JP2017108697A - Production method of bombyx mori larva heart middle silk gland extract, and acellular protein synthesis method using extract thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To increase the synthesis amount to a practical level in Bombyx mori middle silk gland acellular protein synthesis system.SOLUTION: After the fragmentation of extirpated middle silk gland, sericin which is synthetic inhibitor contained in large quantities in a middle silk gland can be removed by the pressurization process on a substrate or the agitation process of the middle silk gland in an aqueous solution, the acellular protein synthesis system having a synthesis amount of about 20 μg/ml is successfully constructed. Thereby production of middle silk gland extract becomes possible, while maintaining improved workability, and can be served as an utilizable technique.SELECTED DRAWING: Figure 1

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.

  At present, expression systems using living cells such as Escherichia coli, yeast, cultured insect cells and cultured mammalian cells (hereinafter referred to as “cell systems”) are widely used as protein production methods. 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 “cell-free systems”) are known. Such cell-free protein synthesis systems are less susceptible to the limitations of cell-based protein synthesis, and can synthesize proteins without losing the lives of organisms. 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 an extract for cell-free protein synthesis, those derived from Escherichia coli, wheat germ, rabbit reticulocyte, cultured insect cells and the like have been known. The applicant has so far proposed a cell-free protein synthesis method using an extract derived from a silkworm tissue (a silkworm extract) (Patent Documents 1 to 3). In the cell-free protein synthesis method using silkworm extract proposed by us, an extract can be produced 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 glands and fat bodies, which are silkworm organs suitable for the extraction liquid production, are thin and fragile, so the silk wire 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 production 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.

  On the other hand, to obtain silkworm extract, the applicant can collect the middle silk gland of a hybrid from a mutant silkworm strain that does not specifically produce fibroin, so that it can be easily removed because it is thicker than the posterior silk gland. It is possible to obtain an extract for cell-free protein synthesis, which is efficient by using the middle silk gland and has less viscoelasticity than general silkworm varieties and is suitable for handling because it does not accompany fibroin accumulation. As a result, the amount of synthesis is small and it has not been put to practical use. (Patent Document 4)

Japanese Patent No. 4244625 Japanese Patent No. 4244628 Japanese Patent No. 4243762 JP 2014-97056 A

  Up to now, the applicant has focused on silkworm larvae's high silk (protein) production ability, and using an extract of the posterior silk gland which is its production organ, the animal-derived system has a high production ability (30 μg / ml or more). ) Has been developed. This silkworm cell-free protein synthesis system, together with the cell-free system of insect culture cells developed by us, has high synthetic ability derived from animals, and it has been clarified that various post-translational modifications occur like mammals. In the future, expansion of use can be expected. However, in the conventional cell-free protein synthesis system using silkworm larvae silkworm gland extract, the silkworm larvae that are thinner and more fragile than silkworm larvae must be removed during the production of the extract. Therefore, it has not been put to practical use. Therefore, in the previous patent application (Japanese Patent Application Laid-Open No. 2014-97056), in order to improve the workability, a hybrid strain of sericin hope that makes a cocoon made only of sericin is used, and the middle silk gland can be very easily obtained by using it. A method of extraction was found (workability improved by 10 times or more). An extract was produced from the middle silk gland extracted by the method, and a new cell-free protein synthesis system having a synthesis amount of 1.0 μg / ml was constructed. However, the amount of synthesis is still small and it has not been put to practical use. Therefore, in the present invention, the problem to be solved is to improve the synthesis amount to a practical level by examining the extract production conditions.

  The sericin component accumulated in the middle silk gland was contained in the middle silk gland extract produced by the conventional technique, and this sericin component was thought to be the cause of reducing the efficiency of the protein synthesis reaction. In the present invention, a process for removing a sericin component is incorporated in the step of producing the middle silk gland extract. Specifically, the extracted middle silk gland is subjected to fragmentation treatment, followed by stirring treatment in an aqueous solution or pressure treatment on a plastic substrate. In the protein synthesis reaction using the produced middle silk gland extract as a material, it was found that the protein synthesis yield was improved to about 20 μg / ml (in the conventional technique, the yield was about 1 μg / ml). The middle silk gland was easy to extract from silkworm larvae, but had a low synthetic ability. According to the present invention, it is possible to produce a middle silk gland extract having a high synthesis ability while maintaining an improvement in workability.

  In a cell-free protein synthesis system using silkworm larvae middle silk glands, by removing the sericin component from the extracted middle silk glands, a significant increase in synthesis amount can be achieved, and there is a barrier to practical use that the synthesis amount is low Could be removed.

FIG. 1 is a view showing a middle silk gland extracted from a silkworm larva of a mutant silkworm strain (sericin hope) that does not specifically produce fibroin used in the present invention.

  In the present specification, “silk” is synonymous with lepidopterous insects (silk insects) belonging to the family Bombycidae. In its lifetime, “egg (embryo)” (between immediately after spawning and just before hatching), “larva” (hatching) Immediately after the end of wing formation (divided into 1st to 5th stages)), "蛹" (between the end of wing formation and immediately before the emergence), and "adult (wing)" (from immediately after the emergence) (Until death) and each of its lifetime 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 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, “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.

  Hereinafter, the present invention will be described in detail. The present invention uses a mutant silkworm strain that does not specifically produce fibroin or a middle silk gland of a hybrid produced from the strain as a material, and further a treatment for removing a sericin component from a middle silk gland extracted from a silkworm larva It is a manufacturing method of the extract for cell-free protein synthesis characterized by having. 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 producing 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, Furthermore, by removing the sericin component, a significant improvement in the amount of synthesis was achieved, and as a result, an efficient method for producing an extract was 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, Nd-sD 蚕, MCS300, and MNS300. 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 production of the middle silk gland extract can be produced 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 a high ability to synthesize proteins, the middle silk gland of silkworm larvae at the age of 5 years, especially silkworms from 3 days to 6 days at the age of 5 years. 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, 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 producing an extract for cell-free protein synthesis according to the present invention, compared with the conventional method for producing a silkworm gland or a fat body, the conventional silk gland strain is extracted as a middle silk gland. 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, scissors and scalpels were cut into the ventral side of the sixth somite of the silkworm larva to expose the middle silk gland from the body. This can be achieved by using a method for extracting the organ used, which is characterized by extracting the exposed middle silk gland with tweezers or the like.

  Moreover, the position where the incision is made may be another place, for example, the internode membrane between the abdominal third and fourth legs, or the abdominal second and third legs. It may be an interstitial membrane. 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.

In the first aspect of the method for removing sericin which forms the center of the present invention, it is preferable to pressurize the middle silk gland obtained by extraction with a flat plate. The middle silk gland is cut to a length of about 10 to 20 mm with a razor and placed on a flat plastic substrate (made of polystyrene) or the like. The silk gland fragment is sandwiched between two plastic substrates and the adhesive substance (sericin) accumulated in the middle silk gland is squeezed out. The silk gland segment is removed from the plastic substrate, transferred to a washing solution, and stirred with a micropipette. Discard the turbid supernatant of the dispersion and replace with a new wash. Further, transfer to a centrifuge tube, and centrifuge using a centrifuge at 1300 × g for 5 minutes at about 4 ° C. After centrifugation, the supernatant is discarded and the precipitated middle silk gland is isolated.
Advantages of the pressurization treatment are that the protein synthesis ability is high, the waste liquid is not produced, and it takes only a short time, but there is a problem that the work is complicated.

In the second aspect of the method for removing sericin that forms the center of the present invention, it is preferable to stir the middle silk gland obtained by extraction in an aqueous solution. In a state where the middle silk gland is immersed in the washing solution, cut it into a length of about 3 to 5 mm using scissors for dissection and stir well with a micropipette or the like. Discard the turbid supernatant and replace with fresh wash solution. Next, transfer to a glass beaker and stir for about 120 revolutions per minute for 30 minutes with a magnetic stirrer. Dispersion C is transferred to a centrifuge tube and centrifuged using a centrifuge at 1,300 × g for 5 minutes at 4 ° C. After centrifugation, the supernatant is discarded and the precipitated middle silk gland is recovered.
As an advantage of the stirring treatment, automation is possible, but the problem is that the protein synthesis ability is low and a certain stirring time (about 30 minutes) is required.

Next, the extracted tissue can be frozen with liquid nitrogen, then ground using 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-5 mM PMSF, 50 mM-200 mM potassium acetate, 0.5 mM-5 mM magnesium acetate, 0.5 mM-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.
This process of freezing and disrupting cells has the advantage of extracting components necessary for protein synthesis without damage.

Moreover, the manufactured extract can be separately frozen and stored in liquid nitrogen. As a result, the extract can be stored frozen while retaining the activity of the extract and thawed (4 ° C.) from the frozen state at the time of use to initiate the protein reaction.
Conventionally, silk gland extract contains a large amount of fibroin or sericin, and these were denatured into a gel state when frozen, and thus could not be cryopreserved. However, fibroin according to the present invention was not produced and sericin was not produced. The removed extract can be collected as a liquid even by freezing and thawing, so it can be stored for a long time after the extract is manufactured, and it can be stored, transported and transferred in the frozen state. It has become a great advantage over the prior art.

Examples of the cleaning solution used in the present invention include salt-containing aqueous solutions (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, magnesium acetate, sodium acetate, potassium acetate, calcium chloride, etc.) and buffer solutions (phosphate buffer, citrate buffer). , Tris buffer, etc.). For example, in the case of a sodium chloride aqueous solution, the concentration of the sodium chloride aqueous solution is not particularly limited, but is preferably 0.1 to 5%, more preferably 0.5 to 2%, and particularly preferably 0.75 to 0.75. 0.8%. If the distance is larger than this range, cell damage may occur due to the difference in salt concentration inside and outside the cell.
Washing is usually performed by stirring the middle silk gland in the washing solution, discarding the turbid solution supernatant and replacing it with a fresh washing solution. Unless otherwise specified, a sodium chloride aqueous solution is used as a cleaning liquid in the present specification.

The above-mentioned 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 manufacturing method of an extract, after performing this centrifugation once, let the obtained supernatant be an 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.
Further, in the method for producing the extract of the present invention, the extract obtained by the above-mentioned 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 manufacture 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 produce an extract that can be used for cell-free protein synthesis compared to the production of an extract using a conventional silkworm posterior silk gland. And efficient cell-free protein synthesis can be realized.

  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 produced 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 produced 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 produced 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. It is more preferable to manufacture so that it may become 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.

  Prior to the description of the examples, a process diagram showing the relationship between each example and experimental example in the cell-free protein synthesis process using the silkworm larvae middle silk gland extract is described.

[Process diagram]

  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.

Experimental example 1: Middle silk gland excision from silkworm larvae of sericin hope hybrids

Silkworm larvae of a hybrid of sericin hope × day 604 that reached the fifth day of age 5 were prepared. A scissor was cut into the ventral side of the sixth body segment of the silkworm larva to expose the middle silk gland from the body. The exposed middle silk gland was removed with tweezers and immersed in a cleaning solution (0.75% aqueous sodium chloride solution). The middle silk gland was stirred in the washing solution, and the supernatant of the cloudy solution was discarded and replaced with a fresh washing solution. The middle silk gland soaked in this washing solution was used in Examples 1, 2, 5, and Comparative Example 1.

Example 1: Production of middle silk gland subjected to pressure treatment

The middle silk gland obtained in Experimental Example 1 was placed on a flat plastic substrate (made of polystyrene), and the middle silk gland was cut to a length of about 10 to 20 mm with a razor (silk gland fragment A). The fragment A was sandwiched between two plastic substrates (made of polystyrene), and the adhesive substance (sericin) accumulated in the middle silk gland was squeezed out from the silk gland fragment A (silk gland fragment B). The silk gland fragment B was taken out from the plastic substrate, transferred to a washing solution (0.75% aqueous sodium chloride solution), and stirred with a micropipette (dispersion D). The cloudy supernatant of Dispersion D was discarded and replaced with a new washing solution (Dispersion E). Dispersion E was transferred to a centrifuge tube and centrifuged using a centrifuge (himac CR22GII, manufactured by Hitachi Koki Co., Ltd.) at 1,300 × g for 5 minutes at 4 ° C. After centrifugation, the supernatant was discarded and the precipitated middle silk gland could be isolated.

Example 2: Production of middle silk gland subjected to stirring in an aqueous solution

The middle silk gland obtained in Experimental Example 1 was cut into a length of about 3 to 5 mm using a dissecting scissors in a state of being immersed in a washing solution (dispersion A). Dispersion A was thoroughly stirred with a micropipette. The cloudy supernatant was discarded and replaced with a new washing solution (dispersion B). Next, Dispersion B was transferred to a glass beaker and stirred with a magnetic stirrer for 30 minutes at 120 revolutions per minute (Dispersion C). Dispersion C was transferred to a centrifuge tube and centrifuged using a centrifuge (himac CR22GII, manufactured by Hitachi Koki Co., Ltd.) at 1,300 × g for 5 minutes at 4 ° C. After centrifugation, the supernatant was discarded and the precipitated middle silk gland was collected.

Experimental Example 2: Production of middle silk gland extract

The middle silk gland obtained in Example 1 or Example 2 was immersed in liquid nitrogen and frozen, and then ground into powder using a mortar and pestle (powder A). Powder A was transferred to a centrifuge tube and the weight of powder A was measured. Powder A and an extraction liquid having the following composition (1/2 amount of powder A weight) were mixed and stirred with a spatula (liquid A). Liquid A was centrifuged at 30,000 × g for 30 minutes at 4 ° C. After centrifugation, the supernatant containing the middle silk gland extract was collected and used as the middle silk gland extract (extract A).

[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

Reference Example 1: Production of template mRNA

The template DNA used for cell-free protein synthesis was pTD1-βgal. pTD1 is an expression vector for insect cell-free protein synthesis (DDBJ / GenBank / EMBL Accession Number: AB194742), and pTD1-βgal is a gene sequence that encodes a β-galactosidase gene immediately below the translation promoting sequence and between the BamHI sites. Has been inserted. This expression plasmid was digested with restriction enzyme BamHI to obtain linear DNA. The linear DNA was purified and subjected to phenol / chloroform extraction treatment, then concentrated by ethanol precipitation, and redissolved in sterilized distilled water to produce template DNA. Next, template mRNA was synthesized from the template DNA by an in vitro transcription reaction using RiboMAX (registered trademark) T7 Large Scale RNA Production System (Promega). The reaction solution containing the synthesized template mRNA is applied to a gel filtration column (illustra (registered trademark) NICK Columns, manufactured by GE Healthcare) to remove unreacted nucleic acid, followed by ethanol precipitation and re-dissolution in sterile distilled water. Thus, a purified mRNA solution was obtained.

Experimental example 3: Protein-free protein synthesis using middle silk gland extract

Extract A prepared in Experimental Example 2, reaction reagent (Reaction Buffer, 4 mM methionine) of insect cultured cell cell-free protein synthesis reagent kit Transdirect insect cell (manufactured by Shimadzu Corporation), mRNA encoding β-galactosidase (reference) Using Example 1), each reagent was mixed so as to have the following composition, and a cell-free protein synthesis (β-galactosidase) synthesis reaction was performed. The synthesis reaction conditions were a final solution volume of 25 μl, a reaction temperature of 25 ° C., and a reaction time of 5 hours (sample solution A).

[Composition of reaction solution]
50 (v / v)% middle silk gland extract (extract A)
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 species)
0.1 mM EGTA
200 μg / ml tRNA
320 μg / ml mRNA

Experimental Example 4: Method for quantifying β-galactosidase

The synthesis amount of β-galactosidase contained in the sample solution A obtained in Experimental Example 3 is quantified using BetaGlo assay system (Promega) capable of quantifying β-galactosidase by luminescence measurement, and is necessary for preparing a calibration curve As a known standard sample, commercially available β-galactosidase (β-Galactosidase from Escherichia coli, Sigma-Aldrich) was used.

Example 3: Cell-free protein synthesis system produced by the method of Example 1

In the case of Example 1, the synthesis amount of β-galactosidase when the operations of Experimental Examples 1 to 4 were performed was 0.7 μg / ml.

Example 4: Cell-free protein synthesis system produced by the method of Example 2

In the case of Example 2, the synthesis amount of β-galactosidase when the operations of Experimental Examples 1 to 4 were performed was 20.2 μg / ml.

Comparative Example 1: Comparison with conventional method (synthesis amount of β-galactosidase)

A middle silk gland extract was produced as follows by a conventional method (Japanese Patent Laid-Open No. 2014-97056). The middle silk gland obtained in Experimental Example 1 was washed with a 0.75% aqueous sodium chloride solution. Next, the middle silk gland was placed on a filter paper to remove water, and then β-galactosidase was synthesized by the same method as in Experimental Examples 2, 3, and 4. Table 1 shows a comparison result of the amount of β-galactosidase synthesized obtained by the conventional method and the methods of Examples 1 and 2. In Example 2, the amount of synthesis was 200 times higher than that of the conventional method.

Comparative Example 2: Comparison of production time of silk gland extract with conventional method

Table 2 shows a comparison of working time required for producing silkworm larvae silk gland extract for 40 silkworm larvae with Patent Document 4 (Japanese Patent Laid-Open No. 2014-97056).

Example 5: Synthesis of β-galactosidase without glycerol in the case of Example 2

In the case of Example 2, Experimental Examples 1 to 4 were performed. Here, a middle silk gland extract obtained by removing the glycerol component from the composition of the extract solution of Experimental Example 2 was also produced. When glycerol was included in the cell-free protein synthesis system, the amount of synthesis of β-galactosidase when not included was compared, and the difference in the amount of synthesis was about 3% (Table 3).

  In a cell-free protein synthesis system using silkworm extract, a method for producing an extract that has a high protein synthesis ability and can be cryopreserved using a thick and efficient middle silk gland, and cell-free using the extract A protein synthesis method is provided.

Claims (9)

A method for producing an extract for cell-free protein synthesis using a mutant silkworm strain that does not specifically produce fibroin or a middle silk gland of a hybrid thereof, characterized by having a step of removing sericin from the extracted middle silk gland Extract liquid manufacturing method. The above sericin removal step is a pressurizing step of pressurizing the middle silk gland between two plates to squeeze out the sericin component accumulated in the middle silk gland, or the middle silk wire in an aqueous solution or buffer containing salt The method for producing an extract according to claim 1, which is an agitation step for agitation treatment. The method for producing an extract according to claim 1, comprising a freeze-thaw step in which the extract is frozen, stored for a necessary period, and thawed before protein synthesis as a final step of the extract-producing method. The method for producing an extract according to claims 1 to 3, further comprising a freeze crushing step of freezing, crushing, and thawing after the sericin removing step. The extract production method according to claims 1 to 4, wherein the cocoon line is sericin hope. The method for producing an extract according to any one of claims 1 to 5, wherein the middle silk thread to be used is extracted from silkworm larvae on the third to fifth days of the fifth age. An extract for cell-free protein synthesis produced by the method according to claim 1. A cell-free protein synthesis reagent kit comprising an extract for cell-free protein synthesis produced by the method according to claim 1. A protein synthesis method using the cell-free protein synthesis extract produced by the method according to claim 1.