JP4243762B2 - Preparation method of cell-free protein synthesis extract and silkworm tissue extraction kit - Google Patents

Description

  The present invention relates to a method for preparing an extract for cell-free protein synthesis and an extraction kit for silkworm tissue.

  In recent years, genetic information of many organisms including the human genome has been decoded. Under such circumstances, functional analysis of proteins corresponding to these genetic information and genome drug discovery are attracting attention as post-genome research. In order to apply and use proteins corresponding to these genetic information in medicines, it is necessary to synthesize huge types of proteins easily in a short time.

  At present, expression systems using living cells such as yeast and insect cells (hereinafter sometimes referred to as “cell systems”) are widely used as protein production methods by genetic recombination techniques. However, living cells tend to exclude foreign proteins in order to maintain self-function, and there are many proteins that are difficult to express, such as when cells express no cytotoxic proteins in living cells.

  On the other hand, as a protein production method that does not use a cell system, a genetic information translation system for organisms is prepared in a test tube by adding a substrate or an enzyme to a cell lysate or extract, and mRNA that encodes the target protein is used. Thus, cell-free protein synthesis is known in which a synthetic system capable of combining amino acids with the required number of residues in the desired order is reconstructed. Such cell-free protein synthesis is less subject to the limitations of the above-mentioned cell-based protein synthesis, and can synthesize proteins without losing the lives of living organisms. Since it is not accompanied, protein synthesis can be performed in a short time compared with the cell system. Furthermore, cell-free protein synthesis is expected to be a promising expression method because it enables mass production of proteins consisting of amino acid sequences that are not utilized by living organisms. As such cell-free protein synthesis, for example, a method using an extract of wheat germ or an extract of Escherichia coli is known.

However, cell-free protein synthesis using an extract of wheat germ has a drawback that the extraction operation of the extract is generally very complicated.
As an example of a method for preparing an extract of wheat germ, for example, Patent Document 1 describes the following procedure. After adding wheat seeds to the mill and crushing, the crude germ fraction is obtained with a sieve, and the germination ability is increased by flotation using a mixture of carbon tetrachloride and cyclohexane (carbon tetrachloride: cyclohexane = 2.5: 1). The germs that have them are collected from the floating fraction, and the organic solvent is removed by drying at room temperature. Impurities such as seed coat mixed in the embryo fraction are adsorbed and removed using an electrostatically charged body. Next, in order to completely remove the wheat endosperm components from this sample, it is suspended in a 0.5% solution of NP40, which is a nonionic surfactant, and washed using an ultrasonic cleaner until the cleaning solution does not become cloudy. repeat. Ultrasonic cleaning is performed once again in the presence of distilled water to purify the wheat germ.
As described above, cell-free protein synthesis using an extract of wheat germ has a problem that the preparation of the extract is complicated and requires a lot of time and labor.

In addition, cell-free protein synthesis using an E. coli extract has the disadvantage that since E. coli is a prokaryotic organism, sugar chain modification to the protein cannot be performed and glycoproteins cannot be synthesized. The sugar chain added to the protein by the above sugar chain modification functions as a signal or ligand involved in recognition and adhesion between substances or cells, as a function regulator of the protein itself, or as a protein protection or stabilization factor. It is thought that. Therefore, in order to analyze the in vivo function of a protein that undergoes sugar chain modification, it is necessary to obtain a protein that has undergone sugar chain modification (glycoprotein). Cell-free protein synthesis that can be performed is desired.
In addition, it is desired that the efficiency of protein synthesis in a cell-free system can be improved as much as possible, and a method for preparing such an extract for cell-free protein synthesis and the development of an extraction solution have also been developed. It is desired.
JP 2000-236896 A

  The present invention has been made to solve the above-mentioned problems, and the object of the present invention is to provide a method for preparing an extract for cell-free protein synthesis that can improve the efficiency of protein synthesis compared to conventional methods. is there.

As a result of intensive studies to solve the above problems, the present inventors have completed the present invention. That is, the present invention is as follows.
(1) A method for preparing an extract for cell-free protein synthesis, wherein extraction from a silkworm tissue is performed using an extraction solution containing a polyhydric alcohol.
(2) The above (1), wherein the polyhydric alcohol is at least one selected from glycerol, diglycerol, ethylene glycol, polyerythritol, erythritol, xylitol, sorbitol, trehalose, sucrose, lactose, xylose, maltose, and inositol. ) Method.
(3) The supernatant obtained by centrifugation after extraction is mixed with the supernatant obtained by further centrifuging after extraction from the precipitate after centrifugation using the above extraction solution. The method according to (1) or (2) above, wherein an extract is prepared.
(4) An extraction kit for an extract for cell-free protein synthesis derived from a silkworm tissue having an extraction solution containing a polyhydric alcohol.
(5) The above (4), wherein the polyhydric alcohol is at least one selected from glycerol, diglycerol, ethylene glycol, polyerythritol, erythritol, xylitol, sorbitol, trehalose, sucrose, lactose, xylose, maltose, and inositol. ) Kit.

In the present specification, “silkworm” is synonymous with lepidopterous insects (silk insects) belonging to the family Bombycidae. In its lifetime, “egg (embryo)” (from just after spawning to just before hatching), “larva” ( Immediately after hatching, immediately before the end of wing formation (divided into 1st to 5th stage)), 蛹 (from the time immediately before wing formation to just before emergence), and "adult (wing)" (immediately after emergence) (Until death), and includes all of its life-long forms.
In the state of larvae after hatching from eggs, silkworms alternate between the period of growing by eating mulberry (age) and the period of preparing for molting without eating (sleeping). In the silkworm larvae, the period from hatching to the first molting is called the 1st instar period, and the period from the 1st molting to the 2nd molting is called the 2nd instar stage. This mature silkworm larva is also called "ripe". Silkworm larvae become transparent when they become mature and spit out silk to form cocoons and hatch. After the pupae, they emerge and become adults.

  The “silk gland” in the present specification is a pair of tubular exocrine glands that continue from the discharge port located at the tip of the lower lip of the head to the blind tube on both sides of the silkworm larvae. It is roughly divided into glands and posterior silk glands. The posterior silk gland secretes fibroin, which forms the center of the silk thread. The middle silk gland also secretes sericin. Fibroin accumulates in the middle silk gland, and its outer periphery is covered with sericin to form a gel-like silk substance. This silk substance is discharged from the outlet through the front silk gland and solidifies into a silk thread.

  In the present specification, the “fatty body” is a white soft flat band-like, string-like or leaf-like tissue that is distributed throughout the body in the silkworm larva. The fat body plays a role of storing nutrients and energy sources similar to the human liver, and therefore contains fat globules, proteins, glycogen, and other substances involved in metabolism in the cells.

  As used herein, “embryo” refers to a tissue in a silkworm egg state.

  In the present specification, “cell-free protein synthesis” refers to protein synthesis (translation system) only by a cell-free translation system that reads mRNA information and synthesizes the protein, and a transcription step that transcribes mRNA from foreign template DNA, Any of protein synthesis (transcription / translation system) including a translation step of reading the information of mRNA obtained in the transcription step to synthesize a protein. Here, the “protein” synthesized in a cell-free system by the synthesis method of the present invention encompasses any molecular weight peptide composed of a plurality of amino acid residues, that is, any of low molecular weight peptides to high molecular weights. Shall. The “protein” as used herein also includes glycoproteins that are modified with a sugar chain.

  ADVANTAGE OF THE INVENTION According to this invention, the preparation method of the extract for cell-free protein synthesis which can improve the synthetic | combination efficiency of protein conventionally, and the kit for silkworm tissue extraction for that can be provided.

Hereinafter, the present invention will be described in detail.
The present invention is a method for preparing an extract for cell-free protein synthesis, wherein extraction from a silkworm tissue is performed using an extraction solution containing a polyhydric alcohol. By using a cell-free protein synthesis reaction using an extract containing a silkworm tissue-derived extract prepared using such an extract, the extract was prepared using an extract containing no polyhydric alcohol. Compared with the case where a cell-free protein synthesis reaction is performed using an extract containing an extract derived from silkworm tissue, the amount of protein synthesis is significantly improved. Although the details are unknown, protein synthesis is because the components involved in protein synthesis are efficiently extracted and stabilized, and the viscosity of the extract itself is reduced by containing polyhydric alcohol. This is thought to be because the reaction is likely to proceed.

The polyhydric alcohol contained in the extraction liquid used in the method of the present invention is not particularly limited as long as it is an alcohol having two or more hydroxyl groups in the same molecule, but is the same from the viewpoint of protein synthesis efficiency and handleability. Those having 2 to 50 hydroxyl groups in the molecule are preferred, and those having 2 to 30 hydroxyl groups in the same molecule are more preferred.
Examples of the polyhydric alcohol that can be used in the method of the present invention include glycerol, diglycerol, polyglycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, polyvinyl alcohol, erythritol, polyerythritol, xylitol, glucose, mannose, and galactose. , Fructose, ribose, inositol, sucrose, lactose, maltose, cellobiose, sorbitol, trehalose, xylose, etc. , Trehalose, sucrose, lactose, xylose, maltose and At least one selected from among the inositol is preferred.

  Among the polyhydric alcohols, those other than polyerythritol can be produced by various conventionally known methods, and commercially available ones may be used as appropriate. Polyerythritol is a dimer to hexamer of erythritol originally developed by one of the present applicants. A production method of tetramer polyerythritol is illustrated in Production Example 1 described later. Polyerythritol other than the tetramer can be appropriately produced by those skilled in the art based on the description in Production Example 1.

  From the viewpoint of protein synthesis efficiency, the concentration of polyhydric alcohol in the extraction liquid used in the present invention (in the case of a mixture, the total concentration thereof) is 1 (v / v)% to 60 (v / v)%. It is preferable that it is 2 (v / v)%-50 (v / v)%.

  The extraction solution used in the method of the present invention is not particularly limited as long as it contains a polyhydric alcohol as described above, but preferably further contains a protease inhibitor. When an extraction solution containing a protease inhibitor is used, the activity of the protease contained in the silkworm tissue-derived extract is inhibited, and unwanted decomposition of the active protein in the extract by the protease can be prevented. There is an advantage that the protein synthesis ability of the tissue-derived extract can be effectively extracted.

  The protease inhibitor is not particularly limited as long as it can inhibit the activity of the protease. For example, phenylmethanesulfonyl fluoride (hereinafter sometimes referred to as “PMSF”), aprotinin, bestatin, leupeptin, pepstatin A, E-64 (L-trans-epoxysuccinyl leucylamide-4-guanidinobutane), ethylenediaminetetraacetic acid, phosphoramidon, etc. can be used, but the extract after extraction from silkworm tissue has serine protease activity. Therefore, it is preferable to use PMSF having high specificity for serine protease. Further, not only one type of protease inhibitor but also several types of mixtures (protease inhibitor cocktail) may be used.

  The content of the protease inhibitor in the extraction solution is not particularly limited, but is preferably 1 μM to 50 mM from the viewpoint of suitably exhibiting the ability to inhibit the degradation of enzymes essential for the action of the present invention. More preferably, it is contained in an amount of 0.01 mM to 5 mM. This is because if the protease inhibitor is less than 1 μM, the protease degradation activity tends not to be sufficiently suppressed, and if the protease inhibitor exceeds 50 mM, the protein synthesis reaction tends to be inhibited.

  The extraction solution used in the present invention further contains a potassium salt, a magnesium salt, dithiothreitol (hereinafter sometimes referred to as “DTT”) and a buffer in addition to the polyhydric alcohol and protease inhibitor. Is preferred.

  The potassium salt is not particularly limited as long as it does not inhibit the action of the present invention. For example, potassium acetate, potassium carbonate, potassium hydrogen carbonate, potassium chloride, dipotassium hydrogen phosphate, dipotassium hydrogen citrate, It can be used in a general form such as potassium sulfate, potassium dihydrogen phosphate, potassium iodide, potassium phthalate, etc. Among them, potassium acetate is preferably used. Potassium salts act as cofactors in protein synthesis reactions.

  Although there is no restriction | limiting in particular in content of the potassium salt in the said liquid for extraction, From a viewpoint of storage stability, when it is monovalent potassium salts, such as potassium acetate, for example, it is preferable that 10 mM-500 mM are contained, 50 mM- More preferably, it contains 200 mM. This is because if the potassium salt is less than 10 mM or more than 500 mM, components essential for protein synthesis tend to become unstable.

  The magnesium salt is not particularly limited as long as it does not inhibit the action of the present invention. For example, magnesium acetate, magnesium sulfate, magnesium chloride, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, It can be used in a general form such as magnesium nitrate and magnesium oxalate, and it is preferable to use magnesium acetate. Magnesium salts also act as cofactors in protein synthesis reactions.

  Although there is no restriction | limiting in particular in content of the magnesium salt in the said liquid for extraction, From a viewpoint of storage stability, when it is bivalent salt, such as magnesium acetate, it is preferable that 0.1-10 mM is contained, More preferably, 5 mM to 5 mM is contained. This is because when the magnesium salt is less than 0.1 mM or more than 10 mM, components essential for protein synthesis tend to become unstable.

  The DTT is blended for the purpose of preventing oxidation, and is preferably contained in the extraction liquid at a concentration of 0.1 mM to 10 mM, more preferably 0.5 mM to 5 mM. This is because when DTT is less than 0.1 mM or more than 10 mM, components essential for protein synthesis tend to become unstable.

The buffering agent is blended for the purpose of imparting buffering capacity to the extraction solution and preventing the denaturation of the extract due to a rapid change in pH caused by the addition of an acidic or basic substance. Such a buffer is not particularly limited, and for example, HEPES-KOH, Tris-HCl, acetic acid-sodium acetate, citric acid-sodium citrate, phosphoric acid, boric acid, MES, PIPES, etc. may be used. it can.
It is preferable to use a buffering agent whose pH of the extraction liquid is maintained at 4 to 10, and more preferably a buffering agent whose pH is maintained at 6 to 8. This is because if the pH of the extraction liquid is less than 4 or exceeds 10, the components essential for the reaction of the present invention may be denatured. From such a viewpoint, it is particularly preferable to use HEPES-KOH (pH 6 to 8) among the above.

  Although there is no restriction | limiting in particular in content of the buffer in the said extract, From a viewpoint of hold | maintaining suitable buffer capacity, it is preferable to contain 5 mM-200 mM, and it is more preferable that 10 mM-50 mM are contained. This is because when the buffer is less than 5 mM, the pH tends to fluctuate due to the addition of an acidic or basic substance, and the extract tends to denature in the extract after preparation, and the buffer exceeds 200 mM. This is because the salt concentration becomes too high and components essential for protein synthesis tend to become unstable.

  That is, the extraction liquid used in the present invention contains 2 (v / v)% to 50 (v / v)% of the polyhydric alcohol, 0.01 mM to 5 mM PMSF, and 50 mM to 200 mM potassium acetate. It is preferably realized to contain 0.5 mM to 5 mM magnesium acetate, 0.5 mM to 5 mM DTT, 10 mM to 50 mM HEPES-KOH (pH 6 to 8).

The silkworm tissue to be extracted in the present invention may be any tissue in any state (egg, larva (1st to 5th instar), pupa, adult) during the lifetime of the silkworm. The silkworm tissue is not limited to a single tissue in a single state (for example, only the posterior silk gland in the silkworm larvae at the age of 5 years), but a plurality of tissues in the single state (eg, silkworm larvae at the age of 5 years of age). Posterior silk gland and fat body), or a single tissue in a plurality of states (eg, posterior silk gland in each silkworm larvae in 3rd, 4th, and 5th stages) Shall. Of course, it may be a plurality of organizations in a plurality of states.
The silkworm tissue need not be the entire silkworm tissue (for example, the entire posterior silk gland).

  The silkworm tissue is preferably at least one selected from the posterior silk gland of the silkworm larva, the fat body, and the silkworm embryo. Whether or not the extract prepared by the method of the present invention contains an extract derived from at least one of silkworm larvae-derived posterior silk gland, fat body and silkworm embryo is determined by, for example, an isozyme for aldolase It can be discriminated by performing an analysis (Nagaoka et al. (1995), Insect Biochem Mol Biol. 25, 819-825).

  Preparation by the method of the present invention from the posterior silk gland of the silkworm larvae is preferable because an extract for cell-free protein synthesis having particularly excellent advantages capable of synthesizing a large amount of protein in a short time can be obtained.

Further, it is preferable to prepare from the fat body of the silkworm larva by the method of the present invention, because the fat body is a soft tissue, the work of grinding is completed in a short time, and as a result, the extract can be easily prepared.
In addition to the above isozyme analysis, the 5-year-old fat pad is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in addition to the above isozyme analysis, such as SP-1, SP-2 which are fat pad-derived proteins. Whether or not it is contained in the extract prepared by the method of the present invention can also be determined.

Preparation from silkworm embryos by the method of the present invention is preferable because the embryo is a single individual and, unlike other tissues, does not require excision and can easily prepare an extract.
For silkworm embryos, in addition to the above isozyme analysis, the extract is subjected to SDS-PAGE to detect embryo-derived proteins such as 30K, ESP, Vitellin (H), Vitellin (L), etc. It can be determined whether or not it is contained in the extract prepared by the method.

In the case of silkworm larvae posterior silk gland or fat body, silkworm larvae can be prepared by the method of the present invention without any particular limitation as long as they are from the 1st to 5th instar stages of silkworm larvae. It is preferably derived from silkworm larvae at the age of 5 years. In the silkworm larvae at the 5th instar stage, the posterior silk gland and the fat body are most matured among the 1st to 5th instar stages. This is because an extract for cell-free protein synthesis capable of synthesizing a large amount of protein in a time can be obtained.
In particular, from the viewpoint of actively producing silk fibroin, which is the main component of silk thread, and having a high protein synthesis ability, the silk gland of the silkworm larvae at the 5th instar stage, especially 3-7 days in the 5th instar stage. It is preferable to perform extraction from the posterior silk gland of the silkworm larvae.

  In order to obtain an extract containing a desired amount of the above extract, it is usually necessary to extract from a plurality of silkworms. The number of silkworms used for extraction varies depending on the condition of silkworms used and individual differences, but for silkworm larvae, it is necessary to obtain the same amount of extract as the tissue matures as it approaches the stage of silkworm formation. The number is small. In particular, since the silk gland matures significantly every day in the 5th instar silkworm larvae, for example, the same amount as about 30 silkworm larvae in 1st day in the 5th infancy, Then, it can be obtained from about 6 to 7 silkworm larvae.

In the method for preparing an extract for cell-free protein synthesis of the present invention, other procedures are not particularly limited as long as extraction is performed from a silkworm tissue using an extraction solution containing a polyhydric alcohol. However, it can be performed by the following procedure, for example.
First, according to a conventional method, a desired tissue is extracted from a silkworm using instruments such as scissors, tweezers, and a scalpel. Although there is no restriction | limiting in particular as a tissue quantity used for the below-mentioned extraction obtained by this extraction, Usually, it exists in the range of 1g-100g.
Next, the extracted tissue is frozen with liquid nitrogen and then ground using a mortar previously cooled with liquid nitrogen. Add the extraction liquid to this, freeze the extraction liquid once, and then dissolve with stirring with a spoonful until it becomes a sherbet (specifically, until it becomes wet yellow crispy ice) . Then, after completely freezing again with liquid nitrogen, until it becomes a sherbet (specifically, until it becomes wet yellow crispy ice), it is melted while stirring with a chemical spoon. Extraction from silkworm tissue.
In this extraction operation, the extracted silkworm tissue may be frozen in, for example, liquid nitrogen and then ground in a mortar frozen at −80 ° C., and an extraction liquid may be added thereto for extraction. By performing the extraction operation as described above, a polyhydric alcohol-containing extraction solution with low viscosity is used, so that the protein synthesis reaction is likely to proceed, and the components involved in protein synthesis are efficient. This has the advantage of being extracted and stabilized.

Next, the liquid obtained by the extraction process is centrifuged. The centrifugation is carried out under conditions usually performed in the art (10000 × g to 50000 × g, 0 ° C. to 10 ° C., 10 minutes to 60 minutes). In the preparation method of the present invention, the supernatant after one centrifugation may be used as an extract as it is, but preferably the supernatant (first supernatant) and the above-mentioned centrifugation (first time) After further extraction using the above-mentioned extraction liquid from the precipitate after the centrifugation), and then mixing with the supernatant (second supernatant) obtained by centrifugation (second centrifugation), It is preferable to prepare it as an extract. The second centrifugation may be performed under the same conditions as the first centrifugation described above. By preparing the extract by mixing the first supernatant and the second supernatant in this way, compared with the case where the first supernatant and the second supernatant are used as the extract alone. There is an advantage that the protein synthesis efficiency is improved. This is because the components involved in the protein synthesis reaction are present in a large amount not only in the first supernatant but also in the second supernatant. It is thought that it is because the component which cannot be supplemented can be complemented.
In addition, the supernatant (third supernatant) obtained by further centrifuging the first supernatant (same conditions as described above) may be mixed with the second supernatant. Then, the said effect is further strengthened and is more preferable. This is considered to be because impurities contained in the first supernatant that could not be removed by one centrifugation can be removed. In addition, you may make it prepare the extract by mixing the said 1st-3rd supernatant.

  In this case, there is no particular limitation on the mixing ratio of the first supernatant and / or the third supernatant (total amount when both are mixed) and the second supernatant in the prepared mixture (extract). However, from the viewpoint of protein synthesis efficiency, the volume ratio is preferably 10:90 to 90:10, and more preferably 20:80 to 80:20.

  In order to purify the first to third supernatants and the mixture, they are subjected to gel filtration, and the vicinity of the fraction having the highest absorbance at 280 nm is extracted from the filtrate after gel filtration. Although it may be prepared as a liquid, from the viewpoint of protein synthesis efficiency, it is preferably prepared as an extract without undergoing gel filtration and fractionation. This is considered to be because the components necessary for the reaction are reduced by the gel filtration and fraction fractionation.

In addition, what is necessary is just to perform in the following procedures, when performing the said gel filtration and fractionating the vicinity of a fraction with the highest light absorbency in 280 nm from the filtrate after gel filtration.
For example, gel filtration is performed using a desalting column PD-10 (manufactured by Amersham Biosciences), and the column is equilibrated with a gel filtration buffer according to a conventional method. Elution is performed under such conditions as elution. The gel filtration buffer is preferably a solution obtained by further adding glycerol to the extraction solution. Glycerol is usually added so as to be 5 (v / v)% to 40 (v / v)% (preferably 20 (v / v)%). The filtrate obtained by gel filtration should be 0.1 mL to 1 mL as one fraction, as is done by normal gel filtration, and efficiently fractions having a high protein synthesis ability. From a viewpoint, it is preferable to make 0.4 mL-0.6 mL into 1 fraction.
Next, a fraction having an absorbance at 280 nm of 10 or more is collected from the filtrate after gel filtration. In the treatment, for example, the absorbance at 280 nm is measured for each fraction using an instrument such as Ultraspec 3300pro (manufactured by Amersham Bioscience), and the vicinity of the fraction having the highest absorbance is fractionated.

  The present invention also provides an extraction kit for silkworm tissue having an extraction liquid containing the polyhydric alcohol described above. The types, concentrations, and other suitable compositions of the suitable polyhydric alcohol in the extraction liquid in the silkworm tissue extraction kit are as described above. The silkworm tissue extraction kit is not particularly limited as long as it is composed of an appropriate container containing the extraction liquid and other appropriate elements. There are no particular restrictions on other components other than the extraction liquid, but for example, scissors, tweezers, and scalpels used for extracting silkworm tissue, and other silkworm dissection tables, tissue washing liquid, and the like may be included. .

As described above, by performing a protein synthesis reaction using the extract obtained by the method of the present invention, any protein, for example, a protein that becomes a cytotoxic agent in living cells, can be synthesized in a short time. Is possible. In addition, because it uses an extract derived from the tissue of the silkworm, a eukaryote, it is possible to synthesize glycoproteins in a cell-free system and to synthesize many types of proteins without any particular restrictions. Can do. The protein synthesis reaction described above is obtained by a protein synthesis reaction (translation system synthesis reaction) using only a cell-free translation system that reads mRNA information and synthesizes a protein, a transcription step that transcribes mRNA from a foreign template DNA, and the transcription step. Any of the protein synthesis reactions (transcription / translation system synthesis reaction) including a translation step of synthesizing the protein by reading the information of the obtained mRNA.
Further, as described later, such an extract can be prepared much more easily as an extract that can be used for cell-free protein synthesis as compared with the conventional preparation of an extract from wheat germ. Efficient cell-free protein synthesis.

  The content of the silkworm tissue-derived extract in the extract obtained by the method of the present invention is not particularly limited, but the protein concentration is preferably 1 mg / mL to 200 mg / mL, and more preferably 10 mg / mL to 100 mg. More preferably, it is / mL. This is because if the content of the extract is less than 1 mg / mL in terms of protein concentration, the concentration of components essential for the action of the present invention will be low, and a sufficient synthesis reaction may not be possible. This is because if the content of the protein exceeds 200 mg / mL in protein concentration, the extract itself has a high viscosity and may be difficult to operate.

  An extract containing the silkworm tissue-derived extract in an amount within the above range can be prepared by measuring the protein concentration of the extract. The protein concentration is measured using, for example, BCA Protein assay Kit (manufactured by PIERCE), 0.1 mL of the sample is added to 2 mL of the reaction reagent, and the reaction is performed at 37 ° C. for 30 minutes. And measuring the absorbance at 562 nm. As a control, bovine serum albumin (BSA) is usually used.

Reaction solutions prepared for carrying out the above-mentioned translation system synthesis reaction and transcription / translation system synthesis reaction using the extract obtained by the method of the present invention (respectively “translation system reaction solution”, “transcription / translation system use”) The composition of the “reaction solution” is not particularly limited, and a conventionally known composition may be appropriately selected.
In either case of the translation system reaction solution or the transcription / translation system reaction solution, the extract obtained by the method of the present invention is 10 (v / v)% to 80 (v / v)%, particularly Is preferably prepared so as to contain 30 (v / v)% to 60 (v / v)%. That is, it is preferable that the content of the silkworm tissue-derived extract is preferably 0.1 mg / mL to 160 mg / mL in terms of protein concentration in the whole reaction solution, and 3 mg / mL to 60 mg / mL. More preferably, it is prepared as follows. This is because when the content of the extract is less than 0.1 mg / mL or exceeds 160 mg / mL in protein concentration, the protein synthesis rate tends to decrease.
Hereinafter, (1) the translation system reaction solution and (2) the transcription / translation system reaction solution will be described.

(1) Translation system reaction solution The translation system reaction solution is a component excluding the extract obtained by the method of the present invention, exogenous mRNA, potassium salt, magnesium salt, DTT, adenosine triphosphate, guanosine triphosphate. Creatine phosphate, creatine kinase, amino acid component, RNase inhibitor, tRNA, and buffer. By carrying out a translation system synthesis reaction using such a translation system reaction solution, a large amount of protein can be synthesized in a short time.

  The foreign mRNA used in the reaction solution for translation system refers to mRNA that does not originate from silkworm tissue. If it is mRNA that does not originate from silkworm tissue, there is no particular limitation on the encoded protein (including peptides) and it is toxic. It may be one that encodes a protein or one that encodes a glycoprotein. Whether mRNA contained in the reaction solution for translation system is foreign mRNA or mRNA derived from silkworm tissue is determined by first isolating and purifying mRNA from the reaction solution for translation system and then using reverse transcriptase. It can be determined by synthesizing cDNA, analyzing the base sequence of the obtained cDNA, and comparing it with the base sequence of a known foreign mRNA.

  The foreign mRNA used in the reaction solution for translation system is not particularly limited in the number of bases, and all mRNAs may not have the same number of bases as long as the target protein can be synthesized. In addition, each mRNA may have a plurality of bases deleted, substituted, inserted or added as long as the sequences are homologous enough to synthesize the target protein.

  In the translation reaction solution, the foreign mRNA is preferably contained at 1 μg / mL to 1000 μg / mL, more preferably 10 μg / mL to 500 μg / mL, from the viewpoint of the rate of protein synthesis. This is because if the mRNA is less than 1 μg / mL or exceeds 1000 μg / mL, the rate of protein synthesis tends to decrease.

  As the potassium salt in the translation reaction solution, the various potassium salts described above as the components of the extraction solution, preferably potassium acetate, can be preferably used. From the same viewpoint as the case of the potassium salt in the extraction solution described above, the potassium salt is preferably contained in the translation reaction solution in an amount of 10 mM to 500 mM, more preferably 50 mM to 150 mM.

  As the magnesium salt in the translation reaction solution, the above-mentioned various magnesium salts, preferably magnesium acetate, can be preferably used as components of the extraction solution. From the same viewpoint as the case of the magnesium salt in the extraction solution described above, the magnesium salt is preferably contained in the translation system reaction solution in an amount of 0.1 mM to 10 mM, and preferably 0.5 mM to 3 mM. Is more preferable.

  The DTT in the translation system reaction solution is preferably contained in an amount of 0.1 mM to 10 mM, more preferably 0.2 mM to 5 mM, from the same viewpoint as in the case of the DTT in the extraction solution described above.

  Adenosine triphosphate (hereinafter sometimes referred to as “ATP”) in the translation system reaction solution is preferably contained in an amount of 0.01 mM to 10 mM, and preferably 0.1 mM to 5 mM, from the viewpoint of the rate of protein synthesis. More preferably it is contained. This is because when ATP is less than 0.01 mM or more than 10 mM, the protein synthesis rate tends to decrease.

  The guanosine triphosphate (hereinafter sometimes referred to as “GTP”) in the reaction solution for translation system is preferably contained in an amount of 0.01 mM to 10 mM, preferably 0.1 mM to 5 mM, from the viewpoint of the rate of protein synthesis. More preferably it is contained. This is because when GTP is less than 0.01 mM or more than 10 mM, the rate of protein synthesis tends to decrease.

  Creatine phosphate in the reaction solution for translation system is a component for continuously synthesizing proteins, and is blended for the purpose of regenerating ATP and GTP. From the viewpoint of protein synthesis rate, creatine phosphate is preferably contained in the reaction solution in an amount of 1 mM to 200 mM, more preferably 10 mM to 100 mM. This is because if creatine phosphate is less than 1 mM, sufficient amounts of ATP and GTP are difficult to regenerate, resulting in a tendency for the protein synthesis rate to decrease, and if creatine phosphate exceeds 200 mM, an inhibitory substance. This is because the protein synthesis rate tends to decrease.

  Creatine kinase in the reaction solution for translation system is a component for continuously synthesizing proteins and is formulated for the purpose of regenerating ATP and GTP together with creatine phosphate. From the viewpoint of protein synthesis rate, creatine kinase is preferably contained in the reaction solution in an amount of 1 μg / mL to 1000 μg / mL, and more preferably 10 μg / mL to 500 μg / mL. This is because when creatine kinase is less than 1 μg / mL, sufficient amounts of ATP and GTP are difficult to be regenerated, and as a result, the protein synthesis rate tends to decrease, and when creatine kinase exceeds 1000 μg / mL, This is because it acts as an inhibitor and tends to decrease the rate of protein synthesis.

The amino acid component in the reaction solution for translation system is 20 kinds of amino acids, that is, valine, methionine, glutamic acid, alanine, leucine, phenylalanine, glycine, proline, isoleucine, tryptophan, asparagine, serine, threonine, histidine, aspartic acid, tyrosine , Lysine, glutamine, cystine and arginine. This amino acid also includes radioisotope labeled amino acids. Furthermore, you may contain the modified amino acid as needed. The amino acid component usually contains approximately equal amounts of each type of amino acid.
In the present invention, from the viewpoint of protein synthesis speed, the amino acid component is preferably contained in the reaction solution in an amount of 1 μM to 1000 μM, more preferably 10 μM to 200 μM. This is because if the amino acid component is less than 1 μM or exceeds 1000 μM, the protein synthesis rate tends to decrease.

  The RNase inhibitor in the reaction solution for the translation system interferes with protein synthesis by undesirably digesting mRNA and tRNA during the cell-free protein synthesis of the present invention by the silkworm-derived RNase mixed in the extract. In the reaction solution, it is preferably contained in an amount of 0.1 U / μL to 100 U / μL, and more preferably 1 U / μL to 10 U / μL. This is because if the RNase inhibitor is less than 0.1 U / μL, the degradation activity of RNase tends not to be sufficiently suppressed, and if the RNase inhibitor exceeds 100 U / μL, the protein synthesis reaction tends to be inhibited. Because.

  The tRNA in the translation reaction solution contains approximately equal amounts of the tRNAs corresponding to the 20 amino acids. In the present invention, from the viewpoint of the rate of protein synthesis, the reaction solution preferably contains 1 μg / mL to 1000 μg / mL, more preferably 10 μg / mL to 500 μg / mL. This is because if the tRNA is less than 1 μg / mL or exceeds 1000 μg / mL, the rate of protein synthesis tends to decrease.

  As the buffering agent contained in the translation system reaction solution, the same one as that used in the extraction solution described above can be suitably used. For the same reason, HEPES-KOH (pH 6-8) is used. Is preferred. Moreover, it is preferable that 5 to 200 mM is contained, and it is more preferable that 10 to 50 mM is contained for a buffering agent from the viewpoint similar to the case of the buffering agent in the liquid for extraction mentioned above.

  The translation system reaction solution is more preferably one to which the polyhydric alcohol is further added. This is because the addition of a polyhydric alcohol has an advantage that components essential for protein synthesis can be stabilized in the translation system synthesis reaction. When polyhydric alcohol is added, it is usually added so as to be 5 (v / v)% to 20 (v / v)%.

  Further, the translation system reaction solution preferably contains ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (hereinafter sometimes referred to as “EGTA”). When EGTA is contained, EGTA forms a chelate with a metal ion in the extract to inactivate ribonuclease, protease, etc., thereby inhibiting degradation of components essential for protein synthesis of the present invention. is there. The EGTA is preferably contained in the reaction solution in an amount of 0.01 mM to 10 mM, more preferably 0.1 mM to 5 mM, from the viewpoint of suitably exhibiting the above-described degradation inhibitory ability. This is because if EGTA is less than 0.01 mM, the degradation activity of essential components tends not to be sufficiently suppressed, and if it exceeds 10 mM, the protein synthesis reaction tends to be inhibited.

  That is, as a translation system reaction solution using the extract obtained by the method of the present invention, the extract contains 30 (v / v)% to 60 (v / v)% and 50 mM to 150 mM. Potassium acetate, 0.5 mM to 3 mM magnesium acetate, 0.2 mM to 5 mM DTT, 5 (v / v)% to 20 (v / v)% polyhydric alcohol, 0.1 mM to 5 mM ATP, 0. 1 mM to 5 mM GTP, 10 mM to 100 mM creatine phosphate, 10 μg / mL to 500 μg / mL creatine kinase, 10 μM to 200 μM amino acid component, 1 U / μL to 10 U / μL RNase inhibitor, 10 μg / mL to 500 μg / mL TRNA, 10 μg / mL to 500 μg / mL foreign mRNA, 10 mM to 50 mM HEPES-KOH (pH 6 to 8) Preference is implemented to have. In addition to the above, it is more preferable that the EGTA is further contained in an amount of 0.1 mM to 5 mM.

  The cell-free protein synthesis reaction (translation system synthesis reaction) using the translation reaction solution is carried out in a conventionally known low temperature thermostat. The reaction temperature is usually in the range of 10 ° C to 40 ° C, preferably 20 ° C to 30 ° C. This is because if the reaction temperature is less than 10 ° C, the protein synthesis rate tends to decrease, and if the reaction temperature exceeds 40 ° C, essential components tend to denature. The reaction time is usually 1 hour to 72 hours, preferably 3 hours to 24 hours.

(2) Reaction solution for transcription / translation system The reaction solution for transcription / translation system is a component excluding the extract obtained by the method of the present invention, and includes exogenous template DNA, RNA polymerase, adenosine triphosphate, guanosine triphosphate. It preferably contains at least cytidine 5′-triphosphate, uridine 5′-triphosphate, creatine phosphate, creatine kinase, amino acid component and tRNA. By carrying out a transcription / translation system synthesis reaction using such a reaction solution for transcription / translation system, a large amount of protein can be synthesized in a short time.

The exogenous template DNA used in the reaction solution for transcription / translation system may be a circular DNA such as a plasmid DNA or a linear DNA such as a PCR product. The foreign template DNA refers to a template DNA not derived from silkworm tissue, and has at least a base sequence encoding the target protein and a promoter sequence located 5 ′ upstream thereof. The foreign template DNA used in the present invention is not particularly limited as long as it is a template DNA not derived from silkworm tissue, and has a base sequence that encodes a protein that becomes a cytotoxic agent in a living cell. It may be one having a base sequence encoding a glycoprotein. Further, the promoter sequence in the exogenous template DNA used in the present invention is not particularly limited, and examples thereof include conventionally known T7 promoter sequence, SP6 promoter sequence, T3 promoter sequence and the like.
The foreign template DNA used in the present invention is not particularly limited in the number of bases, and all template DNAs may not have the same number of bases as long as the target protein can be synthesized. In addition, each foreign template DNA may have a plurality of bases deleted, substituted, inserted or added as long as the sequences are homologous to the extent that the target protein can be synthesized. In the extract, whether the template DNA contained is a foreign template DNA or a template DNA derived from silkworm tissue is extracted from the extract by phenol-chloroform extraction, and the template DNA is extracted. It can be determined by analyzing the base sequence.

  Further, the exogenous template DNA used in the present invention is a terminator sequence having a function of terminating transcription at the 3 ′ downstream side of the base sequence encoding the target protein and / or the stability of the synthesized mRNA. It preferably has a poly A sequence. Examples of the terminator sequence include conventionally known T7 terminator sequences, SP6 terminator sequences, T3 terminator sequences, and the like.

  The exogenous template DNA is preferably contained in the reaction solution for transcription / translation system in an amount of 0.1 μg / mL to 8000 μg / mL, more preferably 3 μg / mL to 600 μg / mL. This is because if the exogenous template DNA is less than 0.1 μg / mL or exceeds 8000 μg / mL, the rate of protein synthesis tends to decrease.

  The RNA polymerase used in the transcription / translation system reaction solution can be appropriately selected according to the promoter sequence of the foreign template DNA. For example, when the foreign template DNA has a T7 promoter sequence, it is preferable to use T7 RNA polymerase that recognizes the sequence. In addition, when the foreign template DNA has an SP6 or T3 promoter sequence, it is preferable to use SP6 RNA polymerase or T3 RNA polymerase, respectively.

  From the viewpoint of the rate of mRNA synthesis and the rate of protein synthesis, RNA polymerase is preferably contained in the reaction solution for transcription / translation system in an amount of 0.01 U / μL to 100 U / μL, preferably 0.1 U / μL to 10 U / μl. More preferably, μL is contained. This is because if RNA polymerase is less than 0.01 U / μL, the amount of mRNA synthesis decreases, and as a result, the rate of protein synthesis tends to decrease, and if RNA polymerase exceeds 100 U / μL, protein synthesis. This is because the reaction tends to be inhibited.

  Adenosine triphosphate (hereinafter sometimes referred to as “ATP”) in the reaction solution for transcription / translation system is preferably contained in an amount of 0.01 mM to 10 mM from the viewpoint of the rate of protein synthesis. More preferably, it is contained at -5 mM. This is because when ATP is less than 0.01 mM or more than 10 mM, the protein synthesis rate tends to decrease.

  The guanosine triphosphate (hereinafter sometimes referred to as “GTP”) in the reaction solution for transcription / translation system is preferably contained in an amount of 0.01 mM to 10 mM, from the viewpoint of the rate of protein synthesis. More preferably, it is contained at -5 mM. This is because when GTP is less than 0.01 mM or more than 10 mM, the rate of protein synthesis tends to decrease.

  The cytidine 5′-triphosphate (hereinafter sometimes referred to as “CTP”) in the reaction solution for transcription / translation system is preferably contained in an amount of 0.01 mM to 10 mM from the viewpoint of the rate of protein synthesis. More preferably, the content is 0.1 mM to 5 mM. This is because if the CTP is less than 0.01 mM or exceeds 10 mM, the protein synthesis rate tends to decrease.

  Uridine 5′-triphosphate (hereinafter sometimes referred to as “UTP”) in the reaction solution for transcription / translation system is preferably contained in an amount of 0.01 mM to 10 mM from the viewpoint of the rate of protein synthesis. More preferably, the content is 0.1 mM to 5 mM. This is because when the UTP is less than 0.01 mM or more than 10 mM, the protein synthesis rate tends to decrease.

  Creatine phosphate in the reaction solution for transcription / translation system is a component for continuously synthesizing proteins, and is blended for the purpose of regenerating ATP and GTP. From the viewpoint of protein synthesis rate, creatine phosphate is preferably contained in an amount of 1 mM to 200 mM, more preferably 10 mM to 100 mM, in the transcription / translation system reaction solution. This is because if creatine phosphate is less than 1 mM, sufficient amounts of ATP and GTP are difficult to regenerate, resulting in a tendency for the protein synthesis rate to decrease, and if creatine phosphate exceeds 200 mM, an inhibitory substance. This is because the protein synthesis rate tends to decrease.

  Creatine kinase in the reaction solution for transcription / translation system is a component for continuously synthesizing proteins, and is formulated for the purpose of regenerating ATP and GTP together with creatine phosphate. From the viewpoint of protein synthesis rate, creatine kinase is preferably contained in the reaction solution for transcription / translation system in an amount of 1 μg / mL to 1000 μg / mL, and more preferably 10 μg / mL to 500 μg / mL. This is because when creatine kinase is less than 1 μg / mL, sufficient amounts of ATP and GTP are difficult to be regenerated, and as a result, the protein synthesis rate tends to decrease, and when creatine kinase exceeds 1000 μg / mL, This is because it acts as an inhibitor and tends to decrease the rate of protein synthesis.

The amino acid component in the reaction solution for transcription / translation system is 20 kinds of amino acids, that is, valine, methionine, glutamic acid, alanine, leucine, phenylalanine, glycine, proline, isoleucine, tryptophan, asparagine, serine, threonine, histidine, aspartic acid , Tyrosine, lysine, glutamine, cystine, arginine at least. This amino acid also includes radioisotope labeled amino acids. Furthermore, you may contain the modified amino acid as needed. The amino acid component usually contains approximately equal amounts of each type of amino acid.
From the viewpoint of the speed of protein synthesis, the amino acid component is preferably contained in the reaction solution for transcription / translation system in an amount of 1 μM to 1000 μM, more preferably 10 μM to 500 μM. This is because if the amino acid component is less than 1 μM or exceeds 1000 μM, the protein synthesis rate tends to decrease.

  The tRNA in the reaction solution for transcription / translation system contains approximately the same amount of tRNA of the types corresponding to the 20 types of amino acids. From the viewpoint of the speed of protein synthesis, tRNA is preferably contained in the transcription / translation system reaction solution in an amount of 1 μg / mL to 1000 μg / mL, and more preferably 10 μg / mL to 500 μg / mL. This is because if the tRNA is less than 1 μg / mL or exceeds 1000 μg / mL, the rate of protein synthesis tends to decrease.

  The reaction solution for transcription / translation system preferably further contains potassium salt, magnesium salt, DTT, RNase inhibitor, spermidine and buffer.

  As the potassium salt in the transcription / translation system reaction solution, the various potassium salts described above as the components of the extraction solution, preferably potassium acetate, can be preferably used. From the same viewpoint as the potassium salt in the extraction solution described above, the potassium salt is preferably contained in the transcription / translation system reaction solution in an amount of 10 mM to 500 mM, more preferably 50 mM to 150 mM. preferable.

  As the magnesium salt in the transcription / translation system reaction solution, the above-described various magnesium salts, preferably magnesium acetate, can be preferably used as components of the extraction solution. From the same viewpoint as the case of the magnesium salt in the extraction solution described above, the magnesium salt is preferably contained in the transcription / translation system reaction solution in an amount of 0.1 mM to 10 mM, preferably 0.5 mM to 3 mM. More preferably.

  DTT in the reaction solution for transcription / translation system is blended for the purpose of preventing oxidation, and is preferably contained in the reaction solution at a concentration of 0.1 mM to 100 mM, and preferably 0.2 mM to 20 mM. Is more preferable. This is because when DTT is less than 0.1 mM or more than 100 mM, components essential for protein synthesis tend to become unstable.

  The RNase inhibitor in the transcription / translation system reaction solution interferes with protein synthesis by undesirably digesting mRNA and tRNA during the transcription / translation system synthesis reaction by the silkworm-derived RNase mixed in the extract. In the reaction solution, it is preferably contained in an amount of 0.1 U / μL to 100 U / μL, and more preferably 1 U / μL to 10 U / μL. This is because if the RNase inhibitor is less than 0.1 U / μL, the degradation activity of RNase tends not to be sufficiently suppressed, and if the RNase inhibitor exceeds 100 U / μL, the protein synthesis reaction tends to be inhibited. Because.

  The spermidine is added for the purpose of promoting an extension reaction in transcription, and is preferably contained in the reaction solution for transcription / translation system in an amount of 0.01 mM to 100 mM, and 0.05 mM to 10 mM. Is more preferable. This is because when the spermidine is less than 0.01 mM, the mRNA synthesis rate decreases and the amount of mRNA produced decreases, and as a result, the protein synthesis rate tends to decrease, and spermidine reduces 100 mM. It is because it tends to inhibit the protein synthesis reaction.

  As the buffer contained in the reaction solution for transcription / translation system, those similar to those used in the extraction solution described above can be suitably used. For the same reason, HEPES-KOH (pH 6-8) is used. It is preferable to do this. In addition, the buffer is preferably contained in an amount of 1 mM to 200 mM, more preferably 5 mM to 50 mM, from the same viewpoint as that of the buffer in the extraction liquid described above.

  The reaction solution for transcription / translation system is more preferably one to which the above polyhydric alcohol is further added. This is because the addition of polyhydric alcohol has the advantage that components essential for protein synthesis can be stabilized in the transcription / translation system synthesis reaction. When polyhydric alcohol is added, it is usually added so as to be 5 (v / v)% to 20 (v / v)%.

  That is, the transcription / translation system reaction solution using the extract obtained by the method of the present invention contains 30 (v / v)% to 60 (v / v)% of the extract, and further contains 0 1 U / μL to 10 U / μL RNA polymerase, 0.1 mM to 5 mM ATP, 0.1 mM to 5 mM GTP, 0.1 mM to 5 mM CTP, 0.1 mM to 5 mM UTP, 10 mM to 100 mM creatine phosphorus It preferably contains an acid, 10 μg / mL to 500 μg / mL creatine kinase, 10 μM to 500 μM amino acid component, 10 μg / mL to 500 μg / mL tRNA. Furthermore, 50 mM to 150 mM potassium acetate, 0.5 mM to 3 mM magnesium acetate, 0.2 mM to 20 mM DTT, 1 U / μL to 10 U / μL RNase inhibitor, 0.05 mM to 10 mM spermidine, 5 mM to 50 mM It is preferably realized to contain HEPES-KOH (pH 7.4), 5 (v / v)% to 20 (v / v)% polyhydric alcohol.

The cell-free protein synthesis reaction (transcription / translation system synthesis reaction) using the reaction solution for transcription / translation system may be carried out in a conventionally known low-temperature thermostatic chamber as in the case of the translation system synthesis reaction. . The reaction temperature in the transfer step is usually in the range of 10 ° C to 60 ° C, preferably 20 ° C to 50 ° C. This is because if the reaction temperature in the transfer step is less than 10 ° C., the transfer speed tends to decrease, and if the reaction temperature in the transfer step exceeds 60 ° C., components essential for the reaction tend to denature. . Moreover, the temperature of a translation process is 10 to 40 degreeC normally, Preferably it exists in the range of 20 to 30 degreeC. If the reaction temperature of the translation process is less than 10 ° C, the protein synthesis rate tends to decrease, and if the reaction temperature of the translation process exceeds 40 ° C, components essential for the reaction tend to denature. is there.
In the transcription / translation system synthesis reaction, it is particularly preferable to perform the reaction in a range of 20 ° C. to 30 ° C. suitable for both steps from the viewpoint that the transcription and translation steps can be carried out continuously. The reaction time is usually 1 hour to 72 hours, preferably 3 hours to 24 hours, in total for all steps.

  There is no particular limitation on the protein that can be synthesized using the above translation system reaction solution and transcription / translation system reaction solution. The amount of the synthesized protein can be measured by measuring enzyme activity, SDS-PAGE, immunoassay, and the like.

  The present invention will be described in more detail with reference to the following examples, but these are merely illustrative and do not limit the scope of the present invention.

Example 1
The silkworm larvae that reached the 6th day of the 5th age were sampled. The posterior silk gland was extracted from the sampled silkworm using scissors, tweezers and a scalpel, and the posterior silk gland was extracted according to the following procedure.
In the extraction, first, the rear silk gland extracted from the silkworm larvae that reached the 6th day of the fifth infancy was frozen with liquid nitrogen and then ground using a mortar previously cooled with liquid nitrogen. An extraction solution having the following composition was added thereto, the extract was once frozen, and then dissolved with stirring with a spoon until it became a sherbet. Then, after completely freezing with liquid nitrogen, extraction from a silkworm tissue was performed by performing an operation of dissolving with stirring with a spoon again until it became a sherbet shape.
[Composition of extraction liquid]
・ 20 mM HEPES-KOH (pH 7.4)
100 mM potassium acetate 2 mM magnesium acetate 19.3 (v / v)% glycerol 1 mM DTT
・ 0.5 mM PMSF
After extraction, the obtained liquid substance was centrifuged with a centrifuge (himacCR20B3 (manufactured by Hitachi Koki Co., Ltd.)) at 30000 × g for 30 minutes at 4 ° C. After centrifugation, only the supernatant (first supernatant) was isolated and centrifuged again at 30000 × g for 30 minutes at 4 ° C. to isolate the supernatant (third supernatant). . On the other hand, after adding the extraction liquid having the above composition to the precipitate after the first centrifugation and performing the same extraction operation as described above, the mixture was centrifuged at 30000 × g for 30 minutes at 4 ° C. Separation was performed and the supernatant (second supernatant) was isolated. 6.25 μl of the second supernatant thus obtained and 6.25 μl of the third supernatant were mixed to prepare an extract.

Example 2
The same procedure as in Example 1 was performed except that 12.5 μl of the third supernatant obtained in Example 1 was directly prepared as an extract.

Example 3
The same procedure as in Example 1 was performed except that 12.5 μl of the second supernatant obtained in Example 1 was directly prepared as an extract.

Experimental example 1
: Protein synthesis in cell-free system using each extract of Examples 1 to 3 Using each extract obtained in Examples 1 to 3 above, a reaction solution having the following composition was prepared.
[Composition of reaction solution]
・ 50 (v / v)% extract ・ 40 μg / mL foreign mRNA
・ 30 mM HEPES-KOH (pH 7.4)
・ 100 mM potassium acetate ・ 1.0 mM magnesium acetate ・ 0.5 mM DTT
・ 10 (v / v)% glycerol ・ 0.5 mM ATP
・ 0.5 mM GTP
・ 0.25 mM EGTA
・ 25 mM creatine phosphate ・ 200 μg / mL creatine kinase ・ 40 μM amino acids (20 types)
2U / μL RNase inhibitor 200μg / mL tRNA
ATP (manufactured by Sigma), GTP (manufactured by Sigma), amino acids (20 types) (manufactured by Sigma), RNase inhibitor (manufactured by Takara Shuzo), and tRNA (manufactured by Roche Diagnostics) were used. As the exogenous mRNA, mRNA encoding luciferase (luciferase control RNA, manufactured by Promega) was used.
Using each prepared reaction solution, a cell-free protein (luciferase) was synthesized using a low-temperature aluminum block thermostatic chamber MG-1000 (manufactured by Tokyo Rika Kikai Co., Ltd.) as a reaction apparatus. The reaction volume was 25 μL. The reaction temperature was 25 ° C., sampling was performed every reaction time, and the amount of synthesized luciferase was measured.
The synthesized luciferase was quantified using a luciferase assay kit (E-1500, manufactured by Promega). The reaction solution (2.5 μL) was added to 50 μL of the luciferase assay reagent, and luminescence by luciferase was measured using a luminometer (Luminescence-JNR AB-2100, manufactured by Ato).
FIG. 1 is a graph showing the results of Experimental Example 1. In FIG. 1, the vertical axis represents the accumulated amount of luciferase luminescence, and the horizontal axis represents the reaction time (hours).

Example 4
The mixture of the second supernatant and the third supernatant prepared in Example 1 was further added to Sephadex G-25 Fine (manufactured by Amersham Bioscience) to equilibrate the column. Gel filtration was performed by supplying the mixture and eluting with the extraction solution. The fraction of the filtrate after gel filtration was fractionated using the spectrophotometer (Ultraspec 3300pro, manufactured by Amersham Biosciences), and the fraction having the highest absorbance at 280 nm was collected and used as an extract. Same as 1.

Example 5
The third supernatant obtained in Example 2 was further added to Sephadex G-25 Fine (manufactured by Amersham Biosciences), the extraction liquid was added to equilibrate the column, and then the above mixture was supplied. Gel filtration was performed by elution with a liquid. The fraction of the filtrate after gel filtration was fractionated using the spectrophotometer (Ultraspec 3300pro, manufactured by Amersham Biosciences), and the fraction having the highest absorbance at 280 nm was collected and used as an extract. Same as 2.

Example 6
The second supernatant obtained in Example 3 was further added to Sephadex G-25 Fine (manufactured by Amersham Biosciences), the extraction liquid was added to equilibrate the column, and then the above mixture was supplied. Gel filtration was performed by elution with a liquid. The fraction of the filtrate after gel filtration was collected using a spectrophotometer (Ultraspec 3300pro, manufactured by Amersham Biosciences), and fractions having an absorbance at 280 nm of 10 or more were collected and used as an extract. Same as Example 3.

Experimental example 2
Cell-free protein synthesis using each extract of Examples 4 to 6 Cell-free protein synthesis using each extract obtained in Examples 4 to 6 in the same manner as in Experimental Example 1. Reaction was performed.
FIG. 2 is a graph showing the results of Experimental Example 2 for Examples 4-6. In FIG. 2, the vertical axis represents the accumulated amount of luciferase luminescence, and the horizontal axis represents the reaction time (hours).

Example 7
The silkworm larvae that reached the 6th day of the 5th age were sampled. The posterior silk gland was extracted from the sampled silkworm using scissors, tweezers and a scalpel, and the posterior silk gland was extracted according to the following procedure.
In the extraction, 3.0 g of the rear silk gland extracted from the silkworm larvae that reached the 6th day of the 5th infancy was frozen with liquid nitrogen, and then ground using a mortar previously cooled with liquid nitrogen. The extraction liquid was added using the extraction liquid.
[Composition of extraction liquid]
・ 20 mM HEPES-KOH (pH 7.4)
100 mM potassium acetate 2 mM magnesium acetate 10 (v / v)% glycerol 1 mM DTT
・ 0.5 mM PMSF
After adding the extraction solution, it was frozen and thawed with liquid nitrogen, and the obtained liquid was centrifugated (himacCR20B3 (manufactured by Hitachi Koki Co., Ltd.)) at 30000 × g for 30 minutes at 4 ° C. Centrifugation was performed at After centrifugation, only the supernatant was isolated and centrifuged again at 30000 × g for 30 minutes at 4 ° C. After this centrifugation, only the supernatant was isolated and used as an extract.

Example 8
An extract solution was prepared in the same manner as in Example 7 except that it contained 20 (v / v)% of glycerol, using the same extraction solution as in Example 7.

Example 9
An extract was prepared in the same manner as in Example 7, except that instead of glycerol, 20 (v / v)% diglycerol was contained, the same extraction liquid as in Example 7 was used.

Example 10
An extraction liquid was prepared in the same manner as in Example 7 except that it contained 10 (v / v)% ethylene glycol instead of glycerol, and the same extraction liquid as in Example 7 was used.

Production Example 1
: Production of polyerythritol (tetramer) 100.0 g of meso-erythritol and 1.0 g of sodium hydroxide were reacted at 240 ° C. and 30 mmHg to 40 mmHg for 2 hours in a carbon dioxide atmosphere. 150 ml of water is added to the obtained brown liquid, neutralized with dilute hydrochloric acid, decolorized with activated carbon, cation exchange resin (Mitsubishi Chemical Corporation, Diaion SK-112) and anion exchange resin (Mitsubishi Chemical Corporation) , Diaion WA30), and water was distilled off to obtain 69.3 g of colorless viscous liquid (water content: 5%).
When the obtained product was subjected to TOF-MS analysis (LCT mass spectrometer manufactured by Micromass, ionization method: ESI, measurement ion: positive ion), 104 Da repeating units derived from meso-erythritol could be observed. Moreover, in addition to the peak of chain polyerythritol from m / z 226 (polymerization degree 2) to 1474 (polymerization degree 14), there are also peaks of dehydrated products with 18 Da and 36 Da smaller than those m / z. Observed. Components were separated by LC (column: TSK-gel Amide-80 (manufactured by Tosoh Corp.)), eluent: (acetonitrile / water = 80/20), 60 ° C., flow rate 1 ml / min) of the same apparatus. As a result, linear and branched polyerythritol were observed even at the same molecular weight. The average degree of polymerization was 4 when calculated from the ionic strength of TOF-MS.

Example 11
An extraction liquid was used in the same manner as in Example 7 except that it contained 10 (v / v)% of polyerythritol obtained in Production Example 1 instead of glycerol. Was prepared.

Comparative Example 1
An extract was prepared in the same manner as in Example 7 except that extraction was performed using the same extraction solution as that used in Example 7 except that glycerol was not contained.

Experimental example 3
: Protein synthesis in cell-free system using each extract of Examples 7 to 11 and Comparative Example 1 Reaction of the following composition using each extract obtained in Examples 7 to 11 and Comparative Example 1 above A liquid was prepared.
[Composition of reaction solution]
・ 50 (v / v)% extract ・ 160 μg / mL foreign mRNA
・ 30 mM HEPES-KOH (pH 7.4)
・ 100 mM potassium acetate ・ 1.75 mM magnesium acetate ・ 2 mM DTT
・ 0.75 mM ATP
・ 0.5 mM GTP
・ 0.25 mM EGTA
・ 25 mM creatine phosphate ・ 50 μg / mL creatine kinase ・ 100 μM amino acids (20 types)
2U / μL RNase inhibitor 100μg / mL tRNA
ATP (manufactured by Sigma), GTP (manufactured by Sigma), amino acids (20 types) (manufactured by Sigma), RNase inhibitor (manufactured by Takara Shuzo), and tRNA (manufactured by Roche Diagnostics) were used. As the exogenous mRNA, mRNA encoding luciferase (luciferase control RNA, manufactured by Promega) was used.
Using each prepared reaction solution, a cell-free protein (luciferase) was synthesized using a low-temperature aluminum block thermostatic chamber MG-1000 (manufactured by Tokyo Rika Kikai Co., Ltd.) as a reaction apparatus. The reaction volume was 25 μL. The reaction temperature was 25 ° C., sampling was performed every reaction time, and the amount of synthesized luciferase was measured.
The synthesized luciferase was quantified using a luciferase assay kit (E-1500, manufactured by Promega). 2.5 μL of the reaction solution was added to 50 μL of the luciferase assay reagent, and luminescence by luciferase was measured using a luminometer (Turner Designs TD-20 / 20, manufactured by Promega).
FIG. 3 is a graph showing the amount of luciferase synthesis 4 hours after the start of the reaction in Experimental Example 3. In FIG. 3, the vertical axis represents the cell-free system when each extract obtained in Examples 7 to 11 is used with respect to the amount of cell-free protein synthesis when the extract obtained in Comparative Example 1 is used. The ratio of protein synthesis amount (relative synthesis amount:%) is shown. As shown in FIG. 3, the reaction liquid using the extract prepared using the extraction liquid containing polyhydric alcohol is a reaction using the extract prepared using the extraction liquid not containing polyhydric alcohol. Compared with the solution, the amount of protein synthesis is about 200% to 350%.

Example 12
An extract was prepared in the same manner as in Example 7, except that it contained 10 (v / v)% erythritol instead of glycerol, and the same extraction liquid as in Example 7 was used.

Example 13
An extract was prepared in the same manner as in Example 7 using the same extraction solution as in Example 7 except that it contained 10 (v / v)% xylitol instead of glycerol.

Example 14
An extraction liquid was prepared in the same manner as in Example 7 except that it contained 10 (v / v)% sorbitol instead of glycerol, and the same extraction liquid as in Example 7 was used.

Example 15
An extract was prepared in the same manner as in Example 7 except that it contained 10 (v / v)% trehalose instead of glycerol, and the same extract solution as in Example 7 was used.

Example 16
An extract solution was prepared in the same manner as in Example 7 except that it contained 10 (v / v)% sucrose instead of glycerol, and the same extract solution as in Example 7 was used.

Example 17
An extract was prepared in the same manner as in Example 7, except that 10 (v / v)% lactose was used instead of glycerol, and the same extraction liquid as in Example 7 was used.

Example 18
An extract was prepared in the same manner as in Example 7 using the same extraction solution as in Example 7 except that it contained 10 (v / v)% xylose instead of glycerol.

Example 19
An extract was prepared in the same manner as in Example 7, except that it contained 10 (v / v)% maltose instead of glycerol, and the same extract solution as in Example 7 was used.

Example 20
An extract was prepared in the same manner as in Example 7, except that it contained 10 (v / v)% inositol instead of glycerol, and the same extraction liquid as in Example 7 was used.

Comparative Example 2
An extract was prepared in the same manner as in Example 7 except that extraction was performed using the same extraction solution as that used in Example 7 except that glycerol was not contained.

Reference example 1
: Vector pT _N T-Luc the pGEM-Luc Vector (manufactured by Promega Corporation) 5 ng having a structural gene encoding the construct luciferase as the template and primers having the nucleotide sequence shown in SEQ ID NO: 1 (Luc T7-F3-Kpn ), A primer (Luc T7-R4-Kpn) having the base sequence shown in SEQ ID NO: 2 and KOD plus (manufactured by Toyobo Co., Ltd.), and the template was denatured at 96 ° C. for 2 minutes. PCR was performed at 15 seconds, 50 ° C. for 30 seconds, 68 ° C. for 120 seconds, and 30 cycles to amplify the open reading frame (ORF) of the structural gene. The PCR product was purified by ethanol precipitation and then digested with KpnI. Apart from this, pT _N T Vector (manufactured by Promega) was digested with KpnI. These reaction solutions were separated by agarose gel electrophoresis and then purified using Gen Elute Gel Purification Kit (manufactured by Sigma). These were ligated using Ligation High (Toyobo Co., Ltd.), and then transformed into E. coli DH5α (Toyobo Co., Ltd.). Plasmid prepared from transformed Escherichia coli by alkaline-SDS method was sequenced using a primer (T7 promoter) having the base sequence shown in SEQ ID NO: 3 in the sequence listing and Big Dye Terminator Cycle Sequencing FS (Applied Biosystems). The reaction (96 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes, 25 cycles) was performed. This reaction solution was subjected to ABI PRISM 310 Genetic Analyzer (Applied Biosystems), and base sequence analysis was performed. A plasmid in which the start codon of the luciferase gene was inserted downstream of the 5′-β globin leader sequence derived from pT _N T Vector was named pT _N T-Luc.

Reference example 2
: Preparation of template DNA (vector pSphd-Luc) Using the plasmid vector pT _N T-Luc prepared in Reference Example 1 as a template, a primer (T7p Rv) having the base sequence shown in SEQ ID NO: 4 in the sequence listing, and a sequence listing PCR was performed at 96 ° C. for 15 seconds, 50 ° C. for 30 seconds, 68 ° C. for 5 minutes, 30 cycles using a primer (Luc-ATG) having the base sequence shown in SEQ ID NO: 5. After completion of the reaction, PCR products were separated by electrophoresis and then purified using Gen Elute Gel Purification Kit (manufactured by Sigma), and used for the ligation reaction. Thus, a plasmid vector in which the SP6 promoter sequence, the 5′-β globin leader sequence and the 5 ′ upstream multicloning site of the structural gene encoding luciferase were deleted from the plasmid vector pT _N T-Luc was obtained.
S5NPR sense strand and antisense strand of SfNPV (Spodoptera frugiperda nucleopolyhedrovirus) SfNPV (Spodoptera frugiperda nucleopolyhedrovirus) having the base sequence shown in SEQ ID NO. To phosphorylate the 5 ′ end. After completion of the reaction, the sense strand and the antisense strand were mixed and heat-treated at 95 ° C. for 5 minutes. This was allowed to stand until it reached room temperature, and the sense strand and the antisense strand were annealed. After purification by ethanol precipitation, it was dissolved in water. Excess ATP was removed by SigmaSpin Post Reaction Purification Columns (manufactured by Sigma), and then purified by ethanol precipitation again. Double-stranded DNA fragment obtained was an insert, the SP6 promoter sequence, and vector and ligation of 5 'from pT _N T-Luc lacking upstream multiple cloning site of β-globin leader sequence and the structural gene, E.coli DH5α Were transformed. After preparing a plasmid from the obtained Escherichia coli, base sequence analysis was performed. In this way, a vector (template DNA) in which one 5 ′ UTR of the polyhedrin gene of SfNPV was incorporated in the forward direction (5 ′ → 3 ′) was selected. Thus, a vector (template DNA) in which one 5′UTR of the polyhedrin gene of SfNPV was integrated in the forward direction between the T7 promoter sequence and the structural gene was prepared. The obtained template DNA was named pSphd-Luc.

Experimental Example 4
: Cell-free protein synthesis using a reaction solution containing an extract derived from silkworm tissue (1) In vitro transcription reaction and purification of mRNA After digesting the vector prepared in Reference Example 2 with BamHI or BglII, phenol- Purification by chloroform extraction and ethanol precipitation. Using 1 μg of the obtained vector as a template, RiboMax Large Scale RNA production System-T7 (manufactured by Promega) was used to perform an in vitro transcription reaction at 20 ° C. for 4 hours at 37 ° C. to synthesize mRNA.
After completion of the transcription reaction, 1 U of RQ1 RNase Free DNase (manufactured by Promega) was added and incubated at 37 ° C. for 15 minutes to digest the template. After removing the protein by phenol-chloroform extraction, ethanol precipitation was performed. The obtained precipitate was dissolved in 100 μl of sterilized water, applied to Nick column (manufactured by Amersham Bioscience), and then eluted with sterilized water. Potassium acetate was added to the elution fraction to a final concentration of 0.3 M, and ethanol precipitation was performed. The synthesized mRNA was quantified by measuring absorbance at 260 nm and 280 nm.
(2) Cell-free protein synthesis reaction Using the silkworm extract prepared in Examples 12 to 20 and Comparative Example 2 and the mRNA obtained in (1) above, a reaction solution having the following composition was prepared, and a cell-free protein was obtained. A synthetic reaction was performed.
[Composition of reaction solution]
・ 50 (v / v)% silkworm extract ・ 160 μg / mL mRNA
・ 30 mM HEPES-KOH (pH 7.4)
・ 100 mM potassium acetate ・ 1.75 mM magnesium acetate ・ 2.0 mM DTT
・ 0.75 mM ATP
・ 0.5 mM GTP
・ 0.25 mM EGTA
・ 25 mM creatine phosphate ・ 200 μg / mL creatine kinase ・ 40 μM amino acids (20 types)
2U / μL RNase inhibitor 100μg / mL tRNA
ATP (manufactured by Sigma), GTP (manufactured by Sigma), amino acids (20 types) (manufactured by Sigma), RNase inhibitor (manufactured by Takara Shuzo), and tRNA (manufactured by Roche Diagnostics) were used.
A low temperature aluminum block thermostatic chamber MG-1000 (manufactured by Tokyo Rika Kikai Co., Ltd.) was used as a reaction apparatus. The translation reaction was carried out at a reaction temperature of 25 ° C. for 6 hours, and the reaction volume was 25 μL. The synthesized luciferase was quantified using a luciferase assay kit (E-1500, manufactured by Promega). 2.5 μL of the reaction solution was added to 50 μL of the luciferase assay reagent, and luminescence by luciferase was measured using a luminometer (Tuner Designs TD-20 / 20, manufactured by Promega).

  FIG. 4 is a graph showing the amount of luciferase synthesis 6 hours after the start of the protein synthesis reaction in Experimental Example 4. In FIG. 4, the vertical axis represents the cell-free system when each extract obtained in Examples 12 to 20 is used with respect to the amount of cell-free protein synthesized when the extract obtained in Comparative Example 2 is used. The ratio of protein synthesis amount (relative synthesis amount:%) is shown. As shown in FIG. 4, the reaction liquid using the extract prepared using the extraction liquid containing polyhydric alcohol is a reaction using the extract prepared using the extraction liquid not containing polyhydric alcohol. Compared with the liquid, the amount of protein synthesis was about 200% to 300%.

Reference example 3
: Preparation of template DNA (vector pAphd-Luc) Same as Reference Example 2 except that 5′UTR of polyhedrin gene of AcNPV (Autographa californica nuclear polyhedrosis virus) having the base sequence shown in SEQ ID NO: 7 was used. Thus, between the T7 promoter sequence and the structural gene, one 5′UTR of the AcNPV polyhedrin gene having the base sequence shown in SEQ ID NO: 7 was inserted in the forward direction (5 ′ → 3 ′). A vector (template DNA) was prepared. The obtained template DNA was named pAphd-Luc.

Experimental Example 5
: Cell-free protein synthesis using a reaction solution containing an extract derived from silkworm tissue (1) In vitro transcription reaction and mRNA purification Experimental Example 4 except that pAphd-Luc prepared in Reference Example 3 was used as a vector. MRNA was prepared by the same method as in (1).
(2) Prepare a reaction solution similar to Experimental Example 4 (2) except that the silkworm extract prepared in Example 7 and Comparative Example 1 was used as the extract, and the mRNA obtained in (1) above was used as the mRNA. Then, a cell-free protein synthesis reaction was performed.
A low temperature aluminum block thermostatic chamber MG-1000 (manufactured by Tokyo Rika Kikai Co., Ltd.) was used as a reaction apparatus. The reaction volume was 25 μL. Sampling was performed over time, and the amount of luciferase synthesized in the same manner as in Experimental Example 4 (2) was quantified.

  FIG. 5 is a graph showing the results of Experimental Example 5. In FIG. 5, the vertical axis represents the amount of luciferase synthesis (μg / mL), and the horizontal axis represents the reaction time (hours). As shown in FIG. 5, a reaction solution using an extract prepared using an extraction solution containing glycerol is compared with a reaction solution using an extract prepared using an extraction solution not containing glycerol. At each time, the amount of protein synthesis was about 200% to 350%.

  As is apparent from the above description, according to the present invention, it is possible to provide a method for preparing an extract for cell-free protein synthesis that can improve protein synthesis efficiency, and a silkworm tissue extraction kit therefor. .

It is a graph which shows the result of Experimental example 1, a vertical axis | shaft shows the amount of luciferase luminescence integration, and a horizontal axis shows reaction time (hour). It is a graph which shows the result of Experimental example 2 about Examples 4-6, a vertical axis | shaft shows luciferase luminescence integrated amount, and a horizontal axis shows reaction time (hour). 4 is a graph showing the amount of luciferase synthesis 4 hours after the start of the reaction in Experimental Example 3. 6 is a graph showing the amount of luciferase synthesis 6 hours after the start of the reaction in Experimental Example 4. It is a graph which shows the result of Experimental example 5, a vertical axis | shaft shows the amount of luciferase synthesis | combination (microgram / mL), and a horizontal axis shows reaction time (hour).

SEQ ID NO: 1: Primer Luc T7-F3-Kpn
SEQ ID NO: 2: Primer Luc T7-R4-Kpn
Sequence number 3: Primer T7 promoter Sequence number 4: Primer T7p Rv
Sequence number 5: Primer Luc-ATG
SEQ ID NO: 6: Base sequence of SfNPV polyhedrin gene 5′UTR SEQ ID NO: 7: Base sequence of AcNPV polyhedrin gene 5 ′ UTR

Claims (5)

  A method for preparing an extract for cell-free protein synthesis, wherein extraction from a silkworm tissue is performed using an extraction solution containing a polyhydric alcohol.   The polyhydric alcohol is at least one selected from glycerol, diglycerol, ethylene glycol, polyerythritol, erythritol, xylitol, sorbitol, trehalose, sucrose, lactose, xylose, maltose, and inositol. Method.   The supernatant obtained by centrifuging after extraction and the supernatant obtained by centrifuging after further extraction from the precipitate after centrifugation using the above extraction solution, A process according to claim 1 or 2, characterized in that is prepared. An extraction kit for an extract for cell-free protein synthesis derived from a silkworm tissue having an extraction solution containing a polyhydric alcohol.
  The polyhydric alcohol is at least one selected from glycerol, diglycerol, ethylene glycol, polyerythritol, erythritol, xylitol, sorbitol, trehalose, sucrose, lactose, xylose, maltose, and inositol. kit.

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