JP2014097056A - Cell-free protein synthesis using silkworm larva middle silk gland extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing extract, in which a middle silk gland as its ingredient can be picked out thickly and easily, and a cell-free protein synthetic method using the extract, in a cell-free protein synthetic system using a silkworm extract.SOLUTION: In the most common silkworm varieties, while the middle silk gland produces sericin which is a component of a cocoon, it is accompanied by accumulation of fibroin (the main protein of silk thread) produced in the posterior silk gland, making it improper to prepare extract due to fibroin's high viscoelasticity. Therefore, a middle silk gland of a crossbreed produced from a mutant silkworm strain which does not specifically produce fibroin, is used. The present inventors found that a middle silk gland can be picked out so easily by making a cut with scissors into a septum between appendages of abdominal third and fourth segments of the silkworm larva, and picking the middle silk gland from the cut with a tweezer. Using this method greatly shortens the time needed for preparation of cell extract, as it enables a silk gland to be picked out in 23 seconds per one larva in average which is one-sixth of the time required for the conventional posterior silk gland extraction, when compared. In addition, an enhanced protein synthetic ability is confirmed in the middle silk gland extract on the second or third day in the fifth instar and using the ability can allow cell-free protein synthesis to be provided, in which the extract is easily prepared.

Description

  The present invention relates to an extract that can be used in a cell-free protein synthesis system, a method for producing the same, and a protein synthesis method in a cell-free system using the extract.

  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 use proteins corresponding to these genetic information for drug development research and the like, it is necessary to easily synthesize a huge variety of proteins in a short time.

  Currently, expression systems using living cells such as E. coli, yeast, cultured insect cells, and cultured mammalian cells (hereinafter sometimes referred to as “cell systems”) are widely used in protein production methods. ing. 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 or genetic information transcription / translation system for organisms is prepared in a test tube by adding a substrate or an enzyme to a cell disruption solution or extract, and a reaction template. Reconstructing a synthetic system that can combine amino acids with the required number of residues in the desired order using DNA (transcription template) or mRNA (translation template) having a structural gene encoding the target protein as Protein synthesis systems (hereinafter sometimes referred to as “cell-free systems”) are known. Such cell-free protein synthesis systems are less susceptible to the limitations of protein synthesis in the above cell systems, can synthesize proteins without losing the lives of living organisms, and can be used for protein production such as culturing. Since it is not accompanied, protein synthesis can be performed in a short time compared with the cell system. Furthermore, in a cell-free system, mass production of a protein having an amino acid sequence that is not used by living organisms is possible, and thus it is expected to be a promising expression method. As an extract (cell-free protein synthesis extract) to be used in such a cell-free system, the use of various organism-derived extracts has been studied and research is being conducted.

As extracts for cell-free protein synthesis, those derived from insect cultured cells in addition to Escherichia coli, wheat germ, rabbit reticulocytes, and the like are conventionally known. We have so far proposed cell-free protein synthesis methods using an extract derived from silkworm tissue (a silkworm extract) (see, for example, Patent Document 1, Patent Document 2, and Patent Document 3). In the cell-free protein synthesis method using silkworm extract proposed by us, an extract can be prepared using an inexpensive material called silkworm, compared to conventional methods. It has the advantage that it has a synthesis ability and can also synthesize glycoproteins, and is very useful. However, the silk gland and fat body, which are silkworm organs suitable for preparing the extract, are thin and fragile, so the silk line is separated by a method of pushing from the head of the cocoon toward the tail with a roller-like object. The conventional method such as removing the silk wire cells by finely cutting the method or the silk wire cannot be removed without being damaged. As a result, the preparation of the extract is complicated, and there is a problem that it takes a lot of time and labor, and finding an improvement method has been an important issue.

Japanese Patent No. 4244625 Japanese Patent No. 4244628 Japanese Patent No. 4243762

  The present invention has been made to solve the above-mentioned problems, and the object thereof is a silkworm larva tissue that is a suitable material for preparing an extract used for cell-free protein synthesis, An object of the present invention is to find a tissue that can be easily extracted, and to provide a method for preparing an extract using the tissue as a material and a cell-free protein synthesis method using the extract.

As a result of intensive studies to solve the above problems, the present inventors have completed the present invention. That is, the middle silk gland of the silkworm larva is a tissue that synthesizes and secretes sericin, which is a component of silk, and it has been suggested that the tissue has a high ability to synthesize proteins. However, in silkworm larvae of conventional general varieties (for example, Nishiki x Kanewa), fibroin is produced in the posterior silk gland, causing migration and accumulation in the middle silk gland, and as a result, the middle silk gland enlarges and is high Since it has viscoelasticity, it is not suitable as a material for preparing an extract, and therefore no preparation of an extract using a middle silk gland as a material has been reported. On the other hand, the mutant silkworm strain that does not specifically produce fibroin or the middle silk gland of a hybrid produced from that strain was expected to have no high viscoelasticity because fibroin accumulation hardly occurred. Therefore, as a result of examining a method for preparing an extract using a middle silk gland of a mutant silkworm strain that does not specifically produce fibroin or a hybrid produced from that strain, the middle silk gland can be easily removed. It has been found that it can be performed, and it has been found that an efficient extract preparation can be realized by using the middle silk gland as a material.
The invention is as follows.
[1] A method for producing an extract for cell-free protein synthesis, comprising using the middle silk gland of a mutant silkworm strain that does not specifically produce fibroin.
[2] A method for producing an extract for cell-free protein synthesis, comprising using a middle silk gland of a hybrid from a mutant silkworm strain that does not specifically produce fibroin.
[3] The method for producing an extract for cell-free protein synthesis according to claim 1 or 2, wherein the strain is a sericin hope.
[4] The method for producing an extract for cell-free protein synthesis according to claim 1, 2 or 3, wherein the middle silk thread of the silkworm larvae on the first day or the second day of the fifth instar period is used.
[5] A cell-free protein synthesis reagent kit containing the extract for synthesis produced by the method described in 1 to 4 above.
[6] A cell-free protein synthesis method using the synthesis extract produced by the method described in 1 to 4 above.

  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 referred to as the 1st instar period, and the period from the first molting to the second molting is referred to as the 2nd infancy. is there. After about a week, silkworm larvae become translucent and begin to spit silk threads to form cocoons (this state is also called “mature cocoon”) and hatch in the cocoons. After the pupae, they emerge and become adults.

  The “silk gland” in the present specification is a pair of tubular exocrine glands continuous from the discharge port located at the tip of the lower lip of the head to the cephalic duct on both sides of the silkworm larva, the front silk gland, the middle silk thread 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, “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 simpler than before, and the silkworm cell-free protein synthesis method using the extract 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, characterized by using as a material a mutant silkworm strain that does not specifically produce fibroin or a middle silk gland of a hybrid produced from the strain. In silkworm larvae of conventional general varieties (for example, Nishikiaki x Kanwa), fibroin is produced in the posterior silk gland, causing migration and accumulation in the middle silk gland, which is accompanied by enlargement of the middle silk gland and high viscoelasticity. Therefore, it was unsuitable as a material for preparing an extract. On the other hand, the mutant silkworm strain that does not specifically produce fibroin or the middle silk gland of a hybrid produced from that strain does not have viscoelasticity because it does not accumulate fibroin, although it is enlarged as in the general variety, As a result, it can be used as a material, and an efficient extract preparation can be realized.

Examples of mutant silkworm strains that do not specifically produce fibroin used in the present invention include sericin hope (sericin C), sericin N, Nd 蚕, Nd-s 蚕, and Nd-s ^D蚕. In addition, from the viewpoint of stable breeding, a hybrid produced from these mutant strains that do not specifically produce fibroin is preferred, and a hybrid produced from sericin hope is more preferred.

  Sericin Hope is a cocoon line produced by crossing the "Nd line" with the Chinese cocoon line "CS83" and backcrossing "CS83" (see Japanese Patent No. 3374177). This sericin hope is characterized by abnormalities in fibroin synthesis and degeneration of the posterior silk gland.

  The middle silk gland, which is the extraction target in the present invention, may be derived from any state of silkworm larvae (1st to 5th instar). Moreover, it is not limited to a single state (for example, silkworm larvae at the 5th instar stage), but may be a plurality of states (for example, silkworm larvae at the 3rd, 4th, and 5th instar stages). In addition to the middle silk gland, other tissues (for example, the posterior silk gland) may be included. Of course, it may contain multiple silk glands and other tissues in multiple states. The middle silk gland need not be the entire tissue.

  The silkworm larvae used for the preparation of the middle silk gland extract can be prepared by the method of the present invention without particular limitation as long as they are in the 1st to 5th instar stages. preferable. In the silkworm larvae at the 5th instar stage, the middle silk gland is the most mature among the 1st to 5th instar stages. This is because an extract for cell-free protein synthesis that can synthesize proteins is obtained. In particular, from the viewpoint of actively producing sericin, a component of silk thread, and having high protein synthesis ability, the middle silk gland of silkworm larvae at the 5th instar stage, especially silkworms from 1st to 2nd day at the 5th instar stage. Extraction from the middle silk gland of larvae is preferred.

  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, the silk gland matures significantly every day in the 5th instar silkworm larvae. Then, it can be obtained from about 6 to 7 silkworm larvae.

  In the method for preparing an extract for cell-free protein synthesis according to the present invention, the conventional method for extracting the middle silk gland of a wild type silkworm strain compared to the conventional case of using the rear silk gland or fat body for the preparation of the extract. Even when extracted by the reported method (see JP2007-210902A), it is characterized in that it is possible to easily extract the middle silk line of a silkworm strain that does not specifically produce fibroin. In addition, as described below, the inventors have developed a method for extracting the middle silk gland of a silkworm strain that does not specifically produce fibroin in a shorter time than the conventional method. Specifically, it is characterized in that a cut is made with scissors, a scalpel, etc. in the internode membrane between the third and fourth legs of the abdomen of the silkworm larvae, and the middle silk gland is removed with tweezers from the cut. This can be realized by using a method for extracting the organ to be used. Further, the position where the cut is made may be another place, for example, the internode membrane between the second leg and the third leg of the abdomen. If extraction is performed from the middle silk gland, the other procedures are not particularly limited, but can be performed by the following procedures, for example. First, the silkworm larvae are stopped by placing them on ice for a few minutes. Next, according to the above-described method, a desired tissue is extracted from a silkworm using instruments such as scissors, a scalpel, and tweezers. 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, ground with a mortar previously cooled with liquid nitrogen, and extracted with an extraction liquid. As the extraction solution used here, a conventionally known buffer for extraction can be used without particular limitation, but preferably contains a protease inhibitor, potassium salt, magnesium salt, DTT, glycerol and a buffer. Is used. Particularly preferably, 0.01 mM to 5 mM PMSF, 50 mM to 200 mM potassium acetate, 0.5 mM to 5 mM magnesium acetate, 0.5 mM to 5 mM DTT, 5-40% (v / v) glycerol, and 10 mM An extraction solution containing ~ 50 mM HEPES-KOH (pH 6-8) is used. In this way, first, a liquid material containing an extract from the silkworm middle silk gland is obtained.

  Next, the liquid obtained by the extraction process is centrifuged. The centrifugation is performed under the conditions normally performed in this field (10,000 × g to 50,000 × g, 0 ° C. to 10 ° C., 10 minutes to 60 minutes). As a method for preparing the extract, after performing the centrifugation once, the obtained supernatant is used as the extract. From the viewpoint of removing insoluble components unnecessary for protein synthesis, the supernatant may be centrifuged again under the above conditions, and the resulting supernatant may be used as an extract.

  Furthermore, as a method for preparing the extract of the present invention, the extract obtained by the above centrifugation once or twice is subjected to gel filtration, and a fraction having an absorbance at 280 nm of 10 or more is separated from the filtrate after gel filtration. You may prepare an extract by performing the process to take. Specifically, the following procedure is performed. First, gel filtration is performed on the supernatant after centrifugation. For the gel filtration, for example, a desalting column PD-10 (manufactured by GE Healthcare) can be suitably used. After equilibrating the column with the buffer, the sample may be supplied and eluted with the extraction solution. 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 consideration. In this process, for example, using a device such as DU800 (manufactured by Beckman Coulter, Inc.), the absorbance at 280 nm is measured for each fraction, and fractions having an absorbance of 10 or more are collected. The fraction obtained in this way may be used as the extract.

  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 middle silk gland of the silkworm, the eukaryote, it is possible to synthesize glycoproteins in a cell-free system, and many types of proteins can be synthesized 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. Furthermore, as will be described later, such an extract is much easier to prepare an extract that can be used for cell-free protein synthesis as compared with the preparation of an extract from a conventional silkworm posterior silk gland. Efficient cell-free protein synthesis.

  The content of the extract derived from the silkworm middle silk gland 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 More preferably, it is -100 mg / 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 an extract derived from the silkworm middle silk gland in an amount in 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 extract derived from the silkworm middle silk gland in the whole reaction solution is preferably 0.1 mg / mL to 160 mg / mL in terms of protein concentration, and 3 mg / mL to 60 mg / mL. More preferably, it is prepared to be mL. 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 the silkworm middle silk gland, and if it is mRNA that does not originate from the silkworm middle silk gland, there is no particular limitation on the encoded protein (including peptides). It may be one that encodes a toxic 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 middle silk gland is determined by first isolating and purifying mRNA from the reaction solution for translation system, then reverse transcription It can be determined by synthesizing cDNA with an enzyme, analyzing the base sequence of the obtained cDNA, and comparing it with a 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 translation system is an undesired digestion of mRNA and tRNA during cell-free protein synthesis of the present invention by the RNase derived from the silkworm middle silk gland 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 glycerol is further added. This is because the addition of glycerol has an advantage that components essential for protein synthesis can be stabilized in the translation system synthesis reaction. When glycerol is added, it is usually added so as to be 5 (v / v)% to 20 (v / v)%. In addition, various polyhydric alcohols having the same effect may be suitably added instead of glycerol.

  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)% glycerol, 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 Contains 10 μg / mL to 500 μg / mL foreign mRNA, 10 mM to 50 mM HEPES-KOH (pH 6 to 8) Preferably implemented in so that. 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 exogenous template DNA refers to a template DNA not derived from silkworm tissue, and has at least a base sequence encoding a target protein and a promoter sequence located 5 'upstream thereof. The exogenous template DNA used in the present invention is not particularly limited as long as it is a template DNA not derived from the silkworm middle silk gland, and there are no particular restrictions on the encoded protein (including peptides). It may also have 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. Whether the template DNA contained in the extract is foreign template DNA or template DNA derived from the silkworm middle silk gland is extracted from the extract by phenol-chloroform extraction. 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 reaction solution for transcription / translation system, 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 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 spermidine is less than 0.01 mM, the synthesis rate of mRNA decreases and the amount of mRNA produced decreases, and as a result, the rate of protein synthesis tends to decrease, and spermidine is 100 mM. This is because exceeding the value 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 glycerol is further added. This is because the addition of glycerol has the advantage that components essential for protein synthesis in the transcription / translation system synthesis reaction can be stabilized. When glycerol is added, it is usually added so as to be 5 (v / v)% to 20 (v / v)%. In addition, various polyhydric alcohols having the same effect may be suitably added instead of glycerol.

  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)% glycerol.

  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. If the reaction temperature in the transfer process is less than 10 ° C, the transfer speed tends to decrease. If the reaction temperature in the transfer process exceeds 60 ° C, components essential for the reaction tend to be denatured. is there. 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: Extraction of middle silk gland of silkworm larvae The middle silk gland of silkworm larvae was isolated using seven kinds of hybrids prepared from mutant silkworm strains that did not specifically produce fibroin that reached the second day of the fifth instar stage. went. First, 20-40 heads of each hybrid were chilled on ice and put into a dead state to stop the movement. The silkworm larvae are transferred to a stainless steel bat that has been chilled on ice, and scissors are cut into the internode membrane between the abdominal legs of the third and fourth abdomen, so as not to damage the middle silk gland. It was removed using tweezers. Table 1 shows the time required for extraction from 20-40 silkworm larvae of each hybrid.

全体の平均は、中部絹糸腺の摘出には１頭あたり、約２３秒を要することがわかった。なお、日６０３号、日６０４号、中６０４号、中６０５号は、人工飼料育に適する広食性蚕品種「はばたき」の原種であり、いずれも、人工飼料を良く摂食する多糸量系統である。また、ｌｅｍは、独立行政法人農業生物資源研究所に保存されているｌｅｍ遺伝子を持つ系統を改良した系統である。これら全ての系統は、独立行政法人農業生物資源研究所から入手可能である。

As a whole, it was found that it took about 23 seconds per head to remove the middle silk gland. Incidentally, No. 603, No. 604, No. 604, No. 604, and No. 605 are prototypical varieties of habitats suitable for artificial feed breeding, “Habaki”, all of which are multi-yarn lines that often feed on artificial feed. It is. In addition, lem is an improved strain of the strain having the lem gene stored in the National Institute for Agrobiological Sciences. All these strains are available from the National Institute for Agrobiological Resources.

Comparative Example 1: Extraction of silkworm larvae of silkworm larvae As a comparison with Example 1, the silkworm larvae of silkworm larvae were extracted using a common silkworm variety (Kinkiaki x Kanwa) that reached the fourth day of the 5th infancy. It was. First, silkworm larvae were chilled on ice and put into a dead state and stopped moving. Next, fix with insect pin to corrugated cardboard placed on stainless steel vat, cut vertically into the abdomen using scissors and scalpel, and remove the trachea distributed in the silk gland with tweezers so as not to damage the glandular cells The posterior silk gland was removed by a method of picking the posterior silk gland near the boundary between the middle silk gland and the posterior silk gland with tweezers and lifting the silk gland forward and taking it out. As a result, it was found that the removal of the posterior silk gland requires 50 minutes per 20 heads (about 150 seconds per head). This required time is 6.5 times that of the first embodiment.

Example 2: Preparation of silkworm larvae middle silk gland extract The silkworm larvae were cultured for 1 day using a sericin hope x lem, a hybrid produced from a mutant silkworm strain that does not specifically produce fibroin reaching the age of 5 years. Sampling was performed every day from the 8th day to the 8th day. The sampled silkworm larva was extracted by the method of Example 1 so as not to damage the middle silk gland. The extracted middle silk gland was washed with a sterilized 0.75% NaCl solution, removed with a filter paper, transferred to a mortar previously cooled to −80 ° C., and then −80 ° C. until the extract was prepared. And cooled and stored.

  The extract was prepared as follows. First, liquid nitrogen was added to the middle silk gland in a mortar temporarily stored at −80 ° C., and the mixture was crushed using a pestle while cooling. Table 2 shows the number of silkworm larvae sampled according to the number of days and the time required for extraction and crushing.

Next, the powdered middle silk gland was transferred to a centrifuge tube, and its weight was measured using an electronic balance. Extraction was performed by adding 2/3 amount of a solution for extraction having the following composition to the mixture and stirring well with a spatula.
[Composition of extraction liquid]
・ 20 mM HEPES-KOH (pH 7.4)
・ 100 mM potassium acetate ・ 2 mM magnesium acetate ・ 1 mM DTT
・ 0.5 mM PMSF
・ 10% glycerol

  After extraction, the obtained liquid was centrifuged with a centrifuge (himacCR22GII (Hitachi Koki Co., Ltd.)) at 30,000 × g for 30 minutes at 4 ° C. After centrifugation, only the supernatant was isolated and centrifuged again at 30,000 × g for 10 minutes at 4 ° C. After centrifugation, only the supernatant was isolated and used as the middle silk gland extract. During this time, the materials and reagents were operated while cooling on ice, and gloves were worn so that impurities would not enter. The instruments and reagents used were sterilized.

Reference Example 1: Preparation of template mRNA As template DNA used for cell-free protein synthesis, the expression vector pTD1 for insect cell-free protein synthesis (DDBJ / GenBank / EMBL Accession Number: AB194742) is directly below the BamHI site. In addition, pTD1-βgal into which the β-galactosidase gene was inserted was used. Using this expression plasmid as a template, a primer (pTD1_161-179) having a base sequence shown in SEQ ID NO: 1 in the sequence listing, a primer (pTD1_845-827) having a base sequence shown in SEQ ID NO: 2 in the sequence listing, and a KAPA Taq Extra PCR Kit ( KAPA Bioscience) was used, and PCR was performed by repeating a three-step reaction 30 times at 94 ° C. for 15 seconds, annealing at 50 ° C. for 15 seconds, and extension at 72 ° C. for 4 minutes 30 times. The fragment was amplified. From the reaction mixture, linear DNA was purified using HiYeld Gel / PCR DNA Fragments Extraction Kit (manufactured by RBC Bioscience), subjected to phenol / chloroform extraction treatment, concentrated by ethanol precipitation, and reconstituted in sterile distilled water. Template DNA was prepared by lysis. Next, in vitro transcription reaction was performed by incubating at 37 ° C. for 2 hours using T7 Large Scale RNA Production System (manufactured by Promega) using linear DNA as a template. The reaction solution is applied to a gel filtration column (illustra NICK Columns Sephadex G-50 DNA Grade, manufactured by GE Healthcare) to remove unreacted nucleic acid, ethanol precipitated, redissolved in sterile distilled water and purified mRNA. A solution was obtained.

Experimental Example 1: Cell-free protein synthesis using the extract of Example 2 Each of the middle silk gland extracts prepared in Example 2 and insect cultured cell-free protein synthesis reagent kit Transdirect insect cell (manufactured by Shimadzu Corporation) Using reagents encoding reaction reagents (Reaction Buffer, 4 mM methionine) and β-galactosidase, each reagent was mixed so as to have the following composition, and a cell-free protein (β-galactosidase) synthesis reaction was performed. As a reaction apparatus, an aluminum block thermostat (CTU-Mini (manufactured by Taitec Corporation)) was used. The reaction volume was 12.5 μl. The reaction temperature was 25 ° C. and incubation was performed for 5 hours.
[Composition of reaction solution]
-50 (v / v)% middle silk gland extract-30 mM HEPES-KOH (pH 7.4)
・ 100 mM potassium acetate ・ 1.5 mM magnesium acetate ・ 2 mM DTT
・ 5 (v / v)% Glycerol ・ 0.25 mM ATP
・ 0.1 mM GTP
・ 20 mM creatine phosphate ・ 200 μg / mL creatine kinase ・ 80 μM amino acids (20 types)
・ 0.1 mM EGTA
・ 200μg / mL tRNA
320 μg / mL mRNA

The synthesized β-galactosidase was quantified using β-Glo assay system (Promega). Specifically, the synthetic solution was diluted and added to 10 μl of the diluted solution and 10 μl of the substrate solution of β-Glo assay system and mixed well. The mixed solution was transferred to a 96-well microplate for luminescence measurement (half-area 96 well white plate, manufactured by Greiner), incubated at 25 ° C. for 30 minutes using a low temperature aluminum block thermostatic chamber CTU-Mini, and then a flexible microplate reader (infinite M200, manufactured by Tecan Corporation), and the emission intensity was measured. The amount of synthesis was calculated from the luminescence intensity based on a calibration curve prepared using a purified β-galactosidase standard.
Table 3 shows the amount of β-galactosidase synthesized in the extract prepared according to the number of days. Synthesis was confirmed in the extract at the 1st and 5th days of the 5th instar period.

Experimental Example 2: Effect of freezing of extract In Experimental Example 1, an extract after freezing storage at −80 ° C. was used. Since the synthesis amount of β-galactosidase was low, the influence of freezing of the extract was examined. In this experimental example, using the hybrid “Sericin Hop × Day 604” in Table 1, the highest synthesis amount in the Hybrid “Sericin Hop × Lem” in Table 3 was confirmed. Was used to prepare an extract according to Example 2. The obtained extract was stored as it was (without cryopreservation) or frozen at −80 ° C. overnight, and then cell-free protein synthesis and the amount of synthesis were calculated according to Experimental Example 1. Table 4 shows the amount of β-galactosidase synthesized in the extract frozen and unfrozen at −80 ° C. A high synthesis amount of 1.0 μg / ml was obtained with the non-frozen extract.

Experimental Example 3: Number of breeding days suitable for preparation of extract The high synthesis amount was obtained in the non-frozen extract in Experimental Example 2. Next, the number of breeding days suitable for preparation of the extract was examined using the non-frozen extract. It was. An extract was prepared according to Example 2 using silkworm larvae on the 1st to 5th days of 5th instar of sericin hope x day 604, which is a hybrid. Using the obtained extract in a state where it was not cryopreserved, cell-free protein synthesis and the amount of synthesis were calculated according to Experimental Example 1. Table 5 shows the amount of β-galactosidase synthesized in the extract prepared according to the number of days. In the extract at the 5th infancy, on the 2nd and 3rd days, high synthesis amounts of 0.72 and 0.73 μg / mL were obtained, respectively.

SEQ ID NO: 1:
pTD1 — 161-179 primer base sequence SEQ ID NO: 2
pTD1_845-827 primer base sequence

Claims (7)

A method for producing an extract for cell-free protein synthesis, comprising using the middle silk gland of a mutant silkworm strain that does not specifically produce fibroin. A method for producing an extract for cell-free protein synthesis, comprising using a middle silk gland of a hybrid produced from a mutant silkworm strain that does not specifically produce fibroin. The method for producing an extract for cell-free protein synthesis according to claim 1 or 2, wherein the strain is a sericin hope. The method for producing an extract for cell-free protein synthesis according to claim 1, 2 or 3, wherein the middle silk filament of the silkworm larvae on the first day or the second day of the 5th infancy is used. A cut is made in the internode membrane between the multiple legs of the abdominal section 3 and 4 of the silkworm larva or between the multiple legs of the abdominal section 2 and 3 and the middle silk gland is cut from the cut. The method for producing an extract for cell-free protein synthesis according to any one of claims 1 to 4, wherein the extract is extracted. A cell-free protein synthesis reagent kit comprising an extract for cell-free protein synthesis produced by the method according to any one of claims 1 to 5. A protein synthesis method using an extract for cell-free protein synthesis produced by the method according to any one of claims 1 to 5.