcription solution containing unpurified mRNA) provided in a reaction system (eg, a commercially available container such as a 96-well titer plate), preferably a PCR reaction solution and a transcription template. Solution containing components necessary for transcription reaction such as RNA polymerase (for example, SP6 RNA polymerase) compatible with the promoter in the medium and substrate for RNA synthesis (4 types of ribonucleoside triphosphates) And about 20 ° C to about 60 ° C, preferably about 30 ° C to about 42 ° C for about 30 minutes to about 16 hours, preferably about 2 hours to about 5 hours. This is done by incubating the mixture. In addition, the GFP gene DNA (Chiu, W. Ed. 211; L "et al., Curr. Biol. 6, 325-330 (1996)) is inserted into the mRNA that is the translation type used in the examples of the present invention. PEU-GFP vector (Sawasaki, T. et al., PNAS, 99 (23), 14652-7 (2002)), the Ω sequence portion is represented by SEQ ID NO: 136 described in WOO 3/056009. Transcription was carried out using SP6 RNApolymerase (Promega) with the circular plasmid DNA replaced with the nucleotide sequence of 铸 as a saddle type.

[0016] For use in the following translation reaction step, a transcription solution containing unpurified mRNA or purified mRNA is directly added to the cell extract for protein synthesis. The cell extract for protein synthesis used here may be any one as long as it can produce a protein encoded by the translation form by translating the translation form. Specifically, Cell extracts such as Escherichia coli, plant seed germs, and rabbit reticulocytes are used. These may be commercially available, and are known per se. Specifically, in the case of an E. coli extract, Pratt, JM et al, franscnption and Translation, Hames, 179-209, BD & Higgins, It can also be prepared according to the method described in SJ, edsno, IRL Press, Oxford (1984).

Commercially available cell extracts for protein synthesis include those attached to E.coli S30 extract sy stem (Promega) and RTS 500 Rapid Translation System (Roche) from E. coli. Examples of the origin include those attached to Rabbit Reticulocyte Lysate System (Promega), and those derived from wheat germ include those attached to PROTEIOS ™ (TOYOBO). Of these, the plant seed germ extract is preferably used as the plant seeds such as wheat, barley, rice, corn, etc., and seeds such as spinach are preferred. Those using an embryo extract are preferred. Further preferred is a wheat seed germ extract from which the endosperm components of germ and low molecular weight protein synthesis inhibitors have been substantially removed. This is because the components and substances involved in protein synthesis inhibition in the extract are reduced as compared with the conventional wheat seed germ extract.

[0017] The best cell extract of the present invention is an extract derived from wheat germ, and further metabolites such as gnolecose that causes protein synthesis inhibition in the mixed endosperm components and germ tissue cells have been substantially removed. Since this is an extract, the method for preparing the raw material will be described below using this as an example. [0018] Usually, the portion of the germ is very small, so in order to obtain the germ efficiently, it is preferable to remove the portion other than the germ as much as possible. Usually, a mechanical force is first applied to wheat seeds to obtain a mixture containing germ, endosperm crushed material, and seed coat crushed material, and from this mixture, a crude embryo fraction (embryo is the main component, endosperm crushed material, To obtain a mixture containing crushed seed coat). The force applied to the seeds only needs to be strong enough to separate the germ from the seeds. Specifically, a seed mixture is pulverized using a known pulverizer to obtain a mixture containing germ, endosperm crushed material, and seed coat crushed material.

The seeds can be pulverized using a generally known pulverizer, but it is preferable to use a pulverizer of a type that can apply impact force to the material to be pulverized, such as a pin mill or a hammer mill. . The degree of pulverization may be appropriately selected according to the size of the seed germ to be used. For example, in the case of wheat, it is usually pulverized to a maximum length of 4 mm or less, preferably a maximum length of 2 mm or less. The pulverization is preferably performed by a dry method.

Next, a crude germ fraction is obtained from the obtained seed flour cake using a generally known classifier, for example, a sieve. For example, in the case of wheat, a crude embryo fraction having a mesh size of 0.5 mm to 2. Om m, preferably 0.7 mm to: 1.4 mm is usually obtained. Further, if necessary, seed coat, endosperm, dust, etc. contained in the obtained crude germ fraction may be removed using wind power or electrostatic force.

In addition, a crude embryo fraction can also be obtained by a method utilizing the difference in specific gravity between embryo, seed coat, and endosperm, for example, heavy liquid sorting. In order to obtain a crude germ fraction containing more germs, a plurality of the above methods may be combined. Furthermore, the germ is selected from the obtained crude germ fraction using, for example, visual observation or a color sorter.

[0019] Since the embryo fraction obtained in this manner may have an endosperm component attached thereto, it is usually preferable to further perform a washing treatment for germ purification. In the washing treatment, the embryo fraction is dispersed and suspended in water cooled to 10 ° C or lower, preferably 4 ° C or lower, or an aqueous solution or an aqueous solution containing a surfactant, and washed until the washing solution does not become cloudy. I prefer to do it. In addition, it is more preferable to disperse and suspend the embryo fraction in an aqueous solution containing a surfactant, usually at 10 ° C or lower, preferably at 4 ° C or lower, and wash until the cleaning solution does not become cloudy. ,. As a surfactant, a nonionic surfactant is preferred which is preferably a nonionic one. As long as it is, it can be widely used. Specifically, for example, bridges (Brij), Triton, Nonidet P40, Tween, etc., which are polyoxyethylene derivatives, are exemplified as preferable examples. Of these, Nonidet P40 is the best choice. These nonionic surfactants can be used at a concentration sufficient to remove the endosperm component and not adversely affect the protein synthesis activity of the germ component, but may be used at a concentration of 0.5%, for example. it can. Either or both of the washing treatment with water or an aqueous solution and the washing treatment with a surfactant may be performed. These cleaning treatments may be performed in combination with ultrasonic treatment.

[0020] In the present invention, the germ extract having the germination ability obtained by selecting from the seed pulverized product and washing as described above is preferably subdivided in the presence of an extraction solvent, and then the germ extract obtained. Is separated and further purified to obtain an extract for cell-free protein synthesis.

[0021] As the extraction solvent, an aqueous solution containing a buffer solution, potassium ions, magnesium ions and / or a thiol group antioxidant can be used. If necessary, you can add more power ions, L-amino acids, etc. For example, a solution containing N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid (HEPES) -KOH, potassium acetate, magnesium acetate, L-type amino acids and / or dithiothreitol, or the method of Patterson et al. A partially modified solution (a solution containing HEPES-KOH, potassium acetate, magnesium acetate, calcium chloride salt, L-type amino acid and / or dithiothreitol) can be used as an extraction solvent. The composition and concentration of each component in the extraction solvent are known per se, and those used for the production of cell extracts for cell-free protein synthesis may be employed.

The required amount of extraction solvent is added to the germ and the embryo is subdivided in the presence of the extraction solvent. The amount of the extraction solvent is usually 0.1 milliliters or more, preferably 0.5 milliliters or more, more preferably 1 milliliter or more, relative to the germ lg before washing. The upper limit of the amount of the extraction solvent is not particularly limited, but is usually 10 milliliters or less, preferably 5 milliliters or less with respect to the germ lg before washing. In addition, the embryos to be subdivided may be frozen as in the past, but it is more preferable to use those that have not been frozen.

[0022] As the fragmentation method, conventionally known grinding methods such as grinding and crushing can be adopted. However, the method of subdividing embryos by impact or cutting developed by the present inventors (WO03 / 06 4671) is preferred. Here, “subdivide by impact or cutting” means the destruction of cell nuclei of plant embryos, organelles such as mitochondria, chloroplasts, cell membranes, cell walls, etc., with conventional grinding or crushing. This means that the plant germ is destroyed under conditions that can be minimized.

The apparatus and method that can be used when subdividing are not particularly limited as long as the above conditions are satisfied. For example, it is preferable to use an apparatus having a blade that rotates at high speed, such as a Warinda blender. . The rotation speed of the blade is usually lOOOOrpm or more, preferably <5000rpm or more, and usually 30000rpm or less, preferably <is 25000i "pm or less. The rotation time of the blade is usually 5 seconds or more. The upper limit of the rotation time is not particularly limited, but is usually 10 minutes or less, preferably 5 minutes or less, and the operation temperature is preferably 10 ° C. or less. Within the possible range, particularly preferably around 4 ° C is suitable.

In such subdivision of the embryo by impact or cutting, the cell nucleus and cell wall of the embryo are not completely destroyed, and at least part of them remains without being destroyed. In other words, since organelles such as embryonic cell nuclei, cell membranes and cell walls are not destroyed more than necessary, the synthesis of proteins localized in the cytoplasm is less contaminated with impurities such as DNA and lipids contained in them. Necessary RNA, ribosome, etc. can be efficiently extracted from the embryo with high purity In such fragmentation by impact or cutting, an extraction solvent can be added after fragmentation, but the extraction solvent More preferably, it is carried out in the presence.

According to such a method, the conventional process of pulverizing the germ and the process of obtaining the germ extract by simultaneously adding the extraction solvent to the pulverized germ can be performed efficiently as one process. A wheat germ extract can be obtained. Hereinafter, the above method may be referred to as a “pender method”.

Then, the embryo extract is centrifuged at 20,000 to 40,000 G, preferably 2.5 to 350,000 G, more preferably 30,000 G, and a centrifugal supernatant is obtained. At this time, it is more preferable to add an inorganic carrier as a precipitation aid to separate the precipitate from the supernatant. In this deposit, It contains a complex of calcium and enzymes such as glycosidases. Preliminarily removing glycosidase helps to minimize glucose production from starch. Examples of suitable inorganic carriers include bentonite, activated carbon, silica gel, sea sand and the like. By introducing this inorganic carrier, it is possible to almost completely prevent the precipitate from being mixed into the supernatant. When the precipitation aid is not added during centrifugation, an insoluble slurry exists at the top of the precipitate, and the protein synthesis activity of the extract prepared with the S-30 fractional force mixed with it becomes low. Therefore, when collecting the S-30 fraction from the centrifuge tube after centrifugation, extreme care must be taken to avoid contamination.

The wheat germ extract can be further purified by gel filtration or the like. The gel filtration can be performed using, for example, a gel filtration device that has been equilibrated in advance with an appropriate solution. The composition / concentration of each component in the gel filtration solution is known per se, and is used for the production of cell germ extract for cell-free protein synthesis (eg, HEPES_KOH, potassium acetate, magnesium acetate, A solvent containing dithiothreitol or L-type amino acid) may be employed.

The germ cell extract thus obtained has extremely reduced RNase activity and phosphatase activity.

Since the germ extract-containing solution after gel filtration may contain microorganisms or spores such as filamentous fungi, it is preferable to exclude these microorganisms. In particular, it is important to prevent the growth of microorganisms during the long-term (one day or longer) cell-free protein synthesis reaction. The means for eliminating microorganisms is not particularly limited, but it is preferable to use a filter sterilization filter. The pore size of the filter is not particularly limited as long as microorganisms that may be mixed can be removed, but it is usually 0.:! To 1 micrometer, preferably 0.2 to 0.5. A micrometer is appropriate. By the way, the spore size of Bacillus subtilis is 0.5 μ πιχ1 μ ΐη, and the use of a 0.20 micrometer filter (such as the Minisart ™ from Sartorius) is also effective for removing spores. Is. When filtering, it is preferable to first filter with a large pore size filter, and then filter with a pore size filter that can remove possible microorganisms. [0025] The cell extract obtained in this way is a substance that suppresses the protein synthesis function contained or retained by the raw wheat itself (acting on various RNAs, translated protein factors, ribosomes, etc. Substances to be suppressed (for example, various ribonucleases, various proteases, tritin, thionine, etc.) That is, the endosperm where these inhibitors are localized is almost completely removed and purified. The degree of endosperm removal can be evaluated by monitoring the activity of tritin contaminated in the wheat germ extract, that is, the activity of deadenating ribosomes. If the ribosome is not substantially deadenified, it is judged that there is no contaminating endosperm-derived component in the germ extract, that is, the endosperm is almost completely removed and purified. The extent to which the ribosome is not substantially deadenylated is the ribosome deadenination rate of 7% or less, preferably 1. / _Indicates that the value is ₀ or less.

[0026] Using such an embryo extract as a raw material, saccharides, phosphorylated saccharides, and phosphorylation of saccharides for the purpose of “eliminating metabolic pathways and translational reaction control mechanisms such as endogenous glycolysis of embryonic tissue cells” Processing for preparing a cell extract for cell-free protein synthesis with reduced enzymes, glycolytic enzymes and the like can also be performed. The outline of the processing steps is as follows. The obtained cell extract or this extract is used as a translation reaction solution by exchanging the solution by gel filtration or adding necessary components. Is eliminated. Alternatively, a substance having a molecular weight of lOkDa or more can be fractionated and recovered. This fractionation treatment is preferably performed a plurality of times, and in particular, it is preferable to substantially remove substances having a molecular weight of 1 kDa or less. The specific number of times is:! To 10 times, preferably 2 to 9 times, more preferably 3 to 8 times, most preferably 4 to 7 times. The cell extract thus prepared has substantially reduced sugar and phosphorylated saccharide to 10 mM or less, preferably 6 mM or less (glucose concentration in the extract with an absorbance of 200 D / ml at 260 nm). As). The extract with reduced glucose concentration obtained by force possesses an unprecedented high cell-free protein synthesis ability.

[0027] (3) Translation reaction process

To the cell synthesis solution for protein synthesis to which the transcription solution obtained as described above is added directly, amino acid as substrate, energy source, various ions, buffer solution, ATP regeneration system, nucleolytic fermentation Containing inhibitors, _t RNA, a reducing agent, polyethylene glycol, 3 ', 5'cAMP, folate, such as antimicrobial agents, referred solution ( "for translation reaction solution" together containing necessary or suitable ingredients to translation reaction ) And incubating at a temperature suitable for the translation reaction for an appropriate time, the translation reaction can be carried out. The substrate amino acids are usually 20 kinds of natural amino acids constituting proteins, but analogs and isomers thereof can be used depending on the purpose. Energy sources include ATP and Z or GTP. Examples of various ions include acetates such as potassium acetate, magnesium acetate, and ammonium acetate, and glutamates. As the buffer, Hepes-KOH, Tris-acetic acid or the like is used. Examples of the ATP regeneration system include a combination of phosphoenolpyruvate and pyruvate kinase, or a combination of creatine phosphate (creatine phosphate) and creatine kinase. Examples of the nucleolytic enzyme inhibitor include a ribonuclease inhibitor and a nuclease inhibitor. Among these, examples of ribonuclease inhibitors include human placenta-derived RNase inhibitors (such as those manufactured by TOYOBO). tRNA can be obtained by the method described in Moniter, R., et al "Biochim. Biophys. Acta., 43, 1 (1960), etc., or a commercially available one can be used. Examples thereof include dithiothreitol, etc. Examples of the antibacterial agent include sodium azide, ampicillin, etc. The amount of these added can be appropriately selected within the range that can be usually used in cell-free protein synthesis. Specifically, it is as follows.

[0028] In the repeated layering method of the present invention, a system including the following processes, means, or a combination thereof is performed.

[0029] (Process leading to synthesis reaction)

One of the essential means is to superimpose the supply phase on the reaction phase and lead it to the protein synthesis reaction system. The synthesis reaction is usually performed for about 10 minutes to 20 hours, more preferably 20 minutes to 10 hours. This time can be changed for each system, and the optimum time can be adjusted by experimental repetition. In the discontinuous repetitive synthesis method of the present invention, it is particularly preferable that the one-cool processing time is relatively short.

In a preferred embodiment of the present invention, protein synthesis is performed by overlaying a translation reaction solution on a protein synthesis cell extract directly added with a transcription solution so as not to disturb the interface. Do. Specifically, for example, a protein synthesis cell extract preincubated for an appropriate period of time as necessary is added to a translational trapezoidal precipitate and dissolved to form a reaction phase. The translation reaction solution (feed phase) is layered on top of this reaction phase so as not to disturb the interface. The interface between both phases does not necessarily have to be formed in a horizontal plane by means of multiple layers. It is also possible to form a horizontal plane by centrifuging the mixed solution containing both phases. When the diameter of the circular interface of both phases is 7 mm, the volume ratio of the reaction phase to the supply phase is suitably from 1: 4 to 1: 8, but 1: 5 is preferred. The larger the interfacial area of both phases, the higher the efficiency of protein synthesis, which increases the mass exchange rate by diffusion. Therefore, the capacity ratio of both phases varies depending on the interface area between both phases. For example, in a system using a wheat germ extract, the translation reaction is performed at about 10 to about 40 ° C, preferably about 18 to about 30 ° C, more preferably about 20 to about 26 ° C under static conditions. Can be done. When using an E. coli extract, the reaction temperature is suitably about 30 ° C to about 37 ° C.

[0030] (Mixing process of supply phase and reaction phase)

Indispensable means 2 is a step of mixing the supply phase and the reaction phase as the above reaction time, before and after substantially reducing the synthesis rate, before and after almost stopping the synthesis reaction, or in the middle thereof. The approximate decrease in the synthesis rate means the timing at which the amount of protein synthesized over time tends to decrease from the maximum amount over time, and is generally understood as a line rather than a point. In addition, the term “before and after substantially stopping the synthesis reaction” refers to a level at which the amount of synthesis falls to a level where it cannot be substantially detected. On the way, it means the period from when the synthesis rate starts to decrease until the synthesis reaction stops. In order to obtain a more favorable synthesis efficiency, it is necessary to perform a mixing process on the reaction system before and after substantially reducing the synthesis rate. This time is optimally 10 minutes to 10 hours.

The mixing treatment can be performed by mixing the reaction phase and the mixed phase by putting a stirring bar in the reaction solution and stirring the reaction solution.

[0031] (Concentration treatment)

The essential means 3 is the concentration treatment after the mixing treatment. Further, the dilution operation can be performed before and after the concentration treatment as necessary. Concentration to the reaction system is performed as follows. Concentration means that when a reaction solution is removed from the reaction system, substances that cannot pass through the reaction solution (for example, synthetic proteins, ribosomes, magnesium ions and / or nucleotides derived from a transcription solution) are contained in the reaction system. Any concentration means known per se, which can be concentrated, can be used. Preferable examples include filtration using an ultrafiltration membrane, treatment using a centrifuge, gel filtration, a suction pump, and a method of generating a pressure difference in the liquid phase or gas phase. In this treatment, the reaction product and reaction by-products are separated and removed by centrifugation or molecular sieve by adjusting the passage diameter of the membrane. The molecular weight cut size of the membrane, the centrifugal speed, and the gel filtration conditions can be optimally adjusted depending on the physical properties of the product to be processed known per se. In the present invention, a membrane having a molecular weight cut of 10,000 to 100,000 Da is preferably used.

By this concentration treatment, the reaction solution is concentrated to 1/5 to 2/3 volume of the original volume, and as a result, the optimum synthesis concentration of each synthesis factor is greatly shifted. By this concentration, the reaction system is remarkably reduced in its synthesis ability. With this state, the present invention is called discontinuity.

The dilution operation is performed by adding about 1 to 20 times, preferably about 2 to 10 times the volume of aqueous solution to the reaction system. The aqueous solution contains a substrate and a reaction solution as desired. Particularly preferably, a solution containing a substrate, an energy source and the like is used.

[0032] (Reactivation of the synthesis reaction by overlaying the supply phase on the reaction phase)

Indispensable means 4 is to reactivate the synthesis reaction by superimposing the supply phase on the reaction phase. Concentration treatment and supply layer superposition are performed. Concentration here means returning the volume of the reaction system increased by dilution to the original volume. The concentration means is not particularly limited, and any concentration means known per se can be used. Further, the method of superposing the supply phase on the reaction phase is as described in (Process leading to synthesis reaction).

[0033] The reaction system that has been restored to the optimum concentration of the reaction system is adjusted to the optimum reaction temperature again, and the reaction system is reactivated. The optimum temperature is 15-25 ° C.

For separation / recovery / removal of reaction products and / or reaction by-products, treatment based on affinity with the treatment object can be suitably performed. Based on affinity, an affinity substance is immobilized, brought into contact with the target substance, and then bound. The method of elution 'recovering is illustrated. Examples of the affinity substance include an antibody against a protein, a ligand for a receptor, a nuclear acid for a transcription factor, and the like if the recovered substance is a protein. The target product is modified with an appropriate tag marker (for example, streptavidin, histidine tag, GST, maltose-binding protein, etc.), and a substance that can specifically bind to the modified substance (for example, piotin, divalent metal ion, etc.) , Gnorethione, maltose, etc.).

In the repeated multi-layer method of the present invention, mixing, concentration, and multi-layer processing can be repeated discontinuously multiple times, and by this repetition, regeneration of the cell-free synthesis system is achieved multiple times (see FIG. 1A). . This regeneration achieves mass protein synthesis.

[0034] As described above, the repeated layering method of the present invention is a novel cell-free protein synthesis method that combines the advantages of the simple and high efficiency of the conventional layering method with the advantages of the large-scale synthesis of the repeated layered method. is there.

[0035] In the repeated feeding batch method of the present invention, a system including the following processes, means, or a combination thereof is performed.

[0036] (Process leading to synthesis reaction)

One essential means is to add the translation reaction solution to the protein synthesis cell extract to which the transcription solution is directly added, and mix them. As an addition method, a supply amount and a supply timing can be adjusted using a peristaltic pump or the like. Alternatively, when the components contained in the translation reaction solution are previously mixed with the protein synthesis cell extract, the addition of the translation reaction solution can be omitted. As a `` translation reaction solution '' obtained by mixing a protein synthesis cell extract directly added with a transcription solution and a translation reaction solution, for example, when a wheat germ extract is used as a protein synthesis cell extract, 10-50 mM HEPES-KOH (pH 7.8), 55-120 mM potassium acetate, 1-5 mM magnesium acetate, 0.1-0.6 mM spermidine, 0.025-lmM L-amino acid, 20-70 μΜ, preferably f 30-30 μ μ DTT, 1–1.5 mM ATP, 0.2–0.5 mM GTP, 10–20 mM creatine phosphate, 0.5–1. OunitsMZ xl ribonuclease inhibitor, 0.01–: ίΟ Those containing μΜ protein disulfide isomerase and 24-75% wheat germ extract are used. When such a translation reaction solution is used, the preincubation is performed at about 10 to about 40 ° C. for about 5 to about 1 Incubation in this reaction (translation reaction) for 0 minute is also carried out at about 10 to about 40 ° C, preferably about 18 to about 30 ° C, more preferably about 20 to about 26 ° C.

[0037] (Process for stopping supply of supply liquid)

The essential means 2 is a step of stopping the supply of the supply liquid as the above reaction time, before or after substantially reducing the synthesis rate, before or after substantially stopping the synthesis reaction, or in the middle thereof. Although the stop process varies depending on the supply method, the stop time can be adjusted using, for example, a peristaltic pump.

[0038] (Concentration treatment)

The essential means 3 is to concentrate the reaction phase after stopping the feed solution. In addition, the dilution operation can be performed before and after the concentration treatment as necessary. Concentration of the reaction phase is performed as follows.

Concentration means that when a reaction solution is removed from the reaction system, substances that cannot pass through the reaction solution (for example, synthetic proteins, ribosomes, magnesium ions and / or nucleotides derived from a transcription solution) are contained in the reaction system. Any concentration means known per se, which can be concentrated, can be used. Preferable examples include filtration using an ultrafiltration membrane, treatment using a centrifuge, gel filtration, a suction pump, and a method of generating a pressure difference in the liquid phase or gas phase. In this treatment, the reaction product and reaction by-products are separated and removed by centrifugation or molecular sieve by adjusting the passage diameter of the membrane. The molecular weight cut size of the membrane, the centrifugal speed, and the gel filtration conditions can be optimally adjusted depending on the physical properties of the product to be processed known per se. In the present invention, a membrane having a molecular weight cut of 10,000 to 100,000 Da is preferably used.

The dilution operation is performed by adding about 1 to 20 times, preferably about 2 to 10 times the volume of aqueous solution to the reaction system. The aqueous solution contains a substrate and a reaction solution as desired. Particularly preferably, a solution containing a substrate, an energy source and the like is used. [0039] (Reactivation of the synthesis reaction by supplying the supply liquid to the reaction phase)

The essential means 4 is reactivation of the synthesis reaction. After the concentration process in the previous stage, the supply liquid is supplied to the reaction phase.

[0040] The reaction system that has been restored to the optimum concentration of the reaction system by supplying the supply liquid is adjusted to the optimum reaction temperature again, and the reaction system is reactivated. The optimum temperature is 15-25 ° C.

For separation / recovery / removal of reaction products and / or reaction by-products, treatment based on affinity with the treatment object can be suitably performed. Based on affinity, a method in which an affinity substance is immobilized, brought into contact with the target substance and bound thereto, and then the target substance is eluted and recovered is exemplified. Examples of the affinity substance include an antibody against a protein, a ligand for a receptor, a nuclear acid for a transcription factor, and the like if the recovered substance is a protein. The target product is modified with an appropriate tag 'marker (for example, streptavidin, histidine tag, GST, maltose-binding protein, etc.), and a substance that can specifically bind to the modified substance (for example, piotin, divalent metal ion, etc.) , Gnorethione, maltose, etc.).

In the repeated feeding batch method of the present invention, it is possible to repeat the process of stopping the feed liquid, concentrating, and feeding the feed liquid a plurality of times discontinuously. By this repetition, the regeneration of the cell-free synthesis system is achieved a plurality of times. (See Figure 1B). This regeneration achieves massive protein synthesis.

[0041] Further, in the repeated feeding batch method of the present invention, there is no restriction on the size of the reaction tank due to liquid feeding from the feeding phase, and further, both liquids, which are important rate-limiting parameters of protein synthesis rate, are used. The mixing speed can be freely controlled and optimized, and highly efficient large-scale protein production becomes possible. Furthermore, the efficiency of the synthesis reaction can be further increased by using a supply phase with added mRNA. The supply rate of the specific feed solution should be such that the same amount as that at the start of the reaction can be continuously or discontinuously supplied for 5 minutes to 15 hours, preferably 10 minutes to 10 hours.

[0042] As described above, the repetitive feeding batch method of the present invention uses a semipermeable membrane developed by Spirin et al. To continuously replenish substrates and energy sources and discard metabolites. The principle is different, and the effect of the synthesis is several tens to several thousand times, and has a great difference in quality.

In the protein synthesis method using this principle, by selecting the fractional molecular weight size of the filtration membrane to be used, it is possible to selectively isolate the synthesized protein from the reaction system simultaneously with the elimination of by-products. It becomes possible.

[0043] An embodiment of a preferred synthesis method of the repeated multi-layer method of the present invention and the repeated feed batch method of the present invention is as described in FIG.

Specifically, a filtration membrane, preferably an ultrafiltration membrane, is present in the reaction vessel. A space is formed between the filtration membrane and the bottom of the reaction vessel. Then, a synthesis reaction (translation) is performed so as to include the following steps. Each reaction vessel is preferably of a size that can be centrifuged with a known centrifuge.

1. Add the reaction solution that is the reaction phase on the filter membrane.

2. Add feed to reaction phase. Here, in the case of the repeated multi-layer method, the supply liquid as the supply phase is overlaid on the upper layer of the reaction phase. In the repeated supply batch method, the supply liquid is added to the reaction phase continuously or discontinuously. The synthesis reaction (translation) begins.

3. Remove the reaction phase by-products under the filter membrane by centrifugation. Here, in the repeated layer method, the reaction phase and the feed phase are mixed and then centrifuged.

4. After centrifugation, add the feed solution. The synthesis reaction (translation) resumes.

5. Repeat steps 3-4.

6. Lower the temperature of the reaction vessel to stop the synthesis reaction (translation).

[0044] In the present invention, the process of layering, mixing, dilution and concentration can be performed as an automatic cell-free protein synthesizer (repetitive multi-layer method, repetitive feeding batch method) as a series of steps in combination with the control means. For this control, the drive source (motor, pneumatic 'hydraulic equipment, other operation-controllable actuator, etc.) and control circuit by computer control, sequence control circuit, etc. The operation speed and operation interval are adjusted. In addition, a signal transfer driver, an operation confirmation sensor, an operation control switch, a timer, and the like can be appropriately provided as desired.

[0045] As described above, the cell-free protein synthesizer of the present invention discontinuously dilutes and concentrates. The regeneration of the cell-free synthesis system is achieved by repeating multiple times. The effect will be explained more specifically as follows.

[0046] The cell-free protein synthesizer according to the present invention provides a method for automatically performing a series of reaction operations of a synthesis method in which protein synthesis is performed by mixing, concentrating, and repeatedly layering discontinuously. In addition, the cell-free protein synthesizer according to the repetitive feeding batch method of the present invention performs a series of reaction operations of a synthetic method in which protein synthesis is repeatedly concentrated and diluted (continuous or discontinuous supply of supply solution) repeatedly. Provide an automatic method.

[0047] Here, "automatic operation" means that the experimenter does not directly apply a manual operation to the reaction system (reaction vessel) during a series of steps. Therefore, when performing each step, it is necessary for the experimenter to manually operate the predetermined operation buttons and switches provided in the automatic synthesizer of the present invention to be used. It is not a loss.

In the present invention, the apparatus for automatically performing the reaction operation from the synthesized transcriptional cage to the production of the protein encoded by the cage comprises at least the following means (a) to (f). It is the feature.

Hereinafter, although each embodiment will be described in detail with specific embodiments, the method of the present invention is not limited thereto as long as it has the feature of repeating dilution and concentration a plurality of times discontinuously.

[0048] (1) Production process of transfer mold

In the automatic synthesizer of the present invention, this process does not necessarily need to be performed automatically. The transfer mold obtained manually can also be used for the following automated process. It is more preferable to automatically perform a series of steps up to the production of the protein encoded in the saddle type.

There are known apparatuses for performing such a series of operations automatically or semi-automatically (in a part of the process, an experimenter can directly apply manual operations to the reaction system). By incorporating this into the automatic synthesizer of the present invention, it is possible to automatically perform from the production of the transcription mold to the production of the target protein. The purpose of the present invention to provide a high-throughput synthesis system for high-throughput analysis, simplification of the apparatus, In consideration of shortening of the required time, it is more preferable to use the following method for preparing a transcription mold by the polymerase chain reaction (PCR) method.

[0049] (2) Transcription reaction process

Dispensing and mixing of the transfer cage solution and the transcription reaction solution into the reaction vessel are carried out by means of the automatic synthesizer described later (for example, using a pipetter (a commercially available 96-well titer plate is used as the reaction vessel). In some cases, it is preferable to use one having 8 or 12 dispensing tips adapted to the well interval). Incubation for the transcription reaction can be performed while the temperature is controlled at a constant temperature by the temperature control means of the synthesizer described later.

[0050] (3) Translation reaction process

Operations such as dispensing of unpurified mRNA-containing transfer solution, supply solution, and reaction solution into the reaction vessel are performed by dispensing means of an automatic synthesizer described later (for example, pipetter (96-well titer commercially available as a reaction vessel). In the case of using a plate, it is preferable to use a plate having 8 or 12 dispensing tips adapted to the well interval. In addition, the reaction phase and the supply phase are mixed by means of a synthesizer described later, the concentration is concentrated by a synthesizer described later, and the incubation for translation is performed at a constant temperature by a synthesizer temperature control means described later. It can be performed while controlling.

[0051] The above-described cell-free protein synthesizer of the present invention using the repeated layering method comprises at least the following means (a) to (f) in order to perform protein synthesis by mixing, concentrating, and repeatedly layering discontinuously. It is characterized by having. In addition, the cell-free protein synthesizer according to the repetitive supply batch method of the present invention performs the following steps (b) in order to perform protein synthesis in a discontinuous and repeated manner of concentration and dilution (continuous or discontinuous supply solution supply). It has at least the means of (f).

(a) means for mixing the feed phase and the reaction phase;

(b) means for variably controlling the temperature in the reaction vessel;

(c) means for dispensing the sample or reagent into the reaction vessel;

(d) means for transporting the reaction vessel;

(e) Concentration filtration means; as well as

(f) Control means for controlling the means (a) to (e) or (b) to (e) to operate in accordance with the method of the present invention described above.

Hereinafter, each configuration will be specifically described in detail.

[0052] (a) Means for mixing the supply phase and the reaction phase

The means for mixing the supply phase and the reaction phase means that the substances contained in both phases are homogenized by stirring the supply phase and the reaction phase. Therefore, any means can be used as long as it can achieve the homogenization of both phases. Specifically, if a stirrer is introduced into the reaction vessel, mixing of both phases can be easily achieved.

[0053] (b) Means for variably controlling the temperature in the reaction vessel

The means for variably controlling the temperature in the reaction vessel is the transcription reaction, the incubation of the translation reaction and the termination of the translation reaction, or the transcription slab-type preparation process by the PCR method using the automatic synthesizer of the present invention. In this case, it is a means for adjusting the liquid temperature in the reaction vessel to an appropriate temperature condition in the amplification reaction of the PCR method. The temperature range to be variably controlled is not particularly limited, but the temperature range normally required in a series of reaction operations for cell-free protein synthesis including preparation of a transcription cage (for example, about 4 ° C to about 100 ° C, Any means capable of variably controlling the liquid temperature in the reaction vessel within a range of preferably about 26 ° C. to about 99 ° C. is not particularly limited. For example, the conventionally known Takara PCR thermal cycler MP (manufactured by Takara Bio Inc.), Gene Amp PCR System 9700 (manufactured by Applied Biosystems In, Inc.) and the like can be mentioned. Specifically, in addition to the work stage where the reaction vessel is placed, which is a place where pipetting is performed, a plurality of stages for placing the reaction vessel are provided in the apparatus, and the entire space on the stage is set. It is realized that the temperature is variably controlled, and as a result, the temperature in the reaction vessel is variably controlled.

[0054] (c) Means for dispensing sample or reagent into reaction vessel

The means for dispensing a sample or reagent into a reaction vessel is to dispense a sample or reagent into a reaction vessel in order to perform a series of cell-free protein synthesis reactions such as transcription, translation, and PCR. It is means to do. Here, the “sample” refers to a transcription type, a translation type, a PCR type plasmid (or a host (eg, E. coli) having the plasmid), etc. “Reagent” refers to a transcription reaction solution, translation reaction solution, translation reaction solution, dilution solution, alcohol, salt solution, PCR reaction solution, supply solution, reaction solution, and the like. As such a dispensing means, a pipette arm (dispensing machine) that can be automatically dispensed as known in the art can be used as long as it can be dispensed by adjusting the amount of sample and reagent according to the process. It can be realized with no particular restrictions. In addition, the pipette arm can have a function of discarding a used tip at the tip disposal port of the synthesizer and a function of discharging the sucked filtrate or the like to the waste solution port.

In addition to the above functions, the dispensing means preferably has a mixing function (eg, pipetting, stirring, etc.) for homogenizing two or more types of solutions and for dissolving the precipitate. Furthermore, by adding a substrate solution or a diluting solution to each cell of the reaction vessel with a pipette arm, a dilution process for the reaction solution can be performed.

[0055] (d) Means for transporting reaction vessel

The means for transporting the reaction container is a means for moving the reaction container to each stage, centrifuge, elevator, and thermostat. The means for transporting the strong reaction container is not particularly limited as long as the reaction container can be transported to the target location, and can be realized by any conventionally known appropriate means. For example, this can be realized by using a robot arm used in a conventional synthesizer.

[0056] (e) Concentration filtration means

Concentration filtration means is means for concentrating and filtering the reaction solution during repeated translation reactions. Such means can be realized by any conventionally known appropriate means without particular limitation as long as the reaction solution can be concentrated during repeated translation reactions. For example, using a conventionally known centrifuge, other appropriate devices that have also been used for filtration and lyophilization, such as Amicon Ultra concentrated membrane (Milipore), Vivaflow (sart orius), etc. Can be realized.

[0057] (f) Control means

As the control means, the above-mentioned means (a) to (e) or (b) to (e) are used for each means to operate. It includes a control device that controls on / off of the operation of the possible actuators, etc.), and the degree and state of the operation. So The control configuration of (a) to () can achieve the purpose of automatically performing the reaction operation from the synthesized transcription mold to the production of the protein encoded by the mold. It is.

The control device may be configured by combining control devices necessary for controlling the operation of each means, such as a control circuit including a computer having a control program, a sequence control circuit, and the like. The control configuration is such that power, air pressure, hydraulic pressure, and the like can be supplied to each means so that each means operates in order. In addition, a driver necessary for directly sending a drive signal to the drive source of each of the above means, various sensors and switches necessary for detecting the operation state of the drive source of each of the above means may be added as appropriate.

The reaction vessel applicable to the synthesizer of the present invention can be any of various well-known reaction vessels that have been used for cell-free protein synthesis reactions without particular limitations. For example, a 6-well plate, 24 Hole plates, 96-well plates, 384-well plates, 96-well PCR plates, 96-well titer plates, 8-strip tubes and tubes (1.5 mL, 15 mL, 50 mL, etc.) are examples. Can be performed in a small reaction system such as a 96-well plate or a 384-well plate, and the transcription reaction can be performed in a small reaction system according to the synthesis apparatus of the present invention. It is possible to perform a series of cell-free protein synthesis methods, including transcription reactions, purification of translation variants, translation reactions, and PCR for the preparation of transcription variants for further transcription reactions, if desired. Can be performed simultaneously for several proteins, can be force S to synthesize a large number of proteins in a short time.

[0058] Furthermore, a synthesis kit containing at least one of the reagents used in the cell-free protein synthesis method of the present invention is useful because it can easily provide the cell-free protein synthesis of the present invention.

[0059] Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited by these examples.

Example 1

[0060] Preparation of wheat germ extract

(1) Preparation of wheat germ Hokkaido chihoku wheat or Ehime chikugozumi seeds were added to a mill (made by Fritsch: Rotor Speed Mill pulverisettel4) at a rate of lOOg per minute, and the seeds were gently pulverized at a rotational speed of 8,000 mm. After collecting the fraction containing germinating ability (mesh size 0.7 to 1.00 mm) in sieve sieve, a mixture of carbon tetrachloride and cyclohexane (volume ratio = carbon tetrachloride: cyclohex The floating fraction containing germinating embryos was collected by flotation using Xan = 2.4: 1), and the organic solvent was removed by drying at room temperature. Was removed to obtain a crude germ fraction.

Next, using a belt type color sorter BLM-300K (manufacturer: Anzai Manufacturing Co., Ltd., distributor: Anzai Sogyo Co., Ltd.), the germs are selected from the crude germ fraction using the color difference as follows. did. This color sorter includes means for irradiating light to the crude germ fraction, means for detecting reflected light and Z or transmitted light from the crude germ fraction, means for comparing the detected value with the reference value, Supply the crude germ fraction to 1000 to 5000 grains / cm ² on the beige belt of the color sorter, which is a device that has a means to sort out and remove those that fall outside or within the reference value. The reflected light was detected by irradiating the upper crude germ fraction with a fluorescent lamp. The belt conveyance speed was 50 m / min. A monochrome CCD line sensor (2048 pixels) was used as the light receiving sensor.

First, in order to remove dark-colored components (seed coat etc.) from the germ, a reference value was set between the brightness of the germ and the brightness of the seed coat, and those that deviated from the reference value were removed by suction. Next, in order to sort out the endosperm, a reference value was set between the brightness of the germ and the brightness of the endosperm, and anything that deviated from the reference value was removed by suction. Suction was performed using 30 suction nozzles (one suction nozzle per 1 cm length) placed approximately 1 cm above the conveyor belt. By repeating this method, the germs were selected until the purity of the germs (the weight ratio of the germs contained in an arbitrary sample lg) reached 98% or more.

The obtained wheat germ fraction was suspended in distilled water at 4 ° C, and washed with an ultrasonic washing machine until the washing solution did not become cloudy. Subsequently, it was suspended in a 0.5% by volume solution of Nonidet P40 (Nonidet: manufactured by Nacalai Tectonics), and washed with an ultrasonic cleaner until the cleaning solution did not become cloudy to obtain wheat germ. 2 times the volume of the extracted germ solution Medium (80mM HEPES—KOH, pH7.8, 200mM potassium acetate, lOmM magnesium acetate, 8mM dithiothreitol, 4mM calcium chloride, 0.6mM, 20 L-type amino acids, and 2.5mM ATP) Using a Waring blender, limited disruption of the embryo was performed three times at 5,000-20, OOOrpm for 30 seconds each.

[0061] (2) Preparation of S-30 fraction using precipitation aid

The homogenate (crushed material) obtained above was mixed with 20% by weight sea sand or swollen Cefdex G25 particles and mixed. Sea sand was subjected to the following treatment in advance before homogenate addition: water washing → 5 volumes of 0.1 N NaOH or KOH washing → water washing → 0.1 N HC1 washing → water washing → 100 ~: 120 ° RNase inactivation treatment by heating C, followed by drying treatment.

Homogenate mixed with sea sand was centrifuged twice at 30,000 xg for 30 minutes and then once for 12 minutes to obtain a translucent centrifugal supernatant (S-30 fraction). When sea sand or cefadex particles were not added before centrifugation, an insoluble slurry was present at the top of the precipitate, and the protein synthesis activity of the extract prepared from the S-30 fraction contaminated with this was low. The obtained S-30 fraction was applied to Sephadex G25 equilibrated with elution solution (40 mM HEPES_KOH, pH 7.8, 200 mM potassium acetate, lOmM magnesium acetate, 4 mM DTT), gel filtered, and molecular weight below 1000 Dalton An embryo extract from which low molecular weight substances were excluded was prepared.

[0062] (3) Transcription reaction process

The transcription reaction was incubated at 37 degrees for 2.5 hours. The transcription reaction solution is as follows.

1. Amplification by PCR method 'Synthesized DNA template to transcription template

Transcription reaction composition: 75 μ \ 5χ transcription buffer (400 mM HEPES, H 7.6; 80 mM Magnesium acetate; 10 mM Spermidine; 50 mM DTT), 37.5 μ 1 25 mM 4NTPs, 3.7 5 μ 1 RNAsin ( 80 Units), 25 μ 1 PCR product (GFP transcription type: pEU_E01_GFP (SP6 promoter-EOl-GFP)), two primers on the 5 'side and 3' side. 30 cycles) was used to construct a transcription mold, and water (85 1.25 μ1) was added to 7.5 μ1 of SP6 polymerase (80 units) to make a total volume of 1 ml.

2. A method of introducing a transcription cage into a plasmid

Transcription reaction composition: 300 5x transcription buffer (400 mM HEPES, pH 7.6; 80 mM Magnesium acetate; 10 mM Spermidine; 50 mM DTT), 150 μ1 25 mM 4NTPs, 15 μ1 RNAsin (1200 Units) ヽ 44 β 1 pEU (including GFP gene clone, 150 μg), 3

461 μ1 of water was added to 0 μ1 of SP6 polymerase (2400 units) to make a total volume of 1 ml.

Note that the transcription solution containing unpurified mRNA was not manipulated after the transcription reaction.

. On the other hand, the transcription solution containing purified mRNA was subjected to ethanol precipitation after the transcription reaction, and the supernatant was removed and dried.

Example 2

Protein synthesis by repeated layering and repeated feeding batch methods

1-1.Protein synthesis by repetitive layering using unpurified mRNA introduced into a plasmid to form a transcriptional cage

A 6-hole titer plate (TPP, Switzerland) was used as a reaction vessel. First, add 5 · 5 ml of a supply solution containing 220 creatine kinase (a solution containing amino acids, ATP, GTP, creatine-phosphate, ions, and HEPES buffer), and add 0.5 ml of a reaction solution (230 μ 1 of 260Α260 nm). Embryo transfer solution), 250 / i 1 transcription solution containing unpurified mRNA, 10 g / zi l ere atine kinase 2 μ 1 (20 / ι _§ ) and 18 / i 1 feed solution mixed Was carefully and gently added to the bottom of the titer plate. The reaction was allowed to stand for 10 hours at 26 degrees and total. After 5 hours of the synthesis reaction, the feed phase and the reaction phase were mixed by pipetting. After mixing, the mixture was concentrated to 0.5 ml, which is the liquid volume of the starting reaction phase, using an Amicon Ultra concentrated membrane (Millipore) having a molecular weight of 10,000 cut-off. After concentrating, 5.5 ml of feed solution (not containing creatine kinase) was overlaid to resume protein synthesis and synthesized for another 5 hours.

1-2. Protein synthesis by repetitive concentration and supply batch method using unpurified mRNA introduced into plasmid and transformed into transcriptional cage

Place a 0.5 ml reaction solution in a test tube (FALCON, USA: a small rotor placed for stirring) with the same capacity as 1-1, and stir with a small rotor at a slow speed while mixing the peristal Was used to continuously feed 5.5 ml of the feed solution to the reaction solution at a rate of 1.2 ml per hour (1.2 ml / hour) (total feed amount: 5.5 ml). After 5 hours of the synthesis reaction, the supply to the reaction phase was stopped. After stopping, concentration was performed to 0.5 ml, which is the liquid volume of the starting reaction phase, using an Amicon Ultra concentration membrane (Millipore) having a molecular weight of 10,000 cut-off. After concentration, supply solution 5.5ml (creatine The protein synthesis was resumed by continuously supplying (1.2 ml / hour) without kinase, and synthesized for 5 hours (Fig. 3: Lane 5). Protein synthesis was also performed with 50% of the mRNA in the reaction solution and the remaining 50% of the mRNA contained in the supply solution (FIG. 3: lane 6).

[0064] 2. Measurement of protein synthesis

Protein synthesis was measured by measuring the incorporation of radioactivity into the acid-insoluble fraction of ¹⁴ C-labeled leucine as follows: 5 microliters of the reaction mixture was spotted onto 3MM Whatman filter paper, 10% After soaking in ice-cold TCA (triclo-mouth acetic acid) for 1 hour, it was boiled in 5% TCA solution for 10 minutes. Take out this filter, remove TCA and water with ethanol ether (50:50 volume), and after drying, measure the radioactivity incorporated into the hot TCA insoluble fraction with a liquid scintillation counter (toluene scintillator). did.

[0065] Fig. 3 shows the results of highly efficient cell-free protein synthesis by the repeated layering method and the repeated feeding batch method. In both synthesis methods, it can be seen from the increase in the intensity of GFP staining that the amount of synthesis is increased by the second synthesis reaction (number of repetitions: one lane 2) compared to the first reaction synthesis (lane 1). (The * mark shown in the figure is an endogenous protein band derived from germ, and this is used as an internal standard to compare the staining intensity of the synthetic GFP band of the arrow to obtain a more reliable determination). Purified GFP was separated and stained by the same gel electrophoresis. Using this staining intensity as a standard, the amount of the synthesized product obtained in the second synthesis reaction (repeated once: lane 2) was determined based on the band staining intensity. As a result, the amount of product per 1 ml (lower layer reaction solution at the start of synthesis) was 1 mg in the repeated layer method and 1.2 mg in the repeated feed batch method. As described above, the repetitive multi-layer method and repetitive supply batch method are more effective than the conventional multi-layer method and repetitive batch method.

As a rate-determining factor for the duration of the synthesis reaction in the cell-free protein synthesis reaction, the stability of the translational type mRNA can be mentioned. RNA molecules are thought to be digested by trace amounts of the enzyme, even in mRNAs that are generally sensitive to ribonucleases, and lose their translational activity. As a result, the decrease in the synthesis reaction accompanying the degradation of mRNA is strongly influenced as the synthesis reaction takes longer. Therefore, the effect of adding mRNA to the supply solution and affecting the amount of protein synthesis was repeatedly examined by the supply batch method (Fig. 3: lanes 5 and 6: the amount of mRNA is the same). According to the results in Figure 3, mRNA is not contained in the feed solution. Rather than the second synthesis reaction (repeated once: small arrow in lane 4), the second synthesis reaction (must be repeated once: large arrow in lane 6) contains mRNA in the feed solution. It can be seen from the CBB staining intensity of the band that the amount of GFP synthesis is high. In the single synthesis reaction (lanes 1 and 3), no significant difference was found in the amount of protein synthesis. Based on the measurement of band color intensity using densitometer, the second synthesis reaction that contains mRNA in the feed solution (repeated once: large arrow in lane 6) does not contain mRNA in the feed solution 2 A protein synthesis amount 1.3 times that of the first synthesis reaction (one iteration: small arrow in lane 4) was obtained (in other words, the amount of product per 1 ml (lower reaction solution at the start of synthesis) was 1.5mg).

As described above, in the repeated layering method and the repeated feeding batch method, by using the supply solution containing mRNA, by supplying the mRNA in addition to the supply of the substrate and the energy source molecule and the dilution of the by-product. By replenishing mRNA that has been degraded and reduced in concentration in the reaction solution, it is possible to achieve efficient protein synthesis. Example 3

[0066] Amplification by PCR method 'Comparison of protein synthesis efficiency between the synthesized DNA template and the transcription template using plasmid

1.Transcription type

Transcription molds are prepared by the two methods described in the transcription reaction step of Example 1 (method of amplification or synthesis of DNA clones synthesized by PCR method or transcription clones by introducing them into plasmids) did. In both cases, mRNA in the transcription solution after the transcription reaction was not purified.

2.Repeated multi-layer method

The multilayer method was repeated by the same method as in Example 2. The number of repetitions is 3 (the synthesis reaction is 4 times).

[0067] As shown in Fig. 4, it was confirmed from the increase in the intensity of staining of the CBB-stained GFP band (arrow in Fig. 4) on polyacrylamide native gel electrophoresis that GFP synthesis lasted up to 4 times. (In Fig. 4: lanes 5 and 9). There is no GFP band before the start of the synthesis reaction (in Fig. 4, 1 lane). In addition, the protein synthesis method using transcripts from PCR is based on the comparison of PCR and the intensity of GFP staining on the plasmid electrophoresis gel in Fig. 4. It was found that the GFP synthesis efficiency was equivalent to or better than the protein synthesis method using the obtained transcription solution. The number of GFP synthesis obtained by the repeated layering method until the 4th reaction was about 1.8 mg per ml of reaction solution at the start of the synthesis reaction, comparing the intensity of the staining band on the electrophoresis gel with that of the standard GFP band. (The sample volume in each lane used for electrophoresis was 0.167 μ 湘 of the reaction solution at the start of the reaction. Therefore, the amount of GFP in the band showing the same staining intensity as standard GFP 0.3 xg was 0.370. .167 1 =

.

Based on the above, it was confirmed that it was possible to synthesize proteins with high efficiency without refining mRNA synthesized using transcripts from PCR from the transcription solution in the repeated layer method.

Industrial applicability

[0068] The cell-free protein synthesis method according to the present invention includes a complicated method such as an ultrafiltration membrane method using a semipermeable membrane, a dialysis membrane method, or a column chromatography method in which a translation cage is immobilized on a resin. By introducing the efficiency of the synthesis reaction into the conventional cell-free protein synthesis system without using it, the synthesis of proteins in the cell-free system using tissue / cell extract is possible by any means. It was shown that it is possible to carry out with high efficiency. The cell-free protein synthesis method according to the present invention described above is a low membrane material strength, a decrease in membrane function due to clogging, and an operation, as observed in the conventional cell-free protein synthesis method using a conventional membrane. Therefore, protein synthesis can be carried out with much higher efficiency than the conventional method. Therefore, the technology according to the present invention will be a basic element technology for automating the production of gene products (proteins) that will serve as the basis for functional analysis and structural analysis of a huge number of genes that will be brought forward with the completion of future genome projects. . In particular, it can be said that it is indispensable as an elemental technology for automating cell-free protein synthesis systems, such as the development of fully automated cell-free protein synthesis robots for multiple specimens.

Brief Description of Drawings

[0069] [FIG. 1] FIG. 1 shows the principle of the repeated multi-layer method (A) and the repeated feed batch method (B).

[FIG. 2] FIG. 2 is a schematic diagram of a synthesis method by a preferred repeated multi-layer method and repeated feed batch method. is there.

3] Fig. 3 shows the results of highly efficient cell-free protein synthesis by the repeated layering method and repeated feeding batch method.

[Fig. 4] Fig. 4 compares the efficiency of protein synthesis between a DNA template amplified and synthesized by PCR and a transcription template using a plasmid.

Claims

The scope of the claims

[1] A cell-free protein synthesis method by an iterative method including at least the following steps;

3) Concentration process

[2] A synthesis method comprising two upper and lower layers through an interface, wherein the upper layer is a supply phase and the lower layer is a reaction phase, and includes a cell-free protein synthesis method by a repeated multi-layer method including the following steps;

1) The process of superposing the supply liquid that becomes the supply phase on the reaction phase that is the reaction liquid to lead to the synthesis reaction

2) A process of mixing the supply phase and the reaction phase before and after substantially lowering the synthesis rate, before and after the synthesis reaction is almost stopped, or in the middle thereof

3) Concentration process

4) Step of resuming the synthesis reaction by overlaying the feed solution as the feed phase on the reaction phase [3] Cell-free protein synthesis method by repeated feed batch method including the following steps;

3) Concentration process

4) The step of resuming the synthesis reaction by continuously or discontinuously supplying the supply liquid to the reaction phase. [4] The synthesis reaction is controlled at the supply addition rate of the supply liquid (feed liquid volume Z seconds). The synthesis method described.

[5] The method according to claim 4, wherein the same amount of the supply liquid as the reaction phase at the start of the reaction is continuously or discontinuously supplied to the reaction phase in 10 minutes to 10 hours.

[6] The synthesis method according to any one of claims 1 to 5, wherein the step 2) —4) is repeated a plurality of times.

[7] The reaction solution is a transcription solution containing unpurified mRNA after the transcription reaction and wheat seed embryo The synthesis method according to any one of claims 16 to 16, comprising a bud-derived extract.

[8] The synthesis method according to [1], [1] or [1], wherein the transcription solution or the purified mRNA containing the unpurified mRNA after the transcription reaction is supplemented and added to the supply phase at the time of use.

[9] The synthesis method according to [1], wherein the by-product of the reaction phase is removed by concentration treatment.

[10] The synthesis method according to any one of [1] to [8], wherein magnesium ions and / or nucleotides derived from the transfer solution are removed by concentration treatment.

[11] The wheat seed germ extract according to any one of claims 1 to 10, wherein the wheat seed germ extract is a wheat seed germ extract from which a mixed wheat endosperm component and a low molecular weight protein synthesis inhibitor are substantially removed. Synthesis method.

[12] The synthesis method according to claim 11, wherein the sugar phosphorylation system via ATP is selected from any one of the following from an extract derived from wheat seed germ:

1) removal of monosaccharides,

2) removal of phosphorylated sugar,

3) control of the production of monosaccharides from polysaccharides,

4) Control of production of phosphorylated saccharide from monosaccharides.

[13] A synthesis kit comprising at least 1 of the reagent used in the synthesis method according to any one of claims 1 to 12.

[14] An apparatus for carrying out the cell-free protein synthesis method by the repeated multi-layer method according to any one of claims 2, 6-12, and for cell-free protein synthesis comprising at least the following control means: Equipment;

(1) Means to overlay the supply phase on the reaction phase

(2) Means for mixing

(3) Means for concentration

[15] An apparatus for carrying out a cell-free protein synthesis method according to the repeated feeding batch method according to any one of claims 3-12, wherein the apparatus for cell-free protein synthesis comprises at least the following control means;

(1) Means for continuously or discontinuously supplying the supply liquid to the reaction phase (2) Means for concentration

[Figure 1]

(A) «Reversed seedling waste method Cell protein synthesis reaction method