JP2003047496A - Method for synthesizing protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for increasing the synthesized amount of an object protein by increasing the stability of a nucleic acid which is a transcription/translation template of the objective protein in an acellular protein synthetic system. SOLUTION: This method synthesizes the objective protein in the presence of a substance which does not encode the objective protein, and has a transcription/translation template activity lower than the template of the objective protein in the acellular protein synthetic system.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently synthesizing a target protein in a cell-free protein synthesis system. Specifically, it relates to a method for enhancing the stability of a nucleic acid serving as a transcription or translation template of a target protein in a cell-free protein synthesis system and consequently increasing the amount of the target protein synthesized.

[0002]

2. Description of the Related Art In a protein synthesis reaction carried out in a cell, first, DNA having genetic information is transcribed into mRNA, and the information of the mRNA is translated on a ribosome to synthesize a protein. The process is in progress. As a method for performing the protein synthesis in the cell in vitro, for example, a component including a ribosome, which is a protein translation device provided in the cell, is extracted from an organism, and is transcribed into the extract, or a translation template, a nucleic acid serving as a substrate. , An amino acid, an energy source, various ions, a buffer solution, and other effective factors are added to the in vitro method (hereinafter, these series of operations may be referred to as "cell-free protein synthesis system") (JP-A-6-987).
Japanese Patent Laid-Open No. 90, Japanese Patent Laid-Open No. 6-225783, Japanese Patent Laid-Open No.
-194, JP 9-291, JP 7-
147992 publication).

The cell-free protein synthesis system has the performance comparable to that of living cells in terms of peptide synthesis rate and accuracy of translation reaction, and does not require complicated chemical reaction steps or complicated cell culture steps. Have. However, in a conventional cell-free protein synthesis cell or tissue extract, a contaminated group of nucleic acid degrading enzymes, translation inhibitory protein factors, protein degrading enzymes (hereinafter referred to as "translation inhibitory factor" Is caused by inactivation or inactivation of the transcription or translation reaction system during the protein synthesis reaction (Ogasawar
a, T. et al., EMBOJ., 18, 6522-6531 (1999)) had a problem that the amount of protein synthesized was small.

In order to solve this problem, a method for inactivating the above-mentioned inhibitory factor mixed in (Japanese Patent Laid-Open No. 7-20)
3984), a method for preventing contamination of inhibitory factors (JP-A No.
000-236896), and RN which is a translation template.
A method of maintaining the amount of A present in the cell-free protein synthesis system at an appropriate concentration (Japanese Patent Laid-Open No. 2000-333673) and the like have been developed. However, even with these methods, it was not possible to obtain sufficient translation efficiency for industrial application of the synthetic system.

[0005]

The present invention is intended to enhance the stability of a nucleic acid serving as a transcription or translation template for a target protein in a cell-free protein synthesis system, and consequently increase the amount of the target protein synthesized. It was made for the purpose.

[0006]

[Means for Solving the Problems] As a result of intensive studies to achieve the above object, the present inventors have found that in a cell-free protein synthesis system, in addition to a translation template for a target protein, a start codon (AUG It was found that the amount of the target protein to be synthesized is remarkably increased by the presence of an appropriate amount of RNA containing no). The present invention has been accomplished based on these findings.

That is, according to the present invention, (1) a method for synthesizing a target protein in a cell-free protein synthesis system, which serves as a substrate for a nucleolytic enzyme, but does not encode the target protein, and A method comprising synthesizing a target protein in the presence of a substance having a transcription / translation template activity lower than that of the template of the target protein in the synthesis system,
(2) The method according to (1) above, wherein the substance that serves as a substrate for the nucleolytic enzyme is a nucleic acid or a derivative thereof, and (3) a translatable base present in the base sequence of the nucleic acid or a derivative thereof. The method according to (2) above, wherein the number is 30 amino acids or less, and (4) the nucleic acid or a derivative thereof does not include a start codon in its nucleotide sequence, (2) above. Or the method described in (3), (5) the cell-free protein synthesis system is a translation system,
The method according to any one of (2) to (4) above, wherein the nucleic acid or its derivative is RNA.
The method according to any one of (2) to (4) above, wherein the cell-free protein synthesis system is a transcription / translation system, and the nucleic acid or its derivative is DNA or RNA.
(7) In any one of the above (1) to (6), wherein the amount of the substance serving as the substrate of the nucleolytic enzyme is 0.1 times or more the weight ratio of the nucleic acid encoding the target protein. (8) A reagent containing a substance used in the method for synthesizing a target protein in a cell-free protein synthesis system, which substance serves as a substrate for a nucleolytic enzyme, but does not encode the target protein. And a reagent characterized by being a substance having lower transcription / translation template activity than the template of the target protein in the synthetic system, (9) at least,
Provided is a cell-free protein synthesis reaction solution, and a kit for carrying out the method according to any one of (1) to (7) above, which comprises the reagent described in (9) above.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. (1) Competitive Substrate The present invention is a method for synthesizing a target protein in a cell-free protein synthesis system, which serves as a substrate for a nucleolytic enzyme, but does not encode the target protein, and the target in the synthesis system is A method for synthesizing a target protein in the presence of a substance having transcription / translation template activity lower than that of a protein template (this may be referred to as "competitive substrate" in the present specification) (hereinafter, referred to as "competitive substrate method"). Sometimes referred to as)) is provided.

The cell-free protein synthesizing system of the present invention means a component containing a protein translation device in a cell, such as ribosome, extracted from an organism and transcribed into this extract, or a translation template, a nucleic acid serving as a substrate, This is a method in which an amino acid, an energy source, various ions, a buffer solution, and other effective factors are added and the method is performed in vitro. Of these, RN as a template
A that uses A (this may be referred to as "cell-free translation system" hereinafter), and that that uses DNA and further adds an enzyme required for transcription such as RNA polymerase (hereinafter referred to as "cell-free translation system") Sometimes referred to as "transcription / translation system"). As the cell-free protein synthesis system that can be used in the present invention, any of the above cell-free translation system and cell-free transcription / translation system can be used.

The features of the competitive substrate of the present invention include (I)
In the cell-free protein synthesis system (III) that does not encode the target protein (II), which serves as a substrate for the nucleolytic enzyme, the transcription / translation template activity is lower than the template of the target protein. Nucleolytic enzyme in the present invention, those existing in the cell-free protein synthesis system, and mixed when preparing a cell extract used in the synthesis system,
It refers to all enzymes capable of degrading nucleic acids. Specifically, for example, a nuclease having a 3'to 5'direction, an exosome existing in yeast (Cell, 99, 347-
350 (1999)), ribonuclease-L that recognizes and cleaves the double-stranded region of RNA, ribonuclease-III-like nuclease (Trends in plant science, 4, 429-438 (199
9)), and polynucleotide phosphorylase and the like. Of the above-mentioned cell-free protein synthesis systems, in a transcription / translation system, a nuclease can decompose DNA serving as a template for a target protein, and in a translation system, ribonuclease can decompose RNA serving as a template for the target protein. , Is one of the causes of the decrease in protein synthesis efficiency in each synthetic system.

Examples of the substance that serves as a substrate for these nucleic acid degrading enzymes include nucleic acids and their derivatives. Specifically, among the above cell-free protein synthesis systems, transcription /
In the translation system, DNA, and in the translation system, RN
A, an oligomer composed of mixed nucleotides of DNA and RNA, an oligonucleotide having 2'-O-methyl, and the like. As the nucleic acid derivative, any substance may be used as long as it has a structure that is a substrate of the above-mentioned degrading enzyme, that is, a structure that can be recognized and attacked by the degrading enzyme. Whether these substances serve as a substrate for the above-mentioned decomposing enzyme depends on, for example, a reaction system suitable for the decomposing enzyme,
A method is used in which the above substance is added after labeling with a radioisotope or a fluorescent reagent, if necessary, and the molecular weight of the substance or its abundance is analyzed by a method such as electrophoresis over time. When the molecular weight is low or the existing amount is low, it can be judged that the substance serves as a substrate for the degrading enzyme used.

[0012] Further, "not encoding the target protein" means that it does not serve as a translation template for the protein of interest in the cell-free protein synthesis system to be used. As long as it does not completely match the sequence of the nucleic acid serving as a template, those having a part thereof can also be used as the competitive substrate of the present invention.

Further, a substance having a transcription / translation template activity lower than the template of the target protein in the cell-free protein synthesis system to be used means a substance which is less likely to be transcribed / translated than the template of the target protein in the synthesis system or is completely transcribed. / Means a substance that is not translated. Specific examples of the nucleic acid having a sequence that is difficult to translate include, for example, in a cell-free translation system, those having a sequence that does not act on the translation apparatus such as ribosomal RNA, translation initiation / elongation / termination factor, or aminoacylase, Polymers, ORFs present in their nucleotide sequences of 30 amino acids or less, start codon (AU
Examples thereof include those having a sequence not containing G), or derivatives thereof. ORF existing in the base sequence is 30
Specific examples of the amino acid or less include those in which a stop codon exists near the start codon and any sequence may be linked 3 ′ downstream thereof.
Further, in the cell-free transcription / translation system, DNA or RNA having the above sequence, or DNA having no promoter sequence can be used as the nucleic acid having a sequence that is difficult to be transcribed. It is also possible to allow RNA that does not encode the above-mentioned target protein together with these DNAs and has a lower transcription / translation template activity than the template of the target protein to be present in the synthesis system.

Further, the fact that the transcription / translation template activity is lower than the template of the target protein in the cell-free protein synthesis system used by these substances means that, for example, the cell-free protein synthesis system to be used may contain a radioisotope or a fluorescent substance if necessary. The substance is allowed to be present together with the template of the target protein labeled with, and the amount of the protein synthesized in the synthesis system and the molecular weight are analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) or the like over time. A method is used. In the synthesis system, the amounts of the target protein and the protein encoded by the competitive substrate are compared, and it can be confirmed that the transcription / translation template activity is lower than the template of the target protein due to the large amount of the target protein synthesized.

Those having all of these characteristics can be used as the competitive substrate of the present invention. However, when protein synthesis is carried out in a cell-free protein synthesis system using a wheat germ extract, the start codon is used. Does not include
It is most preferable to use RNA as a competitive substrate in terms of the efficiency of synthesizing the target protein.

The chain length of these competing substrates is not particularly limited as long as it has a function of being decomposed by a nucleolytic enzyme using these substances as substrates, but specifically, those having a length of 200 to 4000 bases are used. Is preferred. When DNA is used as a competitive substrate, the DNA may be double-stranded or single-stranded, and one suitable as a substrate for the target nucleolytic enzyme can be selected.

These competing substrates can be prepared by appropriately selecting known and commonly used methods. Specifically, for example, when DNA is used as a competitive substrate, an appropriate sequence that does not include a transcriptional regulatory active site such as a promoter is obtained from a library by digestion with a restriction enzyme, polymerase chain reaction (PCR), or the like. However, it can be obtained by purification by a known appropriate method. The DNA may be genomic DNA or cDNA. When RNA is used, for example, a SP6 or T7 promoter and the like and a DNA having an appropriate sequence 3 ′ downstream thereof are prepared in the same manner as above, and specifically recognize the promoter contained in the DNA. RNA can be produced by RNA polymerase. Here, RNA using SP6 RNA polymerase
When synthesizing, an omega sequence (DR Gallie, et al., Nucleic Acids) is inserted between the promoter and an appropriate DNA.
Res., 20, 4631 (1992)) can increase the transcription efficiency. Nucleic acid derivatives and the like can also be produced by an appropriate chemical synthesis method known per se. Furthermore, commercially available products can also be used as the competitive substrate of the present invention.

(2) Competition in cell-free protein synthesis system
Protein synthesis using combined substrate (a) Cell-free protein synthesis system The cell-free protein synthesis system used in the present invention may be the one described in (1) above, and specifically, Escherichia coli, plants Known materials such as seed germs and cell extracts such as rabbit reticulocytes are used. These may be commercially available ones, or methods known per se,
Specifically, the Escherichia coli extract was prepared by Pratt, JM et al., Tran.
scription and Tranlation, Hames, 179-209, BD &
Higgins, SJ, eds), IRL Press, Oxford (1984) and the like. E. coli S30 extract system (Promega) and RTS 500 Rapid Tranlation System (Roche) are commercially available as cell-free protein synthesis systems or cell extracts derived from E. coli.
Rabbit R is derived from rabbit reticulocytes.
eticulocyte Lysate Sytem (manufactured by Promega) and the like, and those derived from wheat germ include PROTEIOS ^™ (manufactured by TOYOBO). Among them, it is preferable to use the system of the germ extract of plant seeds, and as the plant seeds, plants of the Gramineae family such as wheat, barley, rice, corn, and spinach are preferable. Among them, the one using a wheat germ extract is suitable as the cell-free synthesis system of the present invention.

As a method for preparing a wheat germ extract, for example, Johnston, FB et al., Nature, 179, 160-161 (195
7), or Erickson, AH et al., (1996) Meth. I
n Enzymol., 96, 38-50 etc. can be used. Furthermore, a translation inhibitor contained in the extract,
For example, treatment such as removal of endosperm containing tritin, thionine, nucleic acid degrading enzyme, etc. (JP 2000-236896 etc.), or treatment for suppressing activation of translation inhibitor (JP 7-203984 A). Is also preferable. The cell extract obtained in this manner can be used in a protein synthesis system by a method similar to the conventional method.

The composition of the synthetic reaction solution used in the protein synthesis system of the present invention includes the above-mentioned cell extract, DNA or RNA serving as a transcription / translation template, amino acids serving as substrates, energy sources, various ions, buffer solutions, and ATP. Regeneration system, nucleolytic enzyme inhibitor, tRNA, reducing agent, polyethylene glycol,
3 ', 5'-cAMP, folate, antibacterial agent, and DN as template
When A is used, it may contain a substrate for RNA synthesis, RNA polymerase and the like. These are target proteins,
It is appropriately selected and prepared according to the type of protein synthesis system used. The competitive substrate of the present invention can be added to this synthesis reaction system and used in the protein synthesis system of the present invention.
When the protein synthesis system used is a transcription / translation system, a competitive substrate having a sequence that is difficult to be transcribed, for example, a DNA that does not contain a promoter sequence is selected.

The DNA or RNA serving as a transcription / translation template encodes a target protein to be synthesized in a protein synthesis system. The amino acids that serve as substrates are the 20 types of amino acids that make up proteins. Moreover, ATP or GTP is mentioned as an energy source. Examples of various ions include acetates such as potassium acetate, magnesium acetate and ammonium acetate, and glutamate. Hepes-KOH, Tris-Oac, etc. are used as the buffer solution. 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 ribonuclease inhibitor and nuclease inhibitor. Among these, as a specific example of the ribonuclease inhibitor, a human placenta-derived RNase inhibitor (manufactured by TOYOBO, etc.) and the like are used. t
RNA is based on Moniter, R., et al., Biochim. Biophys. Act.
It can be obtained by the method described in a., 43, 1 (1960) or the like, or a commercially available one can be used. Examples of the reducing agent include dithiothreitol. Examples of the antibacterial agent include sodium azide and ampicillin. Furthermore, SP6 RNA polymerase, T7 RNA polymerase, or the like can be used as the RNA polymerase. The addition amount of these is appropriately selected to prepare a synthetic reaction solution.

(B) Protein Synthesis Using Competitive Substrate The amount of the competitive substrate of the present invention added to the above-mentioned synthetic reaction solution varies depending on the kind of the competitive substrate, but is usually a nucleic acid encoding the target protein in the reaction solution. The lower limit is 0.1 in the weight ratio of
˜1 times, preferably 3 to 4 times. Here, the upper limit of the addition amount of the competitive substrate is not particularly limited as long as the protein synthesis reaction can occur in the synthesis system. The order of adding the synthesis reaction solution and the competitive substrate to the reaction system is not particularly limited and can be appropriately selected depending on the protein synthesis system to be used and the target protein. Preferably, a method is used in which a competitive substrate is added to the synthesis reaction solution from which the translation template has been removed, pre-incubated for an appropriate time, and then the translation template is added. The thus obtained protein synthesis reaction solution to which a competitive substrate is added may be referred to as “competitive substrate-added synthesis solution” in the present specification.

The thus obtained competitive substrate-added synthetic solution is put into a selected system or apparatus known per se, and protein synthesis is carried out. As a system or apparatus for protein synthesis, a batch method (Pratt, JM et al., Transcription and Tranla
tion, Hames, 179-209, BD & Higgins, SJ, ed
s), IRL Press, Oxford (1984)) and a continuous cell-free protein synthesis system (Spirin, AS et al., Scienc) that continuously supplies amino acids, energy sources, etc. to the reaction system.
e, 242, 1162-1164 (1988)), dialysis method (Kikawa et al., No. 21)
The Japan Society for Molecular Biology, WID6), the multi-layer method (Japanese Patent Application No. 2000-259186), and the like. Furthermore, a method of supplying a template RNA, an amino acid, an energy source, etc. to a synthetic reaction system when necessary and discharging a synthetic product or a decomposed product when necessary (Japanese Patent Laid-Open No. 2000-333673: hereinafter referred to as "discontinuous gel filtration"). Sometimes referred to as "method") and the like can be used. Among them, in the batch method, the reaction may be stopped if the protein synthesis is carried out for a long time.Therefore, it is possible to maintain the reaction for a long time by using the latter continuous or discontinuous supply system of amino acids and energy sources. It is possible to further improve efficiency.

When protein synthesis is carried out by the batch method, for example, a competitive substrate is added to the above-mentioned synthesis reaction solution from which the translation template has been removed, pre-incubated for an appropriate time, and then the translation template is added and incubated. You can When a wheat germ extract is used, the pre-incubation is performed at 10 to 40 ° C. for 5 to 10 minutes, and the incubation is also performed at 10 to 40 ° C., preferably 18 to 30.
C., more preferably 20 to 26.degree. The reaction time is the time until the reaction is stopped, but it is usually about 10 minutes to 7 hours in the batch method.

When protein synthesis is carried out by the dialysis method, protein synthesis is carried out by using a device which is separated by a dialysis membrane capable of mass transfer from a dialysis inner solution using a synthetic reaction solution containing a competitive substrate. . Specifically, for example, a competitive substrate is added to the above-mentioned synthesis reaction solution from which the translation template has been removed, pre-incubated for an appropriate period of time, and then the translation template is added to the reaction solution, which is then placed in an appropriate dialysis container to give the reaction solution. Examples of the dialysis container include a container having a dialysis membrane added to the bottom (Daiichi Kagaku KK: dialysis cup 12,000) and dialysis tubing (Sanko Junyaku 12,000). As the dialysis membrane, those having a molecular weight limit of 10,000 daltons or more are used, but those having a molecular weight limit of about 12,000 daltons are preferable.

As the external dialysis solution, the one obtained by removing the translation template and the competitive substrate from the above synthetic reaction solution is used. The external dialysis solution can be replaced with a fresh one when the reaction rate has decreased to increase the dialysis efficiency. The reaction temperature and time are appropriately selected depending on the protein synthesis system to be used, but in the system using the wheat germ extract, it is usually 10 to 40 ° C, preferably 18 to 30 ° C, more preferably 20 to 26 ° C for 10 minutes. It can be done for up to 12 days.

When the protein synthesis is carried out using the multi-layer method, the synthetic reaction solution containing a competitive substrate is placed in an appropriate container, and the dialyzing external solution described in the above dialysis method is not disturbed on the solution. Protein synthesis is performed by stacking layers.
Specifically, for example, a competitive substrate is added to the above-mentioned synthesis reaction solution from which the translation template has been removed, pre-incubated for an appropriate period of time, then the translation template is added, and the reaction template is placed in an appropriate container for the reaction phase. Examples of the container include a microtiter plate and the like. The reaction is carried out by layering the outer dialysis solution (supply phase) described in the above dialysis method on the upper layer of this reaction phase without disturbing the interface. The reaction temperature and time are appropriately selected depending on the protein synthesis system used, but are 10 to 40 ° C., preferably 18 to 3 in the system using wheat germ extract.
0 ° C, more preferably 20 to 26 ° C, usually 10 to 1
It can be done for 7 hours.

Further, the interface between both phases does not necessarily have to be formed in a horizontal plane by the superposition, and it is possible to form a horizontal plane by centrifuging a mixed solution containing both phases. When the diameter of the circular interface between both phases is 7 mm, the volume ratio between the reaction phase and the supply phase is 1: 4 to 1: 8, but
1: 5 is preferred. The larger the interface area composed of both phases, the higher the substance exchange rate due to diffusion, and the higher the protein synthesis efficiency. Therefore, the capacity ratio of both phases changes depending on the interfacial area of both phases. The synthesis reaction is performed under static conditions at the reaction temperature,
And time are appropriately selected in the protein synthesis system used, but in the system using the wheat germ extract, the above,
It can be performed in the same manner as described in the batch method or the dialysis method. In addition, when E. coli extract is used,
It can be performed at 37 ° C.

When protein synthesis is carried out using the discontinuous gel filtration method, the synthetic reaction is carried out using a synthetic reaction solution containing a competitive substrate, and when the synthetic reaction is stopped, template RNA, amino acid, energy source, etc. are supplied. Then, the protein synthesis is performed by discharging the synthetic product and the decomposed product. Specifically, for example, a competitive substrate is added to the above-mentioned synthesis reaction solution from which the translation template has been removed, pre-incubated for an appropriate period of time, then the translation template is added, and the mixture is placed in an appropriate container and reacted. Examples of the container include a microplate and the like. Under this reaction, for example, in the case of a reaction solution containing 48% by volume of wheat germ extract, the synthesis reaction is completely stopped within 1 hour of the reaction. This is because polyribosome analysis by measuring incorporation of amino acids into proteins and sucrose density gradient centrifugation (Proc. Natl. Acad. Sci. USA., 97, 559-564 (2000))
Can be confirmed by.

The reaction solution in which the synthesis reaction has stopped is passed through a gel filtration column that has been equilibrated with a feed solution having the same composition as the external dialysate described in the above dialysis method. By keeping the filtered solution again at an appropriate reaction temperature, the synthetic reaction is restarted and the protein synthesis proceeds for several hours. Hereinafter, this reaction and gel filtration operation are repeated. The reaction temperature and time are appropriately selected depending on the protein synthesis system to be used, but in the system using the wheat germ extract, it is preferable to repeat gel filtration at 26 ° C. about every 1 hour.

The protein thus obtained by the competitive substrate method can be confirmed by a method known per se. Specifically, for example, measurement of incorporation of amino acids into proteins, separation by SDS-polyacrylamide electrophoresis and staining with Coomassie Brilliant Blue (CBB),
Autoradiography (Endo, Y. et al., J. Biot
ech., 25, 221-230 (1992); Proc. Natl. Acad. Sci. U
SA., 97, 559-564 (2000)) and the like can be used. Further, since the reaction solution thus obtained contains the target protein in a high concentration, the reaction solution is subjected to a known separation and purification method such as dialysis, ion exchange chromatography, affinity chromatography, gel filtration, etc. Due to
The target protein can be obtained.

(3) Reagent and Kit The competitive substrate of the present invention can be used as a translation enhancing reagent by itself in a cell-free protein synthesis system to increase translation efficiency. The reagent contains at least the above-mentioned competitive substrate, but may further contain a surfactant, a buffer suitable for the synthesis system, and the like. Also provided is a cell-free protein synthesis kit containing the solution excluding the translation template in the synthesis reaction solution described in (2) above and the above-mentioned translation enhancing reagent. In addition to the above, the kit of the present invention contains a cell extract for cell-free protein synthesis, RNA polymerase, a surfactant, a buffer suitable for the synthesis system, a positive control template nucleic acid, and the like. However, it is not necessary to include all these reagents, and any combination of reagents may be used as long as it is a kit that can be used for protein synthesis by the competitive substrate method.

[0033]

Example 1 Wheat germ cell-free protein synthesis system
( 1) Preparation of RNA excluding start codon (AUG) GFP gene DNA (Chiu, W. -L., Et al., Curr. Biol. 6,
325-330 (1996)) was used as a template and a polymerase chain reaction (PC) using primers having the sequences shown in SEQ ID NOs: 1 and 2 was used.
R). The resulting DNA fragment of approximately 300 bp was XhoI / Kp
Digested with nI, X of pBluescriptKS (−) (STRATAGENE)
Inserted into hoI / KpnI site. Transcription using T7 RNA Polymerase (manufactured by TAKARA) using this plasmid DNA as a template, the resulting RNA was extracted with phenol / chloroform, precipitated with ethanol, and then subjected to Nick Column (Amersham Ph
armacia Biotech). This was used in the following experiment as RNA (-AUG RNA) without the start codon. (2) Preparation of Wheat Germ Extract-Containing Liquid Hokkaido Chihoku Wheat Seeds (undisinfected) 100 per minute
Mill at a ratio of g (Fritsch: Rotor Speed Mill pulve
risette14 type), and the seeds were gently crushed at a rotation speed of 8,000 rpm. After collecting a fraction (mesh size 0.7 to 1.00 mm) containing germs capable of germination through a sieve, a mixed solution of carbon tetrachloride and cyclohexane (volume ratio = carbon tetrachloride: cyclohexane = 2.4: 1). ) Is used to collect the floating fraction containing germs with germination ability, remove the organic solvent by drying at room temperature, and remove the impurities such as seed coat mixed by blowing air at room temperature to obtain the crude embryo fraction. Obtained. From this crude germ fraction, wheat germ was visually identified and sorted using a bamboo skewer.

The obtained wheat germ fraction was suspended in distilled water at 4 ° C. and washed with an ultrasonic washing machine until the washing liquid did not become cloudy. Then, it was suspended in a 0.5% by volume solution of Nonidet (Nakarai Tectonics) P40 and washed with an ultrasonic washing machine until the washing liquid did not become cloudy to obtain a wheat germ.

Preparation of a solution containing wheat germ extract is carried out by a conventional method (Er
ickson, AH et al., (1996) Meth. In Enzymol., 9
6, 38-50). The following operations were performed at 4 ° C. First, wheat germ frozen in liquid nitrogen was pulverized in a mortar.
Extraction solvent obtained by partially modifying the method of Patterson et al. With 1 ml per 1 g of the obtained powder (final concentration of 80 mM H 2
EPES-KOH (pH 7.6), 200mM potassium acetate, 2mM magnesium acetate, 4mM calcium chloride, 0.6mM each L-type amino acid 20 kinds, 8mM disiothreotol) are added, taking care not to generate bubbles. It was stirred. Three
The supernatant obtained by centrifugation at 50,000 xg for 15 minutes was collected as an embryo extract, and the solution (40 mM HEPES-KOH (pH 7.6) as final concentration, 100 mM potassium acetate, 5 mM magnesium acetate, 0 mM each) was previously prepared. Sephadex G-25 column (Amersham Pharmacia) equilibrated with 20 mM 3 L-type amino acids and 4 mM disiothreotol
Gel filtration was performed with Biotech. The concentration of the wheat germ extract-containing solution thus obtained had an optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm of 170 to 250 (A ₂₆₀
/ A ₂₈₀ = 1.5).

(3) Protein Synthesis by Wheat Germ Cell-Free Protein Synthesis System (Batch Method) 6 μl of the solution containing wheat germ extract prepared in (2) above
25 μl of a reaction solution for protein synthesis containing 30 mM HEPES-KOH (pH 7.6), 100 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nakarai Tectonics), 0.3 mM each.
20 L-type amino acids, 4 mM disiothreotol, 1.
2 mM ATP (Wako Pure Chemical Industries, Ltd.), 0.25 mM GTP (Wako Pure Chemical Industries, Ltd.), 16 mM creatine phosphate (Wako Pure Chemical Industries, Ltd.),
1U / μl Rnase inhibiter (TAKARA), 50ng / μ
l tRNA (Moniter, R., et al., Biochim. Biophys. Ac
ta., 43, 1- (1960)), 0.4 μg / l creatine kinase (manufactured by Roche), and 2 nCi / μl ¹⁴ C-leu (manufactured by Moravec). This reaction mixture was prepared in (1) -AUG
・ Add 1,2,3,4,5,6,7μg of RNA at 26 ℃
Translation template mRNA after 5 minutes preincubation
1 μg of (ΩGFP) was added, and the mixture was further incubated for 1, 2, 4 hours. The mRNA (ΩGFP) that serves as the translation template is SP6.
Tobacco mosaic virus (TMV) omega (Ω) sequence, which is a translation initiation reaction sequence linked to a promoter sequence,
Further, using the plasmid GFP / pEU (WO01 / 27260) containing the GFP gene DNA linked to the 3'downstream as a template, transcription was performed using SP6 RNA polymerase (manufactured by TOYOBO), and the resulting RNA was phenol / Chloroform extraction and ethanol precipitation followed by Nick Column (Amersham Phar
It was used after being purified by Macia Biotech).

Reaction liquid 5 1, 2 and 4 hours after the start of the reaction
Spot μl onto filter paper and apply liquid
Chilling counter (LS6000IC: Beckman Call
(Made by Tar Co.)¹⁴Uptake of C-leu was measured. This
The results are shown in FIGS. 1 (a) and 1 (b). Figure 1 (b) is shown in (a).
The results were compared with -AUG RNA addition amount after 4 hours.
¹⁴C-leu uptake is plotted. Reaction system
-AUG · RNA present in GFP
Protein increased, and −AUG RNA was superposed on ΩGFP DNA.
Target protein by adding 0.1 times or more in quantitative ratio
The amount of syntheses increased, and when added 4 times, the ΩGFP protein
Expression level increased about 3-fold.

Example 2 Wheat germ cell-free protein synthesis system
Examination of effect of competitive substrate method in (dialysis method) Wheat germ extract-containing liquid 2 prepared in Example 1 (2)
Reaction solution for protein synthesis containing 1.6 μl (3 at final concentration)
0 mM HEPES-KOH (pH 7.8), 100 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nacalai Tectonics Co., Ltd.), 0.3 mM each L-type amino acid 20 types, 2.5 mM disioseoletol, 1.2
mM ATP (manufactured by Wako Pure Chemical Industries, Ltd.), 0.25 mM GTP (manufactured by Wako Pure Chemical Industries, Ltd.), 16 mM creatine phosphate (manufactured by Wako Pure Chemical Industries, Ltd.),
0.005% sodium azide (Wako Pure Chemical Industries) 1U
/ Μl RNase inhibiter (manufactured by TAKARA), 200ug / μl
tRNA (Moniter, R., et al., Biochim. Biophys. Act
a., 43, 1- (1960)), 0.4 μg / l creatine kinase (manufactured by Roche)). Example 1 was added to this reaction solution.
0, 48, 96, 14 of -AUG RNA prepared in (1)
4,192 μg was added, and 60 μl was placed in a dialysis cup (molecular weight cutoff 12,000D: manufactured by Daiichi Pure Chemicals Co., Ltd.), and at 26 ° C. for 5 minutes.
After pre-incubation, 24 μg of the translation template mRNA (ΩGFP) prepared in Example 1 (3), and an external dialysate (30 mM HEPES-KOH (pH 7.8) at the final concentration, respectively),
100 mM potassium acetate, 2.7 mM magnesium acetate,
0.4 mM spermidine (manufactured by Nakarai Tectonics Co., Ltd.), 20 mM each 0.3 mM L-type amino acid, 2.5 mM disiothreitol, 1.2 mM ATP (manufactured by Wako Pure Chemical Industries, Ltd.),
Incubation was performed for 24 to 108 hours against 600 μl of 0.25 mM GTP (manufactured by Wako Pure Chemical Industries, Ltd.), 16 mM creatine phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.005% sodium azide (manufactured by Wako Pure Chemical Industries, Ltd.). The external dialysate was replaced every 24 hours. Also, in the system without the addition of -AUG RNA,
A synthesis system was also performed in which 24 μg of the translation template mRNA (described in Example 1 (1)) was added after 12 hours. From the reaction solution of each synthesis system described above, 0.
25 μl was collected, separated by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by autoradiogram. The result is shown in FIG. -In the synthesis system with AUG / RNA added, the amount of target protein synthesized is
It was significantly increased as compared with the case where no mRNA was added and mRNA was added every 12 hours.

Example 3 Examination of Sequence Specificity of Competing Substrate
(No. 1) (1) Introduction of mutation into RNA excluding the start codon (AUG) -Produced in Example (1) -In order to show that the coding region of -AUG RNA is not a sequence that is not translated as a sequence, A mutation was introduced to introduce ATG into the sequence. Example 1
PCR was carried out using the plasmid DNA prepared in (1) as a template and the primers having the nucleotide sequences of SEQ ID NOs: 3 and 4 and SEQ ID NOs: 5 and 6. Using the obtained plasmid DNA as a template, T7 RNA polimerase (TAKAR
(Manufactured by Company A), and the resulting RNA is extracted with phenol / chloroform and precipitated with ethanol, and then Nick Col
Purified by umn (Amersham Pharmacia Biotech).

(2) Protein Synthesis by Wheat Germ Cell-Free Protein Synthesis System (Batch Method) 6 μm of the wheat germ extract-containing solution obtained in Example 1 (2)
l-containing reaction solution for protein synthesis (3 for each final concentration)
0 mM HEPES-KOH (pH 7.6), 100 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nacalai Tectonics, Inc.), 0.3 mM each L-type amino acid except 19 types, 4 mM disionoseo Thor,
1.2 mM ATP (manufactured by Wako Pure Chemical Industries, Ltd.), 0.25 mM GTP (manufactured by Wako Pure Chemical Industries, Ltd.), 16 mM creatine phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), 1 U / μl RNase inhibiter (manufactured by TAKARA Corporation), 50
ng / μl tRNA (Moniter, R., et al., Biochim. Bioph
ys. Acta., 43, 1 1960)), 0.4 μg / l creatine kinase (Roche), 10 nCi / μl ¹⁴ C-leu (Morabec) 25 μl. After preincubating this reaction solution at 26 ° C. for 5 minutes, 1 μg of the translation template mRNA (ΩGFP) prepared in Example 1 (3) and 4 μg of the ATG-introduced mRNA prepared in (1) above were added. The reaction solution and -AUG RNA as a control (Example 1
4 μg (described in (1)) or 4 μg of -AUG.RNA having another sequence (described in Example 3 (1)), and one to which nothing was added were prepared and incubated for 4 hours. After 4 hours from the start of the reaction, 5 μl of the reaction solution was collected and analyzed by SDS-PAGE using a 12.5% acrylamide gel. The result is shown in FIG. It was confirmed that when ATG was introduced into the sequence of RNA used as a competitive substrate in Examples 1 and 2, the protein encoded by the RNA was expressed. This indicates that the effect of the competitive substrate method is non-specific to the base sequence of RNA used as a competitive substrate.

Example 4 Examination of Sequence Specificity of Competing Substrate
(Part 2) (1) Preparation of RNA excluding Start Codon (AUG) PCR was carried out using pSP65 (GenBank Accession No. X65329) as a template and primers having the nucleotide sequences of SEQ ID NOs: 7 and 8. It was About 500 bp DNA obtained
The fragment was digested with ApaI / BamHI and pBluescriptKS (−) (STRA
(Manufactured by TAGENE) and inserted into the ApaI / BamHI site. Using this plasmid DNA as a template, T7 RNA Polymerase (TAKARA
(Manufactured by K.K.) and the resulting RNA is extracted with phenol / chloroform and precipitated with ethanol, and then Nick Col
Purified by umn (Amersham Pharmacia Biotech). This RNA is an RNA that does not contain the start codon (AUG)
It was used in the following experiments as (-AUG.RNA).

(2) Protein synthesis by wheat germ cell-free protein synthesis system (dialysis method) Wheat germ extract-containing liquid 2 prepared in Example 1 (2)
Reaction solution for protein synthesis containing 1.6 μl (3 at final concentration)
0 mM HEPES-KOH (pH 7.8), 100 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nacalai Tectonics Co., Ltd.), 0.3 mM each L-type amino acid 20 types, 2.5 mM disioseoletol, 1.2
mM ATP (manufactured by Wako Pure Chemical Industries, Ltd.), 0.25 mM GTP (manufactured by Wako Pure Chemical Industries, Ltd.), 16 mM creatine phosphate (manufactured by Wako Pure Chemical Industries, Ltd.),
0.005% sodium azide (Wako Pure Chemical Industries) 1U
/ Μl RNase inhibiter (manufactured by TAKARA), 200ug / μl
tRNA (Moniter, R., et al., Biochim. Biophys. Act
a., 43, 1- (1960)), 0.4 μg / l creatine kinase (manufactured by Roche)).

24 μg of the translation template mRNA (ΩGFP) prepared in Example 1 (3) was added to 60 μl of this reaction solution, and the mixture was placed in a dialysis cup (fraction molecular weight cutoff 12,000 kD: manufactured by Daiichi Kagaku Co.) at 26 ° C. After pre-incubating for 5 minutes, 96 μg of −AUG · RNA prepared in Example 4 (1) was added, and dialyzed external solution (30 mM HEPES-KOH (pH at each final concentration) was added.
7.8), 100 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nakarai Tectonics Co., Ltd.), 0.3 mM each of 20 types of L-type amino acids, 2.5 m
M disiothreotol, 1.2 mM ATP (manufactured by Wako Pure Chemical Industries, Ltd.), 0.25 mM GTP (manufactured by Wako Pure Chemical Industries, Ltd.), 16 mM creatine phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.005% sodium azide ( Incubated for 48 hours with 600 μl of Wako Pure Chemical Industries. The external dialysate was replaced every 24 hours.
In addition, as a control, a reaction system prepared in Example 1 (1) with the same reaction solution as described above, to which 24 μg of AUG RNA was added as a competitive substrate, prepared in Example 1 (1) -AU.
Reaction system in which 96 μg of G / RNA was added as a competitive substrate, 24 μg of translation template mRNA (ΩGFP) instead of adding a competitive substrate
The reaction was carried out under the same conditions for the synthetic system added after 12 hours.

After 36 hours of incubation, 0.25 μl was taken from the reaction solution of each of the above synthesis systems and analyzed by using 12.5% polyacrylamide gel (SDS-PAGE). The result is shown in FIG. In the synthetic system to which -AUG • RNA was added, similar protein synthesis efficiency was observed even if the nucleotide sequences were different. This indicates that the effect of the competitive substrate method is non-specific to the base sequence of RNA used as a competitive substrate.

Example 5 3′UTR chain length of translation template mRNA and
Examination of Effect of Competitive Substrate Method on Protein Synthesis Efficiency (1) Preparation of Translation Template mRNA mRNAs having 3 ′ untranslated regions of different lengths in the GFP gene were prepared by the following method. Plasmid GF containing the tobacco mosaic virus (TMV) omega (Ω) sequence, which is a translation initiation reaction sequence linked to the SP6 promoter sequence, and the GFP gene DNA linked further 3 ′ downstream
Using P / pEU (WO 01/27260) as a template, the primer having the sequence shown in SEQ ID NO: 9 as the 5'side primer and 18-3 '(SEQ ID NO: 1 as the 3'side primer)
0), 549-3 '(SEQ ID NO: 11), and 1626-.
Using the one having the sequence shown in 3 '(SEQ ID NO: 12)
PCR was performed. About 750 bp, 1280 bp, 23 obtained
A 50 bp DNA fragment was purified and SP6 RNA was prepared using this DNA as a template.
Transcribed using polymerase, the resulting mRNA is extracted with phenol / chloroform, precipitated with ethanol, and then Nick C
Purified by olumn (Amersham Pharmacia Biotech).

(2) Protein synthesis by wheat germ cell-free protein synthesis system (dialysis method) Wheat germ extract-containing liquid 2 prepared in Example 1 (2)
Reaction solution for protein synthesis containing 1.6 μl (3 at final concentration)
0 mM HEPES-KOH (pH 7.8), 100 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nacalai Tectonics Co., Ltd.), 0.3 mM each L-type amino acid 20 types, 2.5 mM disioseoletol, 1.2
mM ATP (manufactured by Wako Pure Chemical Industries, Ltd.), 0.25 mM GTP (manufactured by Wako Pure Chemical Industries, Ltd.), 16 mM creatine phosphate (manufactured by Wako Pure Chemical Industries, Ltd.),
0.005% sodium azide (Wako Pure Chemical Industries) 1U
/ Μl RNase inhibitor (TAKARA), 200ng / μl
tRNA (Moniter, R., et al., Biochim. Biophys. Act
a, 43, 1- (1960)), 0.4 μg / l creatine kinase (manufactured by Roche).

To 60 μl of this reaction solution, 96 μg of -AUG RNA prepared in Example 1 (1) was added, and the mixture was placed in a dialysis cup (fraction molecular weight cut-off 12,000 kD: manufactured by Daiichi Kagaku Co., Ltd.) to give 26
Example 5 after pre-incubation at 5 ° C for 5 minutes
M with 3'length 3'untranslated region prepared in (1)
RNA (ΩGFP) was added in an amount of 12 μg, and the dialyzed external solution (30 mM HEPES-KOH (pH 7.8), 100 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nacalai Tectonics Co., Ltd.) at each final concentration. 0.3 mM
20 types of L-type amino acids, 2.5 mM disioseoletol, 1.2 mM ATP (manufactured by Wako Pure Chemical Industries, Ltd.), 0.25 mM GTP
(Manufactured by Wako Pure Chemical Industries, Ltd.), 16 mM creatine phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.005% sodium azide (manufactured by Wako Pure Chemical Industries, Ltd.) 600 μ were incubated for 48 hours. The external dialysate was replaced every 24 hours. In addition, as a control, the same reaction solution as above was used, but instead of adding a competitive substrate, Example 1 was used.

Translation template mRNA (ΩGFP) prepared in (3)
The reaction was performed under the same conditions for the synthetic system to which 12 μg was added after 12 hours. After incubation for 24 hours, 0.25 μl was taken from the reaction solution of each synthesis system described above and analyzed by SDS-PAGE using a 12.5% polyacrylamide gel. This result is shown in FIG.
Shown in. -Synthesis system without adding AUG / RNA (column 1,
In 4 and 7), the highest protein synthesizing efficiency was found to be 1626 bp in the 3'untranslated region of the gene used as the translation template.
Although the template mRNA attached was used, in the synthetic system with added competitive substrate (columns 2, 5, 8, 3, 6, 9), even if the length of the 3'untranslated region was 549 bp, It showed high efficiency similar to that of 1626 bp.

Example 6 Examination of versatility of competitive substrate method (1) Preparation of translation template mRNA As a gene to be a translation template, luciferase (pSP-lu) was used.
c: Promega), and 4 dihydrofolate reductase
(Dhfr: GENBANK Accetion No. AAA87976) using the omega (Ω) sequence of the tobacco mosaic virus (TMV) which is the translation initiation reaction sequence linked to the SP6 promoter sequence, and the above translation template linked to the 3 ′ downstream. Using a plasmid containing the gene DNA
Transcription was performed using RNA polymerase (manufactured by TOYOBO), and the resulting RNA was extracted with phenol / chloroform and precipitated with ethanol, and then the Nick Column (Amersham Pharmacia Biot) was used.
ech) and used.

(2) Wheat germ cell-free protein synthesis system (dialysis method) for protein synthesis Wheat germ extract-containing liquid 18 prepared in Example 1 (2)
Reaction solution for protein synthesis containing μl (30 mM HE at final concentration)
PES-KOH (pH 7.8), 100 mM potassium acetate, 2.7
mM magnesium acetate, 0.4 mM spermidine (manufactured by Nakarai Tectonics), 0.3 mM each L-type amino acid 20
Type, 2.5mM disiothreotol, 1.2mM ATP
(Wako Pure Chemical Industries, Ltd.), 0.25 mM GTP (Wako Pure Chemical Industries, Ltd.),
16 mM creatine phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.005
% Sodium azide (Wako Pure Chemical Industries, Ltd.), 1U / μl RNase
inhibiter (manufactured by TAKARA), 200ug / μl tRNA (Moni
ter, R., et al., Biochim. Biophys. Acta., 43, 1-
(1960)) and 0.4 μg / l creatine kinase (manufactured by Roche).

To 50 μl of this reaction solution, 80 μg of -AUG RNA prepared in Example 1 (1) was added, and this was placed in a dialysis cup (molecular weight cutoff of 12,000 kD: manufactured by Daiichi Kagaku),
After pre-incubating at 26 ° C. for 5 minutes, the luciferase mRNA (Ωluci) prepared in Example 6 (1) above,
And 20 μg of dhfr mRNA (Ωdhfr), external dialysis solution (at a final concentration of 30 mM HEPES-KOH (pH 7.8), 1
00 mM potassium acetate, 2.7 mM magnesium acetate, 0.
4mM spermidine (manufactured by Nakarai Tectonics), 20 kinds of 0.3mM L-type amino acids, 2.5mM disiothreitol, 1.2mM ATP (manufactured by Wako Pure Chemical Industries, Ltd.), 0.25
Incubation was performed for 12 to 96 hours against 600 μl of mM GTP (manufactured by Wako Pure Chemical Industries, Ltd.), 16 mM creatine phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.005% sodium azide (manufactured by Wako Pure Chemical Industries, Ltd.). The external dialysate was replaced every 24 hours. In addition, as a control, the same reaction solution as above was used, and a reaction system in which no competitive substrate was added was also reacted under the same conditions.

After incubation for 12, 24, 48, 72, 96 hours, 0.5 μl from the reaction solution of the synthesis system using Ωluci as the template mRNA, and the reaction of the synthesis system using Ωdhfr as the translation template mRNA. 0.25 μl was collected from the solution, and SD using 12.5% polyacrylamide gel was used.
It was analyzed by S-PAGE. The result is shown in FIG. -AUG ・ RNA
In the synthetic system to which was added, the protein synthesis efficiency was also increased when luciferase and dhfr were used as the translation template mRNA. This means that the effect of the competitive substrate method is
It indicates that the nucleotide sequence of the RNA used as the translation template is non-specific, or that the nucleotide sequence of the mRNA used as the translation template and the nucleotide sequence of the competitive substrate RNA do not need to contain the same portion.

Example 7 Rabbit reticulocyte cell-free protein
Examination of Effect of Competitive Substrate Method in Synthesis System (Part 1) ΩGF described in Example 1 (3) as a translation template mRNA using Rabbit Reticulocyte Lysate System (Promega)
1 μg of P was added, and 0, 1, 2, 3, 4, 5, 6 μg of -AUG RNA prepared in Example 1 (1) was used as a competitive substrate.
Add ¹⁴ C to a final concentration of 2 nCi / μl.
-Leu (manufactured by Moravec) was added, and the mixture was incubated at 30 ° C for 2 hours. 5 μl of this reaction solution was spotted on a filter paper, and ¹⁴ C-leu uptake was measured by a solid support method using a liquid scintillation counter (LS6000IC: manufactured by Beckman Coulter, Inc.). This result is shown in FIG.
Shown in (a) and (b). FIG. 7B shows the result shown in FIG.
¹⁴ is a plot of the amount of ^{14 A} -leu uptake after 4 hours with respect to the amount of -AUG RNA added. The effect of the competitive substrate method was also confirmed in the rabbit reticulocyte cell-free protein synthesis system, and it was shown that the effect was highest when the competitive substrate RNA was added 3 to 4 times the translation template mRNA.

Example 8 Rabbit reticulocyte cell-free protein
Examination of Effect of Competitive Substrate Method in Synthetic System (Part 2) In Example 7, a translation template mRNA having no cap structure was used. Here, however, mRNA having a cap structure was used as a translation template. The effect of the competitive substrate method was analyzed and examined. (1) Preparation of translation template mRNA having Cap structure m7 was added to the translation template mRNA (ΩGFP) prepared in Example 1 (3).
G (5 ') ppp (5') G cap analog (New England Bila
b) was added, and the resulting capped mRNA was added to phenol /
After chloroform extraction and ethanol precipitation, Nick Colum
n (Amersham Pharmacia Biotech). (2) Protein synthesis by rabbit reticulocyte cell-free protein synthesis system Using the Rabbit Reticulocyte Lysate System (Promega), 5'ca prepared in (1) above as a translation template mRNA.
1 μg of ΩGFP having a p structure was added, and 0- 1, 2-AUG RNA prepared in Example 1 (1) was used as a competitive substrate.
Add 3, 4, 5, and 6 μg, and further add 2 nCi / μ at the final concentration.
Add ¹⁴ C-leu (made by Moravec Co.) to give l
Incubation was carried out at 0 ° C for 2 hours. This reaction liquid 5
The μl was spotted on a filter paper, and ¹⁴ C-leu uptake was measured by a solid support method using a liquid scintillation counter (LS6000IC: manufactured by Beckman Coulter, Inc.). The results are shown in FIGS. 8 (a) and 8 (b). FIG. 8 (b) shows the results shown in FIG. 8 (a) for the amount of -AUG RNA added after 2 hours.
¹⁴ is a plot of ¹⁴ C-leu incorporation. 5'cap in rabbit reticulocyte cell-free protein synthesis system
The effect of the competitive substrate method was confirmed even when a structural mRNA was used as a translation template.
It was shown that the highest was obtained when the competitive substrate RNA was added 3 to 4 times to NA.

[0055]

INDUSTRIAL APPLICABILITY According to the present invention, the stability of a nucleic acid serving as a translation template is enhanced in a cell-free protein synthesis system, and as a result, the amount of target protein synthesized is significantly increased.
This greatly improves the applicability of the cell-free protein synthesis system to industry.

[0056]

[Sequence list]                                SEQUENCE LISTING        <110> Endo, Yaeta       Mitsubishi chemical corporation <120> Method for obtaining proteins in cell-free protein synthesis s ystem <160> 10 <170> PatentIn Ver. 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 1 tcctcgaggc ccgaaggc 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 2 caggtacccg cgcttctc 18 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 3 gctctagaac taatggatcc cccg 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 4 ggccgccacc gcggtggagc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 5 gaatgggccc acgcgcgggg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 6 taccggatcc ggcgcagcgg 20 <210> 7 <211> 4 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 20 cgatttaggt gacactatag 20 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 8 atccggatcc agccgtaaat tctatac 27 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 9 ccctcgaggc gtgggcccca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized <400> 10 agcgtcagac cccgtagaaa 20

[Brief description of drawings]

FIG. 1 is a diagram showing the correlation between the amount of competitive substrate present and the amount of target protein synthesized in a wheat embryo cell-free protein synthesis system (batch method).

FIG. 2 shows the amount of target protein synthesized when the amount of competing substrate was changed and the amount of target protein synthesized when a translation template was added in the wheat embryo cell-free protein synthesis system (dialysis method). It is a figure.

FIG. 3 is a schematic diagram of a wheat embryo cell-free protein synthesis system (batch method) in which ATG-introduced RNA is used as a template.
It is a figure which showed the synthetic amount of the protein which RNA codes.

FIG. 4 is a diagram showing a synthetic amount of a target protein when AUG-free RNA having a different base sequence is present in a wheat embryo cell-free protein synthesis system (dialysis method).

FIG. 5 is a diagram showing the amount of target protein synthesized when a competitive substrate is present in a translation template having different lengths of 3 ′ untranslated regions in a wheat embryo cell-free protein synthesis system (dialysis method).

FIG. 6 is a diagram showing the amount of each target protein synthesized when the same competing substrate is present in two different translation templates in the wheat embryo cell-free protein synthesis system (dialysis method).

FIG. 7 is a diagram showing the correlation between the amount of competitive substrate present and the amount of target protein synthesized in a rabbit reticulocyte cell-free protein synthesis system.

FIG. 8 is a diagram showing a correlation between the amount of competitive substrate present and the amount of target protein synthesized in the case of using RNA having a cap structure as a translation template in a rabbit reticulocyte cell-free protein synthesis system.

Claims (9)

[Claims] 1. A method for synthesizing a target protein in a cell-free protein synthesis system, which serves as a substrate for a nucleolytic enzyme, but does not encode the target protein, and is transcribed from the template of the target protein in the synthesis system. / A method comprising synthesizing a target protein in the presence of a substance having a low translation template activity. 2. The method according to claim 1, wherein the substance serving as a substrate for the nucleic acid degrading enzyme is a nucleic acid or a derivative thereof. 3. The method according to claim 2, wherein the number of translatable bases present in the base sequence of the nucleic acid or its derivative is 30 amino acids or less. 4. The method according to claim 2 or 3, wherein the nucleic acid or its derivative does not contain a start codon in its base sequence. 5. The method according to claim 2, wherein the cell-free protein synthesis system is a translation system, and the nucleic acid or its derivative is RNA. 6. The method according to claim 2, wherein the cell-free protein synthesis system is a transcription / translation system, and the nucleic acid or its derivative is DNA or RNA. 7. The abundance of a substance serving as a substrate for a nucleolytic enzyme is 0.1 by weight ratio of a nucleic acid encoding a target protein.
7. The method according to any one of claims 1 to 6, which is more than double. 8. A reagent containing a substance used in a method for synthesizing a target protein in a cell-free protein synthesis system, the substance being a substrate for a nucleolytic enzyme, which does not encode the target protein, and A reagent, which is a substance having a transcription / translation template activity lower than that of a template of a target protein in the synthesis system. 9. A kit for carrying out the method according to any one of claims 1 to 7, which comprises at least a cell-free protein synthesis reaction solution and the reagent according to claim 9.