WO2013183627A1 - Protein synthesis method and protein synthesis kit - Google Patents
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- the present invention relates to a protein synthesis method and a protein synthesis kit, and in particular, to a protein synthesis method using a cell-free protein synthesis system and a protein synthesis kit that can be used in this protein synthesis method.
- a so-called cell-free protein synthesis method is known in which an amino acid serving as a substrate, an energy source such as ATP, and a gene of a target protein are added to a cell extract containing factors necessary for protein synthesis, and the protein is synthesized in vitro. ing.
- an energy source such as ATP
- a gene of a target protein are added to a cell extract containing factors necessary for protein synthesis, and the protein is synthesized in vitro. ing.
- cell-free protein synthesis systems systems using extracts prepared from E. coli, insect cells, wheat germ, animal cells and the like are known, and kits are commercially available from several companies.
- the cell-free protein synthesis system (i) can freely set reaction conditions, (ii) is not limited to circular DNA obtained by cloning a target gene in an expression vector, It has the advantage that linear DNA such as PCR products can be used, and (iii) various labeled proteins can be easily synthesized.
- the cell-free protein synthesis system is more expensive than the cell expression system. This is one factor that hinders the spread as a tool that can be easily used.
- Non-Patent Document 1 Nucleoside triphosphates (ATP, GTP, CTP and UTP.
- ATP nucleoside triphosphates
- GTP GTP
- CTP CTP
- UTP UTP
- NTP expensive reagents
- Non-Patent Document 1 In the case of a conjugated system in which DNA is added to a reaction solution as a template and transcription and translation are performed simultaneously in one reaction solution, it is common to add four types of NTPs.
- ATP and GTP not only act as substrates in the transcription reaction of mRNA, but are essential as an energy source for the translation reaction.
- the cell extract contains various enzymes in addition to factors necessary for transcription and translation. By actively utilizing these enzymes, it is possible to synthesize useful substrates and the like from precursors in the system.
- Non-Patent Document 2 discloses a technique in which an amino acid is synthesized from an amino acid precursor using an enzyme in a cell extract in a cell-free protein synthesis system, and this is used for protein synthesis in the cell-free protein synthesis system. ing.
- 20 kinds of stable isotope-labeled amino acids are added, or 4 kinds of amino acids, Trp, Asn, Gln and Cys, which are not included in an algae-derived amino acid mixture.
- protein synthesis is performed by adding.
- Trp, Asn, Gln, and Cys are compared to the other 16 types of amino acids.
- the problem is that it is very expensive.
- Trp, Asn, Gln, and Cys are synthesized in-system by enzymes in the extract from several inexpensive raw materials labeled with stable isotopes, and cell-free protein is obtained.
- Non-Patent Document 3 nucleoside monophosphates (AMP, GMP, CMP, and UMP. These four are collectively referred to as NMP hereinafter) are added instead of NTP, and phospho is used as an energy source.
- NMP nucleoside monophosphates
- Non-Patent Document 4 describes synthesizing ATP from AMP and GTP from GMP by an enzyme
- Non-Patent Document 5 describes synthesizing CTP from CMP by an enzyme
- Non-Patent Document 6 describes the synthesis of ATP from ADP using pyruvate in a cell-free protein synthesis system
- Non-Patent Document 7 describes that cell-free protein synthesis is performed using S12 extract containing expensive NAD and CoA.
- NMP is cheaper than NTP, but when a large amount of protein needs to be synthesized, such as for structural analysis, a larger amount of substrate is required, so further cost reduction of the synthesis system is desired.
- the present invention has been made in view of the above problems, and an object thereof is to provide a method capable of performing protein synthesis at a lower cost in a cell-free protein synthesis system.
- a protein synthesis method is a protein synthesis method using a cell-free protein synthesis system, and comprises a template DNA encoding a protein and adenosine, guanosine, uridine and cytidine. At least one ribonucleoside is added to a reaction solution containing a cell extract for protein synthesis.
- a protein synthesis kit according to the present invention is a protein synthesis kit used in the above-described protein synthesis method in order to solve the above problems, and is at least one of adenosine, guanosine, uridine and cytidine. It contains a seed ribonucleoside and a cell extract.
- a protein can be synthesized in a cell-free protein synthesis system without adding NTP as a transcription reaction substrate and NMP as a precursor thereof.
- the protein synthesis method according to the present invention is a protein synthesis method using a cell-free protein synthesis system, and comprises a template DNA encoding the protein and at least one ribonucleoside of adenosine, guanosine, uridine and cytidine. Can be added to the reaction solution containing the cell extract for protein synthesis, and other specific steps, and the equipment and apparatus to be used are not particularly limited.
- the “cell-free protein synthesis system” is a system for synthesizing proteins by reconstituting a transcription and translation reaction system in an artificial container such as a test tube using a cell extract.
- a cell extract factors necessary for transcription of RNA contained in cells and DNA as a template, and factors necessary for protein translation are disrupted and taken out as an extract.
- “for protein synthesis” is intended to include not only the synthesis of a protein based on information encoded in RNA but also the synthesis of RNA used for protein synthesis using DNA as a template. is doing.
- cell extracts for constructing a cell-free protein synthesis system cell extracts derived from various species can be used, for example, extracts of bacterial cells such as Escherichia coli and thermophilic bacteria, and wheat germ, Extracts of eukaryotic cells such as rabbit reticulocytes, mouse L-cells, Ehrlich ascites tumor cells, HeLa cells, CHO cells and budding yeast can be used.
- the cell extract of a bacterial cell can be used suitably, The cell extract of E. coli is more preferable.
- E. coli S30 extract contains all the E. coli enzymes and factors required for transcription and translation.
- E. coli is first cultured, and the cells are collected by centrifugation or the like.
- the collected bacterial cells are washed, resuspended in a buffer, and crushed using a French press, glass beads, Waring blender, or the like.
- the disrupted E. coli insoluble material is removed by centrifugation, mixed with the preincubation mixture and incubated.
- Endogenous DNA and RNA are degraded by this operation, but the endogenous nucleic acid may be further degraded by adding a calcium salt, a micrococcal nuclease, or the like.
- endogenous amino acids, nucleic acids, nucleosides and the like are removed by dialysis, and dispensed in appropriate amounts and stored at liquid nitrogen or at -80 ° C.
- rabbit reticulocyte lysate and wheat germ extract are preferable.
- Rabbit reticulocyte lysate can use the method described by Pelham and Jackson (non-patent literature: Pelham, H.R.B. and Jachson, R.J. Eur. J. Biochem., 67, 247-256, 1976).
- a method for producing a wheat germ extract for example, a method described by Roberts and Paterson (Non-patent literature: Roberts, BE and Paterson, BM, Proc. Natl. Acad. Sci. USA, 70, 2330-2334, 1973) Can be used.
- the amount of the cell extract in the reaction solution is preferably 10 to 50%, more preferably 20 to 30%.
- each cell extract contains all the enzymes and factors necessary for transcription and translation, but when used as a reaction solution for protein synthesis, supplementary components and a buffer can be further added.
- At least one ribonucleoside of adenosine, guanosine, uridine and cytidine is added to a reaction solution containing a cell extract for protein synthesis.
- At least any one ribonucleoside of adenosine, guanosine, uridine and cytidine is added to the reaction solution containing the cell extract, more preferably at least any two ribonucleosides are added. More preferably, at least any three ribonucleosides are added.
- reaction formulas (1) to (3) show the ATP biosynthesis pathway, the GTP biosynthesis pathway, and the CTP and UTP biosynthesis pathway of Escherichia coli, respectively.
- E. coli has an enzyme group that sequentially synthesizes NMP, NDP (nucleoside diphosphate) and NTP from ribonucleosides as shown in the following formulas (1) to (3). These enzyme groups are also contained in cell extracts prepared by disrupting bacteria. Therefore, by adding a ribonucleoside such as adenosine or the like instead of NTP to the cell extract, it becomes possible to biosynthesize NTP using these enzyme groups and use it for transcription reaction and translation reaction. (Reaction Formula 1)
- the cell extract is not limited to the cell extract derived from E. coli, but cell extracts derived from other species can also be used in the protein synthesis method using the cell-free protein synthesis system according to the present invention. .
- guanosine As the type of ribonucleoside to be added, at least guanosine is preferably contained, and guanosine and cytidine are more preferably contained. Even when only guanosine is added, a protein synthesis amount comparable to that obtained when NTP is added can be obtained as long as the amount is a certain amount or more. When guanosine and cytidine are added, the amount of synthesis is the same as when all four types of ribonucleosides are added with a smaller amount of addition, and a higher synthesis amount is obtained than when NTP is added. be able to.
- the amount of ribonucleoside to be added is preferably 0.1 to 8 mM, and more preferably 0.2 to 1.6 mM.
- NTP is very expensive because it is a high-purity product produced by an enzymatic method.
- ribonucleosides that are precursors of NTP are very inexpensive compared to NTP. For example, when comparing prices per gram, ribonucleosides are approximately 1/20 to 1/340 of the corresponding NTP.
- the molar amount of NTP (0.8-1.2 mM) added for general cell-free protein synthesis is greater than the preferred molar amount (0.4 mM) of ribonucleoside added in the present invention.
- NMP cell-free protein synthesis system using NMP which is cheaper than NTP instead of NTP
- commercially available NMP is also known as a precursor ribonucleoside. It is expensive in comparison.
- the molar amount (0.8 to 1.2 mM) of NMP added for cell-free protein synthesis is larger than the preferred molar amount of ribonucleoside added in the present invention.
- the template DNA may be a double-stranded DNA containing a gene sequence encoding a desired protein to be expressed and an appropriate expression control region, and may be either linear or circular. Also good.
- the expression control region may include a promoter sequence, terminator sequence, enhancer sequence, poly A addition signal, ribosome binding sequence, and the like.
- the template DNA can be designed so that a fusion protein incorporating an affinity tag (tag) sequence can be synthesized.
- the amount of template DNA to be added is preferably 0.1 to 50 ⁇ g / mL, and more preferably 1 to 10 ⁇ g / mL.
- an energy regeneration system component to the reaction solution containing the cell extract.
- an energy regeneration system component for example, the ATP regeneration system by the combination of creatine kinase and creatine phosphate, the combination of phosphoenolpyruvate and pyruvate kinase, etc. can be used. Creatine kinase and pyruvate kinase are both enzymes that regenerate ADP into ATP, and require creatine phosphate and phosphoenolpyruvate as substrates, respectively.
- Creatine kinase is preferably used at 0.02 to 5 mg / mL, and creatine phosphate is preferably used at 10 to 100 mM.
- ATP using phosphoenolpyruvate and NAD or the like called PANOxSP described in Jewett et al.
- oxygen in the atmosphere is required, so that the reaction solution is in contact with the air. It is necessary to carry out the reaction in a container.
- the ATP regeneration system using a combination of creatine kinase and creatine phosphate there is no influence on the protein productivity due to the presence or absence of oxygen supply. Therefore, in the case of a synthetic system using a combination of creatine kinase and creatine phosphate as the ATP regeneration system, it is not necessary to use a special container for the reaction, and therefore it is easy to scale up for mass preparation. .
- Reaction solutions include cell extracts, template DNA and ribonucleosides, and energy regeneration systems such as creatine kinase and phosphoenolpyruvate, as well as buffers, salts, amino acids that serve as substrates, nucleolytic enzyme inhibitors, antibacterial agents , Reducing agents, polyethylene glycol, cAMP, folic acids, RNA polymerase, tRNA, and the like.
- a buffer such as HEPES-KOH and Tris-OAc can be used.
- salts that can be used include acetates such as potassium acetate, magnesium acetate and ammonium acetate, and glutamates such as potassium glutamate.
- the amino acid used as a substrate is 20 kinds of natural amino acids constituting the protein and derivatives thereof.
- the antibacterial agent for example, sodium azide and ampicillin can be used.
- the RNA polymerase commercially available enzymes such as T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase can be used, and may be appropriately selected so as to match the promoter sequence of the template DNA.
- the protein synthesis kit is a kit used in the above-described protein synthesis method, and comprises at least one ribonucleoside of adenosine, guanosine, uridine and cytidine, and a cell extract. Is included.
- Protein synthesis As a protein synthesis method according to the present invention, a dialysis method and a batch method can be applied.
- the dialysis method is a synthesis method using a closed system that can be shaken or stirred and contains an internal solution as a reaction solution and an external solution containing a reaction substrate separated by a dialysis membrane (ultrafiltration membrane).
- the substrate necessary for synthesis is supplied from the external solution to the reaction solution through the dialysis membrane, and the extra by-product in the reaction solution is diffused into the external solution, so that the reaction can be performed for a longer time. Can last. Therefore, a higher protein synthesis amount can be obtained, and a large amount (milligram unit) of protein can be prepared.
- the batch method is a synthesis method in which all necessary components are uniformly mixed and contained in the reaction solution to perform the reaction.
- the amount of protein synthesis is small compared to dialysis, the reaction time is shorter, results are obtained quickly, and the synthesis reaction can be performed in a general 96-well plate. It can be said that it is a suitable synthesis system.
- the protein synthesis reaction lasts longer than in the synthesis system using NTP. Therefore, when the protein synthesis method according to the present invention is realized by a batch method, not only can a protein synthesis be realized at a lower cost than a synthesis system using NTP, but also a higher protein synthesis amount. Obtainable. Note that such effects are not observed in the synthesis system using NMP.
- the method is not limited to the method of adding all four types of ribonucleosides, and may be a method of adding only one type of guanosine, or a method of adding only guanosine and cytidine.
- the batch method even when only guanosine is added, depending on the addition amount, a protein synthesis amount comparable to that of a synthesis system using NTP can be realized.
- the amount of protein synthesis exceeding the synthesis system using NTP can be realized, which is the same as when all four types of ribonucleosides are added. A degree of protein synthesis can be achieved.
- the amount of guanosine is preferably 0.1 to 8 mM, more preferably 1 to 4 mM.
- the amounts of guanosine and cytidine are each preferably 0.1 to 8 mM, more preferably 0.2 to 1.6 mM.
- the purification of the synthesized protein is relatively easy because it does not need to be disrupted unlike the case of using live cells, and the amount and type of substances mixed in is significantly less than the separation from live cells. It can be carried out.
- the purification method conventionally known methods can be used alone or in appropriate combination depending on the properties of the protein. Examples include conventional techniques such as ammonium sulfate or acetone precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration chromatography, HPLC, electrophoresis, and chromatofocusing. .
- identification and quantification of the synthesized protein can be performed by comparing with a standard sample, if necessary, by activity measurement, immunological measurement, spectroscopic measurement, amino acid analysis, and the like.
- the protein synthesis method according to the present invention is a protein synthesis method using a cell-free protein synthesis system, and is a template DNA encoding the protein and at least one of adenosine, guanosine, uridine and cytidine.
- One type of ribonucleoside is added to a reaction solution containing a cell extract for protein synthesis.
- the protein synthesis method according to the present invention it is preferable to add at least any two ribonucleosides among adenosine, guanosine, uridine and cytidine.
- the cell extract is preferably an extract prepared from bacterial cells.
- the cell extract is preferably an extract prepared from E. coli.
- creatine kinase and creatine phosphate are preferably added to the reaction solution as components of the ATP regeneration system.
- the protein synthesis method according to the present invention may be a dialysis synthesis method.
- the protein synthesis method according to the present invention may be a batch synthesis method.
- a protein synthesis kit according to the present invention is a protein synthesis kit used in the above-described protein synthesis method, and comprises at least one ribonucleoside of adenosine, guanosine, uridine and cytidine, and cell extraction. Liquid.
- Example 1 Synthesis of CAT protein by batch method
- the reaction solution composition of the cell-free synthesis system by the batch method is as follows: 60 mM HEPES-KOH (pH 7.5), 230 mM potassium D-glutamate, 2% PEG8000, 3 mM DTT, 0.4 mM adenosine, 0.4 mM Guanosine, 0.4 mM cytidine, 0.4 mM uridine, 36 ⁇ g / mL folinic acid, 80 mM creatine phosphate, 150 ⁇ g / mL E.
- coli tRNA 13 mM magnesium acetate, each 1.5 mM protein constituent amino acids (however, Cys, Ser, Arg, 4.5 mM for Trp, Asn, and Gln), 50 ⁇ g / mL creatine kinase, 100 ⁇ g / mL T7 RNA polymerase, 4 ⁇ g / mL template DNA, and 24% E. coli S30 extract.
- PK7-CAT was used as template DNA (Non-patent literature: Kim DM, Kigawa T, Choi CY, Yokoyama S (1996) EurJ Biochem 239: 881-886).
- This plasmid is composed of a T7 promoter, a ribosome binding sequence, a CAT (chloramphenicol acetyltransferase) gene, and a T7 terminator.
- the Escherichia coli S30 extract was prepared by the method of Zubay (non-patent literature: Zubay G., Ann Rev Genet, (1973) 7, 267-287) and the method of Kigawa et al. (Non-patent literature: Kigawa T, Yabuki T, , Matsuda T, Nakajima R, Tanaka A, Yokoyama S. (2004) J Struct Funct Genomics 5, 63-68), and prepared from Escherichia coli BL21 codon-plus RIL strain (Stratagene).
- Cytidine (Sigma) and uridine (Sigma) were dissolved in MilliQ water to prepare a 100 mM solution. Since adenosine (Nacalai Tesque) and guanosine (Wako Pure Chemical Industries, Ltd.) have low solubility in water (about 0.2 mM), MilliQ water was added to prepare a 100 mM suspension and used in the experiment. .
- 1.3 mM ATP, 0.9 mM GTP, 0.9 mM CTP, and 0.9 mM UTP are added instead of adenosine, guanosine, cytidine, and uridine.
- the reaction was performed at 30 ° C.
- FIG. 1 is a diagram showing the relationship between reaction time and synthesis amount.
- the protein synthesis reaction was stopped in about 1 hour.
- the protein synthesis reaction lasted for 2 hours.
- the synthetic system to which ribonucleoside was added about 1.4 times as much CAT protein was synthesized as compared to the synthetic system to which NTP was added. From the above, it was shown that by using ribonucleoside instead of NTP, not only protein synthesis in a cell-free protein synthesis system is possible, but also the amount of synthesis can be increased as compared with the case of using NTP.
- Example 2 Synthesis of EGFP protein, UBA protein, Ras protein, and ⁇ -galactosidase protein
- EGFP protein molecular weight 34 kDa
- UBA protein molecular weight 10 kDa
- Ras protein molecular weight 24 kDa
- ⁇ -galactosidase protein molecular weight 119 kDa
- the reaction was performed at 30 ° C. for 4 hours.
- the protein of interest is bound to the TALON resin.
- a washing step of adding 150 ⁇ L of A buffer and centrifuging at 1500 rpm for 1 minute was performed three times.
- 50 ⁇ L of B buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 300 mM imidazole) was added, and the mixture was shaken and stirred for 3 minutes, and then centrifuged at 1500 rpm for 1 minute to carry out an elution step twice to obtain a crude protein. .
- Quantification of EGFP protein, UBA protein, Ras protein, and ⁇ -galactosidase protein Quantification of each crude protein of EGFP protein, UBA protein, Ras protein, and ⁇ -galactosidase protein was performed by the Bradford method using Protein assay reagent (Bio-Rad) with bovine serum albumin (BSA) as a standard substance. It was. The crude protein was subjected to SDS-polyacrylamide gel electrophoresis to confirm the molecular weight. The results are shown in FIGS.
- FIG. 2 is a graph showing the yield of each protein quantified by the Bradford method.
- FIG. 3 shows the results of electrophoresis.
- Lanes A to D show pCR2.1 NHis-EGFP, pCR2.1 N11-UBA, pK7b2 NHis-Ras and pUC19 N11- ⁇ -galactosidase as template DNAs, respectively.
- Lane E is a crude product derived from a control synthesis system that does not contain template DNA in the synthesis system.
- the protein yield was about 1.2 to 1.4 times that of the synthetic system to which NTP was added. A high yield was obtained. Further, as shown in FIG.
- the protein synthesized in the synthesis system to which ribonucleoside was added was a protein having the molecular weight of the target protein, as in the case of the synthesis system to which NTP was added. From the above, various proteins ranging from very small proteins to high molecular weight proteins exceeding 100 kDa can be synthesized correctly by using ribonucleosides instead of NTP, and also increase the yield compared to using NTP. It was shown that can be made.
- Example 3 Evaluation by type of ribonucleoside to be added
- Example 1 (Synthetic reaction conditions) Example 1 except that pCR2.1 NHis GFP-S1 was used as the template DNA and any one, two or three of the four ribonucleosides were added at 0.1 to 1.6 mM each. The same.
- the plasmid pCR2.1 NHis GFP-S1 has a sequence encoding the GFP-S1 protein, has a T7 promoter and a ribosome binding sequence upstream of this sequence, and has a T7 terminator downstream.
- Non-patent literature See Seki E, Matsuda N, Yokoyama S, Kigawa T. (2008) Anal Biochem. 377 (2): 156-161.).
- the reaction was performed at 30 ° C. for 3 hours.
- FIG. 4 is a diagram showing the fluorescence intensity derived from the GFP-S1 protein in each synthetic system, and (a) shows the results in the synthetic system to which at least adenosine and guanosine are added as ribonucleosides, (b) Shows the result in a synthetic system in which at least adenosine is added as a ribonucleoside, (c) shows the result in a synthetic system in which at least guanosine is added as a ribonucleoside, (d) The results are shown in a synthetic system in which neither adenosine nor guanosine is added as a ribonucleoside.
- Example 4 Synthesis of CAT protein by dialysis
- CAT protein was synthesized by a batch method, but synthesis of CAT protein was also attempted by a dialysis method.
- the internal solution composition of the cell-free synthesis system by dialysis is as follows: 60 mM HEPES-KOH (pH 7.5), 230 mM potassium D-glutamate, 4% PEG8000, 3 mM DTT, 0.05% NaN 3 , adenosine Guanosine, cytidine, uridine, 36 ⁇ g / mL folinic acid, 80 mM creatine phosphate, 175 ⁇ g / mL E.
- coli tRNA 10 mM magnesium acetate, each 1.5 mM protein constituent amino acid, 100 ⁇ g / mL creatine kinase, 60 ⁇ g / mL T7 RNA polymerase, 1 ⁇ g / mL template DNA and 30% E. coli S30 extract.
- composition of the external solution is as follows: 60 mM HEPES-KOH (pH 7.5), 230 mM potassium D-glutamate, 4% PEG8000, 3 mM DTT, 0.05% NaN 3 , adenosine, guanosine, cytidine, uridine 36 ⁇ g / mL folinic acid, 80 mM creatine phosphate, 10 mM magnesium acetate, 1.5 mM protein constituent amino acids, 30% E. coli S30 buffer.
- the concentrations of adenosine, guanosine, cytidine, and uridine are 0.2 mM, 0.4 mM, 0.8 mM, 1.2 mM, or 1.6 mM for both the internal solution and the external solution.
- 1.3 mM ATP, 0.9 mM GTP, 0.9 mM CTP, and 0.9 mM UTP were used instead of adenosine, guanosine, cytidine, and uridine in both the inner and outer solutions. It has been added.
- PK7-CAT was used as the template DNA.
- E. coli S30 extract and cytidine, uridine, adenosine and guanosine were prepared in the same manner as in Example 1.
- the reaction was carried out at 30 ° C. for 14 hours.
- FIG. 5 is a diagram comparing the amount of CAT protein synthesis. As shown in FIG. 5, when 0.8 to 1.6 mM ribonucleoside was added, 80 to 103% of CAT protein was synthesized when NTP was added. That is, it was shown that the same degree of protein can be synthesized using ribonucleosides even in the dialysis method used for mass preparation in milligram units.
- Example 5 Confirmation of NTP synthesis
- a reaction solution to which neither template DNA nor T7 RNA polymerase was added was prepared, and 30 ⁇ l each of a sample before the start of the reaction and a sample after 30 minutes from the start of the reaction were collected.
- An equal amount of% TCA was added and ice-cooled. This was centrifuged at 12,000 rpm for 5 minutes, and 1 ⁇ L of 2 mM NAD was added as an internal standard to 39 ⁇ L of the obtained supernatant.
- Each nucleoside and each NTP contained in 20 ⁇ L of this solution were detected by high performance liquid chromatography (HPLC) connected to a C18 column. The results are shown in FIG.
- FIG. 7 (a) shows the result in the sample before the start of the reaction
- FIG. 7 (b) shows the result in the sample 30 minutes after the start of the reaction.
- the peaks of adenosine, guanosine, cytidine and uridine detected immediately after the start of the reaction are greatly reduced 30 minutes after the start of the reaction, while ATP, GTP, CTP and The UTP peak increased. This confirmed that ATP, GTP, CTP and UTP were synthesized from adenosine, guanosine, cytidine and uridine in the synthesis system.
- Components of the energy regeneration system include (i) creatine phosphate (CP) and creatine kinase (CK), (ii) 30 mM PEP, 0.33 mM NAD, 0.26 mM CoA and 2.7 mM sodium oxalate, or (iii) Example 1 except that 30 mM glucose, 0.33 mM NAD, 0.26 mM CoA and 10 mM K 2 HPO 4 were used, and pCR2.1 NHis-EGFP was used instead of pK7-CAT. Protein synthesis was performed. Since pyruvate kinase is contained in the E.
- the obtained fluorescence value was about 90% of that when NTP was used in a system containing CP and CK, whereas CP and CK were included.
- the fluorescence value obtained was about 1.2 times that obtained when NTP was used in the system containing CP and CK.
- the present invention can be widely applied to industries in the life science field including the pharmaceutical field and the health medicine field, which require protein synthesis, functional analysis, and structural analysis.
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Abstract
To provide a less expensive, simpler cell-free protein synthesizing system, this protein synthesis method synthesizes protein by a cell-free protein synthesizing system; wherein template DNA that encodes a protein, and at least any one ribonucleotide among adenosine, guanosine, uridine, and cytidine, are added to a reaction solution containing cell extract for protein synthesis.
Description
本発明は、タンパク質の合成方法、およびタンパク質の合成キットに関し、詳細には、無細胞タンパク質合成系によるタンパク質の合成方法、およびこのタンパク質の合成方法に利用できるタンパク質の合成キットに関する。
The present invention relates to a protein synthesis method and a protein synthesis kit, and in particular, to a protein synthesis method using a cell-free protein synthesis system and a protein synthesis kit that can be used in this protein synthesis method.
タンパク質合成に必要な因子を含む細胞抽出液に、基質となるアミノ酸、ATPなどのエネルギー源、および目的タンパク質の遺伝子を加え、試験管内でタンパク質を合成する、いわゆる無細胞タンパク質合成方法が従来知られている。無細胞タンパク質合成系には、大腸菌、昆虫細胞、小麦胚芽および動物細胞等から調製した抽出液を用いた系が知られており、キット化されたものが数社から市販されている。
A so-called cell-free protein synthesis method is known in which an amino acid serving as a substrate, an energy source such as ATP, and a gene of a target protein are added to a cell extract containing factors necessary for protein synthesis, and the protein is synthesized in vitro. ing. As cell-free protein synthesis systems, systems using extracts prepared from E. coli, insect cells, wheat germ, animal cells and the like are known, and kits are commercially available from several companies.
細胞を用いた合成系である細胞発現系と比較すると、無細胞タンパク質合成系では、(i)反応条件を自由に設定できる、(ii)発現ベクターに目的遺伝子をクローニングした環状DNAに限らず、PCR産物等の直鎖状DNAも利用できる、および(iii)様々な標識タンパク質を容易に合成できる、といった強みがある。その反面、一部に高価な試薬類を用いるため、無細胞タンパク質合成系は、細胞発現系よりも高コストであることが弱みとされている。この点が、気軽に使用できるツールとしての広がりを妨げるひとつの要因となっている。
Compared with the cell expression system, which is a cell-based synthesis system, the cell-free protein synthesis system (i) can freely set reaction conditions, (ii) is not limited to circular DNA obtained by cloning a target gene in an expression vector, It has the advantage that linear DNA such as PCR products can be used, and (iii) various labeled proteins can be easily synthesized. On the other hand, since some expensive reagents are used, it is considered that the cell-free protein synthesis system is more expensive than the cell expression system. This is one factor that hinders the spread as a tool that can be easily used.
無細胞タンパク質合成系における反応液の構成成分のうち、高価な試薬類の一つとして、ヌクレオシド3リン酸(ATP、GTP、CTPおよびUTP。以下、この4つを総称してNTPと呼ぶ。)が挙げられる(非特許文献1)。反応液にDNAを鋳型として添加し、転写および翻訳をひとつの反応液中で同時に行う共役系の場合では、4種類のNTPを加えることが一般的である。また、ATPおよびGTPについては、mRNAの転写反応における基質として働くだけではなく、翻訳反応のエネルギー源としても必須である。そのため、予め調製したmRNAを鋳型として反応液に加えて翻訳のみを行う合成系であっても、4種類のNTPのうち、通常ATPおよびGTPを添加する必要がある。すなわち、転写および翻訳の両方が含まれる合成系ならびに翻訳のみを行う合成系いずれにおいても、市販の高価なNTPを用いることによるコスト高という共通の課題が存在する。
Among the components of the reaction solution in the cell-free protein synthesis system, nucleoside triphosphates (ATP, GTP, CTP and UTP. Hereinafter, these four are collectively referred to as NTP) as one of expensive reagents. (Non-Patent Document 1). In the case of a conjugated system in which DNA is added to a reaction solution as a template and transcription and translation are performed simultaneously in one reaction solution, it is common to add four types of NTPs. In addition, ATP and GTP not only act as substrates in the transcription reaction of mRNA, but are essential as an energy source for the translation reaction. Therefore, even in a synthetic system that performs translation only by using pre-prepared mRNA as a template, it is usually necessary to add ATP and GTP among the four types of NTP. That is, both the synthesis system including both transcription and translation and the synthesis system that performs only translation have a common problem of high cost by using commercially available expensive NTP.
ところで細胞抽出液には、転写および翻訳に必要な因子以外にも様々な酵素が含まれている。これらの酵素を積極的に利用することによって、前駆体から有用な基質等を系内合成することが可能である。
By the way, the cell extract contains various enzymes in addition to factors necessary for transcription and translation. By actively utilizing these enzymes, it is possible to synthesize useful substrates and the like from precursors in the system.
例えば、非特許文献2には、無細胞タンパク質合成系において細胞抽出液中の酵素を用いてアミノ酸前駆体からアミノ酸を合成し、これを無細胞タンパク質合成系におけるタンパク質合成に利用した技術が開示されている。NMR解析用のタンパク質試料の調製には、20種類の安定同位体標識アミノ酸を加えるか、もしくは藻類由来のアミノ酸混合物に対して、それには含まれないTrp、Asn、GlnおよびCysの4種類のアミノ酸を加えてタンパク質合成を行うことが一般的である。20種類の安定同位体標識アミノ酸を用いる方法よりも、藻類由来のアミノ酸混合物に4種類のアミノ酸を加える方法がより安価であるものの、Trp、Asn、GlnおよびCysは他の16種類のアミノ酸に比べて非常に高価であることが問題となっていた。この問題を解決するために、非特許文献2では、安定同位体標識された安価な数種類の原料から、Trp、Asn、GlnおよびCysを抽出液中の酵素によって系内合成して、無細胞タンパク質合成系におけるタンパク質合成に供する技術を報告している。
For example, Non-Patent Document 2 discloses a technique in which an amino acid is synthesized from an amino acid precursor using an enzyme in a cell extract in a cell-free protein synthesis system, and this is used for protein synthesis in the cell-free protein synthesis system. ing. For preparing a protein sample for NMR analysis, 20 kinds of stable isotope-labeled amino acids are added, or 4 kinds of amino acids, Trp, Asn, Gln and Cys, which are not included in an algae-derived amino acid mixture. In general, protein synthesis is performed by adding. Although the method of adding 4 types of amino acids to an algae-derived amino acid mixture is cheaper than the method of using 20 types of stable isotope-labeled amino acids, Trp, Asn, Gln, and Cys are compared to the other 16 types of amino acids. The problem is that it is very expensive. In order to solve this problem, in Non-Patent Document 2, Trp, Asn, Gln, and Cys are synthesized in-system by enzymes in the extract from several inexpensive raw materials labeled with stable isotopes, and cell-free protein is obtained. We have reported the technology used for protein synthesis in the synthesis system.
また例えば、非特許文献3には、NTPの代わりにヌクレオシド一リン酸(AMP、GMP、CMPおよびUMP。以下、この4つを総称してNMPと呼ぶ。)を添加するとともに、エネルギー源としてホスホエノールピルビン酸の代わりにグルコースを反応液に加えて、代謝酵素を利用することによってNTPを系内合成して、これを無細胞タンパク質合成系に利用したタンパク質合成方法が報告されている。
Further, for example, in Non-Patent Document 3, nucleoside monophosphates (AMP, GMP, CMP, and UMP. These four are collectively referred to as NMP hereinafter) are added instead of NTP, and phospho is used as an energy source. There has been reported a protein synthesis method in which glucose is added to a reaction solution instead of enolpyruvic acid and NTP is synthesized in the system by using a metabolic enzyme, and this is used in a cell-free protein synthesis system.
また、酵素によってAMPからATPを、GMPからGTPを合成することが非特許文献4に記載されており、酵素によってCMPからCTPを合成することが非特許文献5に記載されている。また、非特許文献6には、無細胞タンパク質合成系において、ピルビン酸塩を用いてADPからATPを合成することが記載されている。非特許文献7には、高価なNADおよびCoAが含まれているS12 extractを用いて無細胞タンパク質合成を行うことが記載されている。
Further, Non-Patent Document 4 describes synthesizing ATP from AMP and GTP from GMP by an enzyme, and Non-Patent Document 5 describes synthesizing CTP from CMP by an enzyme. Non-Patent Document 6 describes the synthesis of ATP from ADP using pyruvate in a cell-free protein synthesis system. Non-Patent Document 7 describes that cell-free protein synthesis is performed using S12 extract containing expensive NAD and CoA.
NMPはNTPと比較して安価であるものの、構造解析など大量のタンパク質を合成する必要がある場合には、より多くの基質が必要であるため、合成系のさらなる低コスト化が望まれている。
NMP is cheaper than NTP, but when a large amount of protein needs to be synthesized, such as for structural analysis, a larger amount of substrate is required, so further cost reduction of the synthesis system is desired. .
そこで、本発明は上記の問題点に鑑みてなされたものであり、その目的は、無細胞タンパク質合成系において、より安価にタンパク質合成を行うことができる方法を提供することにある。
Therefore, the present invention has been made in view of the above problems, and an object thereof is to provide a method capable of performing protein synthesis at a lower cost in a cell-free protein synthesis system.
本願発明者らが鋭意検討した結果、細胞抽出液を含む反応液により安価なリボヌクレオシドを添加した合成系であっても、さらには、1種類のリボヌクレオシドのみを添加した場合であっても、NTPを添加した合成系と同程度のタンパク質合成量を得ることができることを見出し、本願発明を完成させるにいたった。
As a result of intensive studies by the inventors of the present application, even in a synthesis system in which an inexpensive ribonucleoside is added by a reaction solution containing a cell extract, or even when only one type of ribonucleoside is added, It was found that the same amount of protein synthesis as in the synthesis system to which NTP was added could be obtained, and the present invention was completed.
本発明に係るタンパク質の合成方法は、上記課題を解決するために、無細胞タンパク質合成系によるタンパク質の合成方法であって、タンパク質をコードする鋳型DNAと、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドとを、タンパク質合成用の細胞抽出液を含む反応液に添加することを特徴とする。
In order to solve the above problems, a protein synthesis method according to the present invention is a protein synthesis method using a cell-free protein synthesis system, and comprises a template DNA encoding a protein and adenosine, guanosine, uridine and cytidine. At least one ribonucleoside is added to a reaction solution containing a cell extract for protein synthesis.
本発明に係るタンパク質の合成キットは、上記課題を解決するために、上述のタンパク質の合成方法に使用されるタンパク質の合成キットであって、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドと、細胞抽出液とを含むことを特徴とする。
A protein synthesis kit according to the present invention is a protein synthesis kit used in the above-described protein synthesis method in order to solve the above problems, and is at least one of adenosine, guanosine, uridine and cytidine. It contains a seed ribonucleoside and a cell extract.
本発明に係るタンパク質の合成方法によれば、無細胞タンパク質合成系において、転写反応の基質としてのNTP、およびその前駆体のNMPを添加することなく、タンパク質を合成することができる。
According to the protein synthesis method of the present invention, a protein can be synthesized in a cell-free protein synthesis system without adding NTP as a transcription reaction substrate and NMP as a precursor thereof.
本発明に係るタンパク質の合成方法の一実施形態について、以下説明する。
An embodiment of the protein synthesis method according to the present invention will be described below.
本発明に係るタンパク質の合成方法は、無細胞タンパク質合成系によるタンパク質の合成方法であって、タンパク質をコードする鋳型DNAと、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドとを、タンパク質合成用の細胞抽出液を含む反応液に添加するものであればよく、その他の具体的な工程、ならびに使用する器具および装置は特に限定されるものではない。
The protein synthesis method according to the present invention is a protein synthesis method using a cell-free protein synthesis system, and comprises a template DNA encoding the protein and at least one ribonucleoside of adenosine, guanosine, uridine and cytidine. Can be added to the reaction solution containing the cell extract for protein synthesis, and other specific steps, and the equipment and apparatus to be used are not particularly limited.
(無細胞タンパク質合成系)
本明細書において「無細胞タンパク質合成系」とは、細胞抽出液を用いて、試験管等の人工容器内で転写および翻訳の反応系を再構成してタンパク質を合成させる系である。細胞抽出液は、細胞内に含まれる、DNAを鋳型としたRNAの転写に必要な因子、およびタンパク質の翻訳に必要な因子を、細胞を破砕して抽出液として取り出したものである。すなわち、本明細書において「タンパク質合成用」とは、RNAにコードされた情報に基づくタンパク質の合成のみならず、DNAを鋳型とした、タンパク質の合成に用いられるRNAの合成をも含むことを意図している。 (Cell-free protein synthesis system)
In the present specification, the “cell-free protein synthesis system” is a system for synthesizing proteins by reconstituting a transcription and translation reaction system in an artificial container such as a test tube using a cell extract. In the cell extract, factors necessary for transcription of RNA contained in cells and DNA as a template, and factors necessary for protein translation are disrupted and taken out as an extract. That is, in this specification, “for protein synthesis” is intended to include not only the synthesis of a protein based on information encoded in RNA but also the synthesis of RNA used for protein synthesis using DNA as a template. is doing.
本明細書において「無細胞タンパク質合成系」とは、細胞抽出液を用いて、試験管等の人工容器内で転写および翻訳の反応系を再構成してタンパク質を合成させる系である。細胞抽出液は、細胞内に含まれる、DNAを鋳型としたRNAの転写に必要な因子、およびタンパク質の翻訳に必要な因子を、細胞を破砕して抽出液として取り出したものである。すなわち、本明細書において「タンパク質合成用」とは、RNAにコードされた情報に基づくタンパク質の合成のみならず、DNAを鋳型とした、タンパク質の合成に用いられるRNAの合成をも含むことを意図している。 (Cell-free protein synthesis system)
In the present specification, the “cell-free protein synthesis system” is a system for synthesizing proteins by reconstituting a transcription and translation reaction system in an artificial container such as a test tube using a cell extract. In the cell extract, factors necessary for transcription of RNA contained in cells and DNA as a template, and factors necessary for protein translation are disrupted and taken out as an extract. That is, in this specification, “for protein synthesis” is intended to include not only the synthesis of a protein based on information encoded in RNA but also the synthesis of RNA used for protein synthesis using DNA as a template. is doing.
無細胞タンパク質合成系を構築する細胞抽出液としては、さまざまな生物種に由来する細胞抽出液を利用することができ、例えば、大腸菌および好熱性細菌等の細菌細胞の抽出液、ならびに小麦胚芽、ウサギ網状赤血球、マウスL-細胞、エールリッヒ腹水癌細胞、HeLa細胞、CHO細胞および出芽酵母等の真核細胞の抽出液を用いることができる。なかでも、細菌細胞の細胞抽出液を好適に用いることができ、大腸菌の細胞抽出液がより好ましい。
As cell extracts for constructing a cell-free protein synthesis system, cell extracts derived from various species can be used, for example, extracts of bacterial cells such as Escherichia coli and thermophilic bacteria, and wheat germ, Extracts of eukaryotic cells such as rabbit reticulocytes, mouse L-cells, Ehrlich ascites tumor cells, HeLa cells, CHO cells and budding yeast can be used. Especially, the cell extract of a bacterial cell can be used suitably, The cell extract of E. coli is more preferable.
大腸菌の抽出液としては、Zubay(非特許文献:Zubay G., Ann Rev Genet, (1973) 7, 267-287)またはPrattら(非特許文献:Pratt, J.M. et al., Transcription and Translation-A Practical Approach, (1984), pp.179-209, Henes, B.D. et al. eds., IRL Press, Oxford)に記載された方法により調製されたS30抽出液を用いることができる。大腸菌S30抽出液は、転写および翻訳に必要な大腸菌の全ての酵素と因子を含んでいる。具体的な調製方法としては、まず最初に大腸菌を培養し、菌体を遠心分離等により回収する。回収された菌体は、洗浄後、緩衝液に再懸濁し、フレンチプレス、ガラスビーズ、およびワーリングブレンダー等を用いて破砕する。破砕された大腸菌の不溶物質を遠心分離で除去し、プレインキュベーション混合液と混合してインキュベーションする。この操作によって内在性のDNAおよびRNAが分解されるが、さらに、カルシウム塩およびマイクロコッカスのヌクレアーゼ等を添加して内在性の核酸を分解してもよい。続いて、透析により内在性のアミノ酸、核酸およびヌクレオシド等を除き、適量ずつ分注して液体窒素又は-80℃にて保存する。
As an extract of E. coli, Zubay (non-patent literature: Zubay G., Ann Rev Genet, (1973) 7, 267-287) or Pratt et al. (Non-patent literature: Pratt, JM et al., Transcription and Translation-A S30 extract prepared by the method described in Practical (Approach, 1984), pp.179-209, Henes, BD et al. Eds, IRL Press, Oxford) can be used. The E. coli S30 extract contains all the E. coli enzymes and factors required for transcription and translation. As a specific preparation method, E. coli is first cultured, and the cells are collected by centrifugation or the like. The collected bacterial cells are washed, resuspended in a buffer, and crushed using a French press, glass beads, Waring blender, or the like. The disrupted E. coli insoluble material is removed by centrifugation, mixed with the preincubation mixture and incubated. Endogenous DNA and RNA are degraded by this operation, but the endogenous nucleic acid may be further degraded by adding a calcium salt, a micrococcal nuclease, or the like. Subsequently, endogenous amino acids, nucleic acids, nucleosides and the like are removed by dialysis, and dispensed in appropriate amounts and stored at liquid nitrogen or at -80 ° C.
真核細胞抽出液としては、ウサギ網状赤血球溶解物および小麦胚芽抽出液が好ましい。ウサギ網状赤血球溶解物は、PelhamおよびJachson(非特許文献:Pelham, H.R.B. and Jachson, R.J. Eur. J. Biochem., 67, 247-256, 1976)によって記載された方法を用いることができる。小麦胚芽抽出液の作製方法としては、例えばRobertsおよびPaterson(非特許文献:Roberts, B.E. and Paterson, B.M., Proc. Natl. Acad. Sci. USA, 70, 2330-2334, 1973)によって記載された方法を用いることができる。
As the eukaryotic cell extract, rabbit reticulocyte lysate and wheat germ extract are preferable. Rabbit reticulocyte lysate can use the method described by Pelham and Jackson (non-patent literature: Pelham, H.R.B. and Jachson, R.J. Eur. J. Biochem., 67, 247-256, 1976). As a method for producing a wheat germ extract, for example, a method described by Roberts and Paterson (Non-patent literature: Roberts, BE and Paterson, BM, Proc. Natl. Acad. Sci. USA, 70, 2330-2334, 1973) Can be used.
反応液における細胞抽出液の量は、10~50%であることが好ましく、20~30%がより好ましい。
The amount of the cell extract in the reaction solution is preferably 10 to 50%, more preferably 20 to 30%.
なお、いずれの細胞抽出液においても、市販の細胞抽出液を使用することも可能である。また、いずれの細胞抽出液も転写および翻訳に必要な全ての酵素と因子を含んでいるが、タンパク質合成の反応液として用いる際に、さらに補充的な成分および緩衝液を添加することができる。
In any cell extract, a commercially available cell extract can be used. Each cell extract contains all the enzymes and factors necessary for transcription and translation, but when used as a reaction solution for protein synthesis, supplementary components and a buffer can be further added.
(リボヌクレオシド)
本発明に係るタンパク質の合成方法では、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドが、タンパク質合成用の細胞抽出液を含む反応液に添加される。 (Ribonucleoside)
In the protein synthesis method according to the present invention, at least one ribonucleoside of adenosine, guanosine, uridine and cytidine is added to a reaction solution containing a cell extract for protein synthesis.
本発明に係るタンパク質の合成方法では、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドが、タンパク質合成用の細胞抽出液を含む反応液に添加される。 (Ribonucleoside)
In the protein synthesis method according to the present invention, at least one ribonucleoside of adenosine, guanosine, uridine and cytidine is added to a reaction solution containing a cell extract for protein synthesis.
細胞抽出液を含む反応液には、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドが添加されればよく、より好ましくは、少なくとも何れか2種のリボヌクレオシドが添加され、さらに好ましくは、少なくとも何れか3種のリボヌクレオシドが添加される。
It is sufficient that at least any one ribonucleoside of adenosine, guanosine, uridine and cytidine is added to the reaction solution containing the cell extract, more preferably at least any two ribonucleosides are added. More preferably, at least any three ribonucleosides are added.
下記反応式(1)~(3)は、それぞれ大腸菌のATPの生合成経路、GTPの生合成経路ならびにCTPおよびUTPの生合成経路を示している。大腸菌は、下記式(1)~(3)中に示されているような、リボヌクレオシドから、NMP、NDP(ヌクレオシド二リン酸)およびNTPを順次合成する酵素群を有している。これらの酵素群は菌を破砕した調製した細胞抽出液にも含まれている。そのため、NTPの代わりにアデノシン等のリボヌクレオシドを細胞抽出液に添加することによって、これらの酵素群を利用してNTPを生合成して、転写反応および翻訳反応に供することが可能になる。
(反応式1) The following reaction formulas (1) to (3) show the ATP biosynthesis pathway, the GTP biosynthesis pathway, and the CTP and UTP biosynthesis pathway of Escherichia coli, respectively. E. coli has an enzyme group that sequentially synthesizes NMP, NDP (nucleoside diphosphate) and NTP from ribonucleosides as shown in the following formulas (1) to (3). These enzyme groups are also contained in cell extracts prepared by disrupting bacteria. Therefore, by adding a ribonucleoside such as adenosine or the like instead of NTP to the cell extract, it becomes possible to biosynthesize NTP using these enzyme groups and use it for transcription reaction and translation reaction.
(Reaction Formula 1)
(反応式1) The following reaction formulas (1) to (3) show the ATP biosynthesis pathway, the GTP biosynthesis pathway, and the CTP and UTP biosynthesis pathway of Escherichia coli, respectively. E. coli has an enzyme group that sequentially synthesizes NMP, NDP (nucleoside diphosphate) and NTP from ribonucleosides as shown in the following formulas (1) to (3). These enzyme groups are also contained in cell extracts prepared by disrupting bacteria. Therefore, by adding a ribonucleoside such as adenosine or the like instead of NTP to the cell extract, it becomes possible to biosynthesize NTP using these enzyme groups and use it for transcription reaction and translation reaction.
(Reaction Formula 1)
(反応式2)
(Reaction Formula 2)
(反応式3)
(Reaction Formula 3)
なお、上記反応式(1)~(3)は、大腸菌における生合成経路を示したものであるが、他の生物種においても同様の代謝経路が存在する。そのため、上述の通り、細胞抽出液は大腸菌由来の細胞抽出液に限らず、他の生物種由来の細胞抽出液も、本発明に係る無細胞タンパク質合成系によるタンパク質の合成方法に用いることができる。
The above reaction formulas (1) to (3) show biosynthetic pathways in E. coli, but similar metabolic pathways exist in other species. Therefore, as described above, the cell extract is not limited to the cell extract derived from E. coli, but cell extracts derived from other species can also be used in the protein synthesis method using the cell-free protein synthesis system according to the present invention. .
添加するリボヌクレオシドの種類としては、少なくともグアノシンが含まれていることが好ましく、グアノシンおよびシチジンが含まれていることがより好ましい。グアノシンのみを添加した場合であっても、一定の添加量以上であれば、NTPを添加した場合と同程度のタンパク質合成量を得ることができる。また、グアノシンおよびシチジンを添加した場合には、より少ない添加量で、4種類すべてのリボヌクレオシドを添加した場合と同程度の合成量であって、NTPを添加した場合よりも高い合成量を得ることができる。
As the type of ribonucleoside to be added, at least guanosine is preferably contained, and guanosine and cytidine are more preferably contained. Even when only guanosine is added, a protein synthesis amount comparable to that obtained when NTP is added can be obtained as long as the amount is a certain amount or more. When guanosine and cytidine are added, the amount of synthesis is the same as when all four types of ribonucleosides are added with a smaller amount of addition, and a higher synthesis amount is obtained than when NTP is added. be able to.
添加するリボヌクレオシドの量は、それぞれ、0.1~8mMであることが好ましく、0.2~1.6mMであることがより好ましい。
The amount of ribonucleoside to be added is preferably 0.1 to 8 mM, and more preferably 0.2 to 1.6 mM.
なお、市販品のNTPは、酵素法によって生産された高純度品であるため非常に高価である。一方、NTPの前駆体であるリボヌクレオシドはNTPに比べて非常に安価である。例えば、それぞれ1gあたりの価格を比較すると、リボヌクレオシドは、対応するNTPのおよそ1/20~1/340の価格である。さらに、一般的な無細胞タンパク質合成に添加されるNTPのモル量(0.8~1.2mM)は、本発明において添加されるリボヌクレオシドの好適なモル量(0.4mM)よりも多い。その結果、1mLの無細胞タンパク質合成系反応液に要するNTPおよびリボヌクレオシドの価格を比較すると、リボヌクレオシドを用いる場合には、NTPを用いる場合と比較して、およそ1/470となる。
Note that a commercially available NTP is very expensive because it is a high-purity product produced by an enzymatic method. On the other hand, ribonucleosides that are precursors of NTP are very inexpensive compared to NTP. For example, when comparing prices per gram, ribonucleosides are approximately 1/20 to 1/340 of the corresponding NTP. Furthermore, the molar amount of NTP (0.8-1.2 mM) added for general cell-free protein synthesis is greater than the preferred molar amount (0.4 mM) of ribonucleoside added in the present invention. As a result, when the prices of NTP and ribonucleoside required for 1 mL of the cell-free protein synthesis system reaction solution are compared, when ribonucleoside is used, it becomes approximately 1/470 compared with the case of using NTP.
また、NTPの代わりに、NTPよりも安価であるNMPを用いる無細胞タンパク質合成系が従来知られているが(上記非特許文献3参照)、市販品のNMPも、前駆体であるリボヌクレオシドと比較すると高価である。また、NMPを用いる合成系において、無細胞タンパク質合成に添加されるNMPのモル量(0.8~1.2mM)は、本発明において添加されるリボヌクレオシドの好適なモル量よりも多い。そのため、1mLの無細胞タンパク質合成系反応液に要するNMPおよびリボヌクレオシドの価格を比較すると、リボヌクレオシドを用いる場合には、安価な形態(ナトリウム塩、水和物、またはナトリウム塩の水和物)のNMPを用いる場合と比較しても、およそ1/12となる。
In addition, a cell-free protein synthesis system using NMP which is cheaper than NTP instead of NTP is known (see Non-Patent Document 3 above), but commercially available NMP is also known as a precursor ribonucleoside. It is expensive in comparison. Further, in the synthesis system using NMP, the molar amount (0.8 to 1.2 mM) of NMP added for cell-free protein synthesis is larger than the preferred molar amount of ribonucleoside added in the present invention. Therefore, when comparing the price of NMP and ribonucleoside required for 1 mL of a cell-free protein synthesis reaction solution, when ribonucleoside is used, an inexpensive form (sodium salt, hydrate, or sodium salt hydrate) Compared to the case where NMP is used, it is about 1/12.
また、NMPを用いた場合には、タンパク質の合成量がNTPを用いた場合よりも低くなることが示されている(上記非特許文献3および7参照)。このことは後述する実施例(実施例6)においても示されている。これらの結果からすると、リボヌクレオシドを使用した場合には、さらに合成量が低くなることが予想される。しかしながら驚くべきことに、リボヌクレオシドを使用した場合には、NTPを添加した場合よりも高い合成量を得ることができる。
It has also been shown that when NMP is used, the amount of protein synthesis is lower than when NTP is used (see Non-Patent Documents 3 and 7 above). This is also shown in an example (Example 6) described later. From these results, it is expected that the amount of synthesis will be further reduced when ribonucleosides are used. However, surprisingly, when ribonucleosides are used, a higher synthesis amount can be obtained than when NTP is added.
さらに、NMPをNTPにするには2分子のATPが必要であるのに対し、リボヌクレオシドをNTPにするには3分子のATPが必要である。すなわち、リボヌクレオシドを使用した場合には、1分子のATP分だけ余計にエネルギーを消費することになる。このことから、リボヌクレオシドを用いることは、タンパク質合成に不利に働くと想定される。また、リボヌクレオシドを使用した場合には、最終的にタンパク質が合成されるまでに関与する酵素数が増加するため、系の不安定化が想定される。これらのことからしても、リボヌクレオシドを使用した場合には、NTPを添加した場合よりも高い合成量を得ることができることは驚くべきことである。
Furthermore, two molecules of ATP are required to convert NP into NTP, whereas three molecules of ATP are required to convert ribonucleoside into NTP. That is, when a ribonucleoside is used, an extra energy is consumed by ATP of one molecule. From this, it is assumed that the use of ribonucleosides adversely affects protein synthesis. In addition, when ribonucleosides are used, the number of enzymes involved until the final synthesis of the protein increases, so that the system may be destabilized. Even from these facts, it is surprising that when ribonucleoside is used, a higher synthesis amount can be obtained than when NTP is added.
(鋳型DNA)
鋳型DNAとしては、発現させたい所望のタンパク質をコードする遺伝子配列と、適当な発現制御領域とが含まれている二本鎖DNAであればよく、直鎖状および環状の何れの形態であってもよい。 (Template DNA)
The template DNA may be a double-stranded DNA containing a gene sequence encoding a desired protein to be expressed and an appropriate expression control region, and may be either linear or circular. Also good.
鋳型DNAとしては、発現させたい所望のタンパク質をコードする遺伝子配列と、適当な発現制御領域とが含まれている二本鎖DNAであればよく、直鎖状および環状の何れの形態であってもよい。 (Template DNA)
The template DNA may be a double-stranded DNA containing a gene sequence encoding a desired protein to be expressed and an appropriate expression control region, and may be either linear or circular. Also good.
発現制御領域としては、プロモーター配列、ターミネーター配列、エンハンサー配列、ポリA付加シグナルおよびリボソーム結合配列などを含み得る。
The expression control region may include a promoter sequence, terminator sequence, enhancer sequence, poly A addition signal, ribosome binding sequence, and the like.
また、合成されたタンパク質を迅速に精製、または検出するために、アフィニティー標識(タグ)配列を組み込んだ融合タンパク質が合成できるように鋳型DNAを設計することもできる。
Also, in order to quickly purify or detect the synthesized protein, the template DNA can be designed so that a fusion protein incorporating an affinity tag (tag) sequence can be synthesized.
添加する鋳型DNAの量は、0.1~50μg/mLであることが好ましく、1~10μg/mLであることがより好ましい。
The amount of template DNA to be added is preferably 0.1 to 50 μg / mL, and more preferably 1 to 10 μg / mL.
(エネルギー再生系)
本発明の方法において、細胞抽出液を含む反応液には、さらにエネルギー再生系の成分を添加することが好ましい。エネルギー再生系の成分に制限はなく、例えば、クレアチンキナーゼとクレアチンリン酸との組合せ、およびホスホエノールピルビン酸とピルビン酸キナーゼとの組合せ等によるATP再生系が使用可能である。クレアチンキナーゼおよびピルビン酸キナーゼは何れもADPをATPに再生する酵素であり、それぞれクレアチンリン酸およびホスホエノールピルビン酸を基質として必要とする。ホスホエノールピルビン酸とピルビン酸キナーゼとの組合せ等の系を効率的に動かすにはNADおよびCoAの2つの補酵素を加える必要がある。一方、クレアチンキナーゼとクレアチンリン酸との組合せの系では、これらの補酵素は不要である。そのため、クレアチンキナーゼとクレアチンリン酸との組合せを用いることが特に好ましい。 (Energy regeneration system)
In the method of the present invention, it is preferable to further add an energy regeneration system component to the reaction solution containing the cell extract. There is no restriction | limiting in the component of an energy regeneration system, For example, the ATP regeneration system by the combination of creatine kinase and creatine phosphate, the combination of phosphoenolpyruvate and pyruvate kinase, etc. can be used. Creatine kinase and pyruvate kinase are both enzymes that regenerate ADP into ATP, and require creatine phosphate and phosphoenolpyruvate as substrates, respectively. Two coenzymes, NAD and CoA, need to be added to efficiently move the system such as a combination of phosphoenolpyruvate and pyruvate kinase. On the other hand, these coenzymes are unnecessary in the combination system of creatine kinase and creatine phosphate. Therefore, it is particularly preferable to use a combination of creatine kinase and creatine phosphate.
本発明の方法において、細胞抽出液を含む反応液には、さらにエネルギー再生系の成分を添加することが好ましい。エネルギー再生系の成分に制限はなく、例えば、クレアチンキナーゼとクレアチンリン酸との組合せ、およびホスホエノールピルビン酸とピルビン酸キナーゼとの組合せ等によるATP再生系が使用可能である。クレアチンキナーゼおよびピルビン酸キナーゼは何れもADPをATPに再生する酵素であり、それぞれクレアチンリン酸およびホスホエノールピルビン酸を基質として必要とする。ホスホエノールピルビン酸とピルビン酸キナーゼとの組合せ等の系を効率的に動かすにはNADおよびCoAの2つの補酵素を加える必要がある。一方、クレアチンキナーゼとクレアチンリン酸との組合せの系では、これらの補酵素は不要である。そのため、クレアチンキナーゼとクレアチンリン酸との組合せを用いることが特に好ましい。 (Energy regeneration system)
In the method of the present invention, it is preferable to further add an energy regeneration system component to the reaction solution containing the cell extract. There is no restriction | limiting in the component of an energy regeneration system, For example, the ATP regeneration system by the combination of creatine kinase and creatine phosphate, the combination of phosphoenolpyruvate and pyruvate kinase, etc. can be used. Creatine kinase and pyruvate kinase are both enzymes that regenerate ADP into ATP, and require creatine phosphate and phosphoenolpyruvate as substrates, respectively. Two coenzymes, NAD and CoA, need to be added to efficiently move the system such as a combination of phosphoenolpyruvate and pyruvate kinase. On the other hand, these coenzymes are unnecessary in the combination system of creatine kinase and creatine phosphate. Therefore, it is particularly preferable to use a combination of creatine kinase and creatine phosphate.
クレアチンキナーゼは0.02~5mg/mLで使用されることが好ましく、クレアチンリン酸は10~100mMで使用されることが好ましい。
Creatine kinase is preferably used at 0.02 to 5 mg / mL, and creatine phosphate is preferably used at 10 to 100 mM.
また、Jewettら(非特許文献:Jewett MC and Swartz JR, Biotechnol. Bioeng., 2004, 86(1), 19-26.)に記載のPANOxSPと呼ばれる、ホスホエノールピルビン酸およびNAD等を用いたATP再生系、あるいは上記非特許文献3に記載のホスホエノールピルビン酸の代わりにグルコースを用いているPANOxSPの改良系では、大気中の酸素を必要とするため、反応液が空気に接触するように工夫した容器で反応を行う必要がある。一方、クレアチンキナーゼとクレアチンリン酸との組合せを用いたATP再生系では、タンパク質の生産性に関し、酸素供給の有無による影響はない。したがって、ATP再生系として、クレアチンキナーゼとクレアチンリン酸との組合せを用いた合成系の場合には、反応に際して特殊な容器を用いる必要はなく、そのため、大量調製のためのスケールアップが容易となる。
In addition, ATP using phosphoenolpyruvate and NAD or the like called PANOxSP described in Jewett et al. (Non-patent literature: Jewett MC and Swartz JR, Biotechnol. Bioeng., 2004, 86 (1), 19-26.) In the regeneration system or the improved system of PANOxSP using glucose in place of the phosphoenolpyruvate described in Non-Patent Document 3 above, oxygen in the atmosphere is required, so that the reaction solution is in contact with the air. It is necessary to carry out the reaction in a container. On the other hand, in the ATP regeneration system using a combination of creatine kinase and creatine phosphate, there is no influence on the protein productivity due to the presence or absence of oxygen supply. Therefore, in the case of a synthetic system using a combination of creatine kinase and creatine phosphate as the ATP regeneration system, it is not necessary to use a special container for the reaction, and therefore it is easy to scale up for mass preparation. .
(補充的な成分および緩衝液)
反応液には、細胞抽出液、鋳型DNAおよびリボヌクレオシド、ならびにクレアチンキナーゼおよびホスホエノールピルビン酸等のエネルギー再生系のほかに、緩衝液、塩類、基質となるアミノ酸、核酸分解酵素阻害剤、抗菌剤、還元剤、ポリエチレングリコール、cAMP、葉酸類、RNAポリメラーゼおよびtRNA等を含むことができる。 (Supplementary ingredients and buffers)
Reaction solutions include cell extracts, template DNA and ribonucleosides, and energy regeneration systems such as creatine kinase and phosphoenolpyruvate, as well as buffers, salts, amino acids that serve as substrates, nucleolytic enzyme inhibitors, antibacterial agents , Reducing agents, polyethylene glycol, cAMP, folic acids, RNA polymerase, tRNA, and the like.
反応液には、細胞抽出液、鋳型DNAおよびリボヌクレオシド、ならびにクレアチンキナーゼおよびホスホエノールピルビン酸等のエネルギー再生系のほかに、緩衝液、塩類、基質となるアミノ酸、核酸分解酵素阻害剤、抗菌剤、還元剤、ポリエチレングリコール、cAMP、葉酸類、RNAポリメラーゼおよびtRNA等を含むことができる。 (Supplementary ingredients and buffers)
Reaction solutions include cell extracts, template DNA and ribonucleosides, and energy regeneration systems such as creatine kinase and phosphoenolpyruvate, as well as buffers, salts, amino acids that serve as substrates, nucleolytic enzyme inhibitors, antibacterial agents , Reducing agents, polyethylene glycol, cAMP, folic acids, RNA polymerase, tRNA, and the like.
緩衝液としては、例えば、HEPES-KOH、およびTris-OAc等の緩衝剤を使用できる。塩類としては、酢酸カリウム、酢酸マグネシウムおよび酢酸アンモニウム等の酢酸塩、ならびにグルタミン酸カリウム等のグルタミン酸塩等を使用できる。基質となるアミノ酸は、タンパク質を構成する天然の20種類のアミノ酸およびこれらの誘導体である。抗菌剤としては、例えば、アジ化ナトリウムおよびアンピシリン等を使用できる。RNAポリメラーゼとしては、T7RNAポリメラーゼ、T3RNAポリメラーゼおよびSP6RNAポリメラーゼ等の市販の酵素を使用でき、鋳型DNAのプロモーター配列に適合するように適宜選択すればよい。
As the buffer, for example, a buffer such as HEPES-KOH and Tris-OAc can be used. Examples of salts that can be used include acetates such as potassium acetate, magnesium acetate and ammonium acetate, and glutamates such as potassium glutamate. The amino acid used as a substrate is 20 kinds of natural amino acids constituting the protein and derivatives thereof. As the antibacterial agent, for example, sodium azide and ampicillin can be used. As the RNA polymerase, commercially available enzymes such as T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase can be used, and may be appropriately selected so as to match the promoter sequence of the template DNA.
(タンパク質の合成キット)
細胞抽出液およびリボヌクレオシド、ならびに必要に応じて、緩衝液および塩類等のその他の成分は、使用しやすいように一定量ごと分注して製品として配送することができる。これらの製品は凍結または乾燥状態で保存することができ、保存および輸送に適した容器に収容してキットとして販売することができる。キットには取扱説明書および陽性コントロールDNA等を添付することができる。すなわち、本発明に係るタンパク質の合成キットは、上述のタンパク質の合成方法に使用されるキットであって、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドと、細胞抽出液とを含むものである。 (Protein synthesis kit)
Cell extracts and ribonucleosides, and other components such as buffers and salts as needed, can be dispensed into a certain quantity and delivered as a product for easy use. These products can be stored frozen or dried and can be sold in kits in containers suitable for storage and transport. An instruction manual and positive control DNA can be attached to the kit. That is, the protein synthesis kit according to the present invention is a kit used in the above-described protein synthesis method, and comprises at least one ribonucleoside of adenosine, guanosine, uridine and cytidine, and a cell extract. Is included.
細胞抽出液およびリボヌクレオシド、ならびに必要に応じて、緩衝液および塩類等のその他の成分は、使用しやすいように一定量ごと分注して製品として配送することができる。これらの製品は凍結または乾燥状態で保存することができ、保存および輸送に適した容器に収容してキットとして販売することができる。キットには取扱説明書および陽性コントロールDNA等を添付することができる。すなわち、本発明に係るタンパク質の合成キットは、上述のタンパク質の合成方法に使用されるキットであって、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドと、細胞抽出液とを含むものである。 (Protein synthesis kit)
Cell extracts and ribonucleosides, and other components such as buffers and salts as needed, can be dispensed into a certain quantity and delivered as a product for easy use. These products can be stored frozen or dried and can be sold in kits in containers suitable for storage and transport. An instruction manual and positive control DNA can be attached to the kit. That is, the protein synthesis kit according to the present invention is a kit used in the above-described protein synthesis method, and comprises at least one ribonucleoside of adenosine, guanosine, uridine and cytidine, and a cell extract. Is included.
(タンパク質の合成)
本発明に係るタンパク質の合成方法としては、透析法およびバッチ法を適用することができる。 (Protein synthesis)
As a protein synthesis method according to the present invention, a dialysis method and a batch method can be applied.
本発明に係るタンパク質の合成方法としては、透析法およびバッチ法を適用することができる。 (Protein synthesis)
As a protein synthesis method according to the present invention, a dialysis method and a batch method can be applied.
透析法は、反応液である内液と反応基質を含む外液とを透析膜(限外濾過膜)によって隔離して含む、振とうまたは攪拌可能な閉鎖系による合成法である。透析法では、透析膜を介して、合成に必要な基質が外液から反応液に供給されるとともに、反応液中の余計な副産物を外液中に拡散させることで、より長時間、反応を持続させることができる。そのため、より高いタンパク質合成量を得ることができ、大量(ミリグラム単位)のタンパク質を調製することができる。
The dialysis method is a synthesis method using a closed system that can be shaken or stirred and contains an internal solution as a reaction solution and an external solution containing a reaction substrate separated by a dialysis membrane (ultrafiltration membrane). In the dialysis method, the substrate necessary for synthesis is supplied from the external solution to the reaction solution through the dialysis membrane, and the extra by-product in the reaction solution is diffused into the external solution, so that the reaction can be performed for a longer time. Can last. Therefore, a higher protein synthesis amount can be obtained, and a large amount (milligram unit) of protein can be prepared.
透析法によってタンパク質を合成する場合には、4種類すべてのリボヌクレオシドを添加することが好ましい。
When protein is synthesized by dialysis, it is preferable to add all four types of ribonucleosides.
一方、バッチ法は、必要な成分すべてを反応溶液中に均一に混合して含ませて、反応を行う合成法である。透析法と比較するとタンパク質合成量は少ないものの、反応時間がより短く、結果がすぐに得られること、および一般的な96穴プレート中で合成反応を行えることから、バッチ法は、ハイスループットスクリーニングに適した合成系といえる。
On the other hand, the batch method is a synthesis method in which all necessary components are uniformly mixed and contained in the reaction solution to perform the reaction. Although the amount of protein synthesis is small compared to dialysis, the reaction time is shorter, results are obtained quickly, and the synthesis reaction can be performed in a general 96-well plate. It can be said that it is a suitable synthesis system.
バッチ法においては、NTPを用いた合成系よりも、より長時間、タンパク質合成反応が持続する。そのため、本発明に係るタンパク質の合成方法をバッチ法にて実現した場合には、NTPを用いた合成系と比較して、より安価にタンパク質合成を実現できるばかりでなく、より高いタンパク質合成量を得ることができる。なお、NMPを用いた合成系では、このような効果はみられない。
In the batch method, the protein synthesis reaction lasts longer than in the synthesis system using NTP. Therefore, when the protein synthesis method according to the present invention is realized by a batch method, not only can a protein synthesis be realized at a lower cost than a synthesis system using NTP, but also a higher protein synthesis amount. Obtainable. Note that such effects are not observed in the synthesis system using NMP.
バッチ法によってタンパク質を合成する場合には、4種類すべてのリボヌクレオシドを添加する方法に限らず、グアノシン1種類のみを添加する方法、またはグアノシンおよびシチジンのみを添加する方法であってもよい。バッチ法によれば、グアノシンのみを添加した場合であっても、添加量によっては、NTPを用いた合成系と同程度のタンパク質合成量を実現することができる。また、グアノシンおよびシチジンのみを添加した場合には、添加量によっては、NTPを用いた合成系を超える量のタンパク質合成量を実現することができ、4種類すべてのリボヌクレオシドを添加した場合と同程度のタンパク質合成量を実現することができる。グアノシンのみを添加する場合には、グアノシンの量は0.1~8mMが好ましく、1~4mMがより好ましい。また、グアノシンおよびシチジンのみを添加する場合には、グアノシンおよびシチジンの量はそれぞれ、0.1~8mMが好ましく、0.2~1.6mMがより好ましい。
When a protein is synthesized by the batch method, the method is not limited to the method of adding all four types of ribonucleosides, and may be a method of adding only one type of guanosine, or a method of adding only guanosine and cytidine. According to the batch method, even when only guanosine is added, depending on the addition amount, a protein synthesis amount comparable to that of a synthesis system using NTP can be realized. In addition, when only guanosine and cytidine are added, depending on the amount added, the amount of protein synthesis exceeding the synthesis system using NTP can be realized, which is the same as when all four types of ribonucleosides are added. A degree of protein synthesis can be achieved. When only guanosine is added, the amount of guanosine is preferably 0.1 to 8 mM, more preferably 1 to 4 mM. When only guanosine and cytidine are added, the amounts of guanosine and cytidine are each preferably 0.1 to 8 mM, more preferably 0.2 to 1.6 mM.
合成されたタンパク質の精製は、生細胞を用いる場合と異なり破砕する必要がないことに加えて、生細胞からの分離と比べて混在する物質の量および種類が格段に少ないため、比較的容易に行うことができる。精製法は、タンパク質の性質に応じて従来公知のものを単独にまたは適宜組み合わせて使用できる。例えば硫酸アンモニウムもしくはアセトン沈殿、酸抽出、アニオンもしくはカチオン交換クロマトグラフィー、疎水性相互作用クロマトグラフィー、アフィニティークロマトグラフィー、ゲルろ過クロマトグラフィー、HPLC、電気泳動、およびクロマトフォーカシングなどの慣用の技術を挙げることができる。
The purification of the synthesized protein is relatively easy because it does not need to be disrupted unlike the case of using live cells, and the amount and type of substances mixed in is significantly less than the separation from live cells. It can be carried out. As the purification method, conventionally known methods can be used alone or in appropriate combination depending on the properties of the protein. Examples include conventional techniques such as ammonium sulfate or acetone precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration chromatography, HPLC, electrophoresis, and chromatofocusing. .
また、合成されたタンパク質の同定および定量は、活性測定、免疫学的測定、分光学的測定、およびアミノ酸分析などによって、必要に応じて標準サンプルと比較しながら行うことができる。
In addition, identification and quantification of the synthesized protein can be performed by comparing with a standard sample, if necessary, by activity measurement, immunological measurement, spectroscopic measurement, amino acid analysis, and the like.
(まとめ)
以上のように、本発明に係るタンパク質の合成方法は、無細胞タンパク質合成系によるタンパク質の合成方法であって、タンパク質をコードする鋳型DNAと、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドとを、タンパク質合成用の細胞抽出液を含む反応液に添加することを特徴とする。 (Summary)
As described above, the protein synthesis method according to the present invention is a protein synthesis method using a cell-free protein synthesis system, and is a template DNA encoding the protein and at least one of adenosine, guanosine, uridine and cytidine. One type of ribonucleoside is added to a reaction solution containing a cell extract for protein synthesis.
以上のように、本発明に係るタンパク質の合成方法は、無細胞タンパク質合成系によるタンパク質の合成方法であって、タンパク質をコードする鋳型DNAと、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドとを、タンパク質合成用の細胞抽出液を含む反応液に添加することを特徴とする。 (Summary)
As described above, the protein synthesis method according to the present invention is a protein synthesis method using a cell-free protein synthesis system, and is a template DNA encoding the protein and at least one of adenosine, guanosine, uridine and cytidine. One type of ribonucleoside is added to a reaction solution containing a cell extract for protein synthesis.
本発明に係るタンパク質の合成方法においては、少なくともグアノシンを添加することが好ましい。
In the protein synthesis method according to the present invention, it is preferable to add at least guanosine.
本発明に係るタンパク質の合成方法においては、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか2種のリボヌクレオシドを添加することが好ましい。
In the protein synthesis method according to the present invention, it is preferable to add at least any two ribonucleosides among adenosine, guanosine, uridine and cytidine.
本発明に係るタンパク質の合成方法においては、少なくともグアノシンおよびシチジンを添加することが好ましい。
In the protein synthesis method according to the present invention, it is preferable to add at least guanosine and cytidine.
本発明に係るタンパク質の合成方法において、上記細胞抽出液は細菌細胞から調製した抽出液であることが好ましい。
In the protein synthesis method according to the present invention, the cell extract is preferably an extract prepared from bacterial cells.
本発明に係るタンパク質の合成方法において、上記細胞抽出液は大腸菌から調製した抽出液であることが好ましい。
In the protein synthesis method according to the present invention, the cell extract is preferably an extract prepared from E. coli.
本発明に係るタンパク質の合成方法においては、ATP再生系の成分を上記反応液に添加することが好ましい。
In the protein synthesis method according to the present invention, it is preferable to add components of the ATP regeneration system to the reaction solution.
本発明に係るタンパク質の合成方法においては、上記ATP再生系の成分として、クレアチンキナーゼおよびクレアチンリン酸を上記反応液に添加することが好ましい。
In the protein synthesis method according to the present invention, creatine kinase and creatine phosphate are preferably added to the reaction solution as components of the ATP regeneration system.
本発明に係るタンパク質の合成方法は、透析法による合成方法であり得る。
The protein synthesis method according to the present invention may be a dialysis synthesis method.
本発明に係るタンパク質の合成方法は、バッチ法による合成方法であり得る。
The protein synthesis method according to the present invention may be a batch synthesis method.
本発明に係るタンパク質の合成キットは、上述のタンパク質の合成方法に使用されるタンパク質の合成キットであって、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドと、細胞抽出液とを含むことを特徴とする。
A protein synthesis kit according to the present invention is a protein synthesis kit used in the above-described protein synthesis method, and comprises at least one ribonucleoside of adenosine, guanosine, uridine and cytidine, and cell extraction. Liquid.
以下に実施例を示し、本発明の実施の形態についてさらに詳しく説明する。もちろん、本発明は以下の実施例に限定されるものではなく、細部については様々な態様が可能であることはいうまでもない。さらに、本発明は上述した実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、それぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された文献の全てが参考として援用される。
Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. Moreover, all the literatures described in this specification are used as reference.
〔実施例1:バッチ法によるCATタンパク質の合成〕
(合成反応条件)
バッチ法による無細胞合成系の反応液組成は、次の通りである:60mM HEPES-KOH(pH7.5)、230mM D-グルタミン酸カリウム、2% PEG8000、3mM DTT、0.4mM アデノシン、0.4mM グアノシン、0.4mM シチジン、0.4mM ウリジン、36μg/mL フォリン酸、80mM クレアチンリン酸、150μg/mL E.coli tRNA、13mM 酢酸マグネシウム、各1.5mM タンパク質構成アミノ酸(ただしCys、Ser、Arg、Trp、Asn、およびGlnについては4.5mM)、50μg/mL クレアチンキナーゼ、100μg/mL T7RNAポリメラーゼ、4μg/mL 鋳型DNA、および24% 大腸菌S30抽出液。 [Example 1: Synthesis of CAT protein by batch method]
(Synthetic reaction conditions)
The reaction solution composition of the cell-free synthesis system by the batch method is as follows: 60 mM HEPES-KOH (pH 7.5), 230 mM potassium D-glutamate, 2% PEG8000, 3 mM DTT, 0.4 mM adenosine, 0.4 mM Guanosine, 0.4 mM cytidine, 0.4 mM uridine, 36 μg / mL folinic acid, 80 mM creatine phosphate, 150 μg / mL E. coli tRNA, 13 mM magnesium acetate, each 1.5 mM protein constituent amino acids (however, Cys, Ser, Arg, 4.5 mM for Trp, Asn, and Gln), 50 μg / mL creatine kinase, 100 μg / mL T7 RNA polymerase, 4 μg / mL template DNA, and 24% E. coli S30 extract.
(合成反応条件)
バッチ法による無細胞合成系の反応液組成は、次の通りである:60mM HEPES-KOH(pH7.5)、230mM D-グルタミン酸カリウム、2% PEG8000、3mM DTT、0.4mM アデノシン、0.4mM グアノシン、0.4mM シチジン、0.4mM ウリジン、36μg/mL フォリン酸、80mM クレアチンリン酸、150μg/mL E.coli tRNA、13mM 酢酸マグネシウム、各1.5mM タンパク質構成アミノ酸(ただしCys、Ser、Arg、Trp、Asn、およびGlnについては4.5mM)、50μg/mL クレアチンキナーゼ、100μg/mL T7RNAポリメラーゼ、4μg/mL 鋳型DNA、および24% 大腸菌S30抽出液。 [Example 1: Synthesis of CAT protein by batch method]
(Synthetic reaction conditions)
The reaction solution composition of the cell-free synthesis system by the batch method is as follows: 60 mM HEPES-KOH (pH 7.5), 230 mM potassium D-glutamate, 2% PEG8000, 3 mM DTT, 0.4 mM adenosine, 0.4 mM Guanosine, 0.4 mM cytidine, 0.4 mM uridine, 36 μg / mL folinic acid, 80 mM creatine phosphate, 150 μg / mL E. coli tRNA, 13 mM magnesium acetate, each 1.5 mM protein constituent amino acids (however, Cys, Ser, Arg, 4.5 mM for Trp, Asn, and Gln), 50 μg / mL creatine kinase, 100 μg / mL T7 RNA polymerase, 4 μg / mL template DNA, and 24% E. coli S30 extract.
鋳型DNAには、pK7-CATを用いた(非特許文献:Kim DM, Kigawa T, Choi CY, Yokoyama S (1996) EurJ Biochem 239:881-886)。このプラスミドはT7プロモーター、リボソーム結合配列、CAT(クロラムフェニコールアセチルトランスフェラーゼ)遺伝子、およびT7ターミネーターから構成されている。
PK7-CAT was used as template DNA (Non-patent literature: Kim DM, Kigawa T, Choi CY, Yokoyama S (1996) EurJ Biochem 239: 881-886). This plasmid is composed of a T7 promoter, a ribosome binding sequence, a CAT (chloramphenicol acetyltransferase) gene, and a T7 terminator.
大腸菌S30抽出液は、Zubayの方法(非特許文献:Zubay G., Ann Rev Genet, (1973) 7, 267-287)、およびKigawaらの方法(非特許文献:Kigawa T, Yabuki T, Matsuda N, Matsuda T, Nakajima R, Tanaka A, Yokoyama S. (2004) J Struct Funct Genomics 5, 63-68)に従って、大腸菌BL21 codon-plus RIL株(Stratagene社)から調製した。
The Escherichia coli S30 extract was prepared by the method of Zubay (non-patent literature: Zubay G., Ann Rev Genet, (1973) 7, 267-287) and the method of Kigawa et al. (Non-patent literature: Kigawa T, Yabuki T, , Matsuda T, Nakajima R, Tanaka A, Yokoyama S. (2004) J Struct Funct Genomics 5, 63-68), and prepared from Escherichia coli BL21 codon-plus RIL strain (Stratagene).
シチジン(Sigma社)、およびウリジン(Sigma社)はミリQ水に溶解して、100mMの溶液として調製した。アデノシン(ナカライテスク社)、およびグアノシン(和光純薬社)は水への溶解度が低い(約0.2mM)ため、ミリQ水を加えて、100mMの懸濁液を調製して実験に用いた。
Cytidine (Sigma) and uridine (Sigma) were dissolved in MilliQ water to prepare a 100 mM solution. Since adenosine (Nacalai Tesque) and guanosine (Wako Pure Chemical Industries, Ltd.) have low solubility in water (about 0.2 mM), MilliQ water was added to prepare a 100 mM suspension and used in the experiment. .
なお、NTPを用いた対照としては、アデノシン、グアノシン、シチジン、およびウリジンの代わりに、1.3mM ATP、0.9mM GTP、0.9mM CTP、および0.9mM UTPが添加されている。
As a control using NTP, 1.3 mM ATP, 0.9 mM GTP, 0.9 mM CTP, and 0.9 mM UTP are added instead of adenosine, guanosine, cytidine, and uridine.
反応は、30℃で行った。
The reaction was performed at 30 ° C.
(CATタンパク質の定量)
CATタンパク質の合成量は、Kigawaらの方法(非特許文献:Kigawa T, YabukiT, Matsuda N, Matsuda T, Nakajima R, Tanaka A, Yokoyama S. (2004) J Struct Funct Genomics 5, 63-68)に従って、比活性の値から求めた。結果を図1に示す。 (Quantification of CAT protein)
The amount of CAT protein synthesized is according to the method of Kigawa et al. (Non-patent literature: Kigawa T, YabukiT, Matsuda N, Matsuda T, Nakajima R, Tanaka A, Yokoyama S. (2004) JStruct Funct Genomics 5, 63-68). The specific activity value was obtained. The results are shown in FIG.
CATタンパク質の合成量は、Kigawaらの方法(非特許文献:Kigawa T, YabukiT, Matsuda N, Matsuda T, Nakajima R, Tanaka A, Yokoyama S. (2004) J Struct Funct Genomics 5, 63-68)に従って、比活性の値から求めた。結果を図1に示す。 (Quantification of CAT protein)
The amount of CAT protein synthesized is according to the method of Kigawa et al. (Non-patent literature: Kigawa T, YabukiT, Matsuda N, Matsuda T, Nakajima R, Tanaka A, Yokoyama S. (2004) J
図1は、反応時間と合成量との関係を示す図である。図1に示されるように、NTPを添加した合成系の場合には、タンパク質合成反応は1時間程度で停止していた。一方、リボヌクレオシドを添加した合成系の場合には、タンパク質合成反応は2時間持続していた。その結果、リボヌクレオシドを添加した合成系では、NTPを添加した合成系と比較して、約1.4倍量のCATタンパク質が合成された。以上から、NTPの代わりにリボヌクレオシドを用いることによって、無細胞タンパク質合成系におけるタンパク質合成が可能であるばかりでなく、NTPを用いた場合よりも合成量を増加させることができることが示された。
FIG. 1 is a diagram showing the relationship between reaction time and synthesis amount. As shown in FIG. 1, in the case of a synthetic system to which NTP was added, the protein synthesis reaction was stopped in about 1 hour. On the other hand, in the case of the synthetic system to which ribonucleoside was added, the protein synthesis reaction lasted for 2 hours. As a result, in the synthetic system to which ribonucleoside was added, about 1.4 times as much CAT protein was synthesized as compared to the synthetic system to which NTP was added. From the above, it was shown that by using ribonucleoside instead of NTP, not only protein synthesis in a cell-free protein synthesis system is possible, but also the amount of synthesis can be increased as compared with the case of using NTP.
〔実施例2:EGFPタンパク質、UBAタンパク質、Rasタンパク質、およびβガラクトシダーゼタンパク質の合成〕
実施例1と同様にして、N末端にHisタグを融合させたEGFPタンパク質(分子量34kDa)、UBAタンパク質(分子量10kDa)、Rasタンパク質(分子量24kDa)およびβガラクトシダーゼタンパク質(分子量119kDa)の合成について比較を行った。 [Example 2: Synthesis of EGFP protein, UBA protein, Ras protein, and β-galactosidase protein]
In the same manner as in Example 1, the synthesis of EGFP protein (molecular weight 34 kDa), UBA protein (molecular weight 10 kDa), Ras protein (molecular weight 24 kDa) and β-galactosidase protein (molecular weight 119 kDa) fused with a His tag at the N-terminus was compared. went.
実施例1と同様にして、N末端にHisタグを融合させたEGFPタンパク質(分子量34kDa)、UBAタンパク質(分子量10kDa)、Rasタンパク質(分子量24kDa)およびβガラクトシダーゼタンパク質(分子量119kDa)の合成について比較を行った。 [Example 2: Synthesis of EGFP protein, UBA protein, Ras protein, and β-galactosidase protein]
In the same manner as in Example 1, the synthesis of EGFP protein (molecular weight 34 kDa), UBA protein (
(合成反応条件)
EGFPタンパク質、UBAタンパク質、Rasタンパク質およびβガラクトシダーゼタンパク質の鋳型DNAとして、それぞれpCR2.1 NHis-EGFP、pCR2.1 N11-UBA、pK7b2 NHis-RasおよびpUC19 N11-β-galactosidaseを用いた以外は、実施例1と同じである。これらのプラスミドは、構造遺伝子の上流に、T7プロモーターおよびリボソーム結合配列を有しており、下流に、T7ターミネーターを有している(非特許文献:Matsuda T, Kigawa T, KoshibaS, Inoue M, Aoki M, Yamasaki K, Seki M, Shinozaki K, Yokoyama S. (2006) J Struct Funct Genomics. 7(2), 93-100.参照)。 (Synthetic reaction conditions)
Except that pCR2.1 NHis-EGFP, pCR2.1 N11-UBA, pK7b2 NHis-Ras and pUC19 N11-β-galactosidase were used as template DNAs for EGFP protein, UBA protein, Ras protein and β-galactosidase protein, respectively. Same as Example 1. These plasmids have a T7 promoter and a ribosome binding sequence upstream of the structural gene, and a T7 terminator downstream (Non-patent literature: Matsuda T, Kigawa T, Koshiba S, Inoue M, Aoki). M, Yamasaki K, Seki M, Shinozaki K, Yokoyama S. (2006) J Struct Funct Genomics. 7 (2), 93-100.
EGFPタンパク質、UBAタンパク質、Rasタンパク質およびβガラクトシダーゼタンパク質の鋳型DNAとして、それぞれpCR2.1 NHis-EGFP、pCR2.1 N11-UBA、pK7b2 NHis-RasおよびpUC19 N11-β-galactosidaseを用いた以外は、実施例1と同じである。これらのプラスミドは、構造遺伝子の上流に、T7プロモーターおよびリボソーム結合配列を有しており、下流に、T7ターミネーターを有している(非特許文献:Matsuda T, Kigawa T, KoshibaS, Inoue M, Aoki M, Yamasaki K, Seki M, Shinozaki K, Yokoyama S. (2006) J Struct Funct Genomics. 7(2), 93-100.参照)。 (Synthetic reaction conditions)
Except that pCR2.1 NHis-EGFP, pCR2.1 N11-UBA, pK7b2 NHis-Ras and pUC19 N11-β-galactosidase were used as template DNAs for EGFP protein, UBA protein, Ras protein and β-galactosidase protein, respectively. Same as Example 1. These plasmids have a T7 promoter and a ribosome binding sequence upstream of the structural gene, and a T7 terminator downstream (Non-patent literature: Matsuda T, Kigawa T, Koshiba S, Inoue M, Aoki). M, Yamasaki K, Seki M, Shinozaki K, Yokoyama S. (2006) J Struct Funct Genomics. 7 (2), 93-100.
反応は、30℃で4時間行った。
The reaction was performed at 30 ° C. for 4 hours.
(EGFPタンパク質、UBAタンパク質、Rasタンパク質、およびβガラクトシダーゼタンパク質のHisタグアフィニティ精製)
合成反応後の反応液を、12,000rpmで5分間、室温で遠心した。その上清80μLを、96穴フィルタープレート(ミリポア社)に加え、そこに60μLのAバッファー(20mM Tris-HCl pH7.5、300mM NaCl)、および予めAバッファーで平衡化した60μLの50% TALON(登録商標)樹脂(TAKARABIO社)を加えて、10分間振とうした。フィルタープレートを1500rpmで1分間遠心し、抽出液由来の夾雑タンパク質を除いた。ここで、目的のタンパク質はTALON樹脂に結合している。次に、150μLのAバッファーを加えて1500rpmで1分間遠心する洗浄工程を、3回行った。最後に、50μLのBバッファー(20mM Tris-HCl pH7.5、300mM NaCl、300mM イミダゾール)を加えて3分間振とう撹拌した後に1500rpmで1分間遠心して粗精製タンパク質を得る溶出工程を2回行った。 (His tag affinity purification of EGFP protein, UBA protein, Ras protein, and β-galactosidase protein)
The reaction solution after the synthesis reaction was centrifuged at 12,000 rpm for 5 minutes at room temperature. 80 μL of the supernatant was added to a 96-well filter plate (Millipore), and 60 μL of A buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl) and 60 μL of 50% TALON (equilibrated with A buffer in advance) (Registered trademark) resin (TAKARA BIO) was added and shaken for 10 minutes. The filter plate was centrifuged at 1500 rpm for 1 minute to remove contaminant proteins derived from the extract. Here, the protein of interest is bound to the TALON resin. Next, a washing step of adding 150 μL of A buffer and centrifuging at 1500 rpm for 1 minute was performed three times. Finally, 50 μL of B buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 300 mM imidazole) was added, and the mixture was shaken and stirred for 3 minutes, and then centrifuged at 1500 rpm for 1 minute to carry out an elution step twice to obtain a crude protein. .
合成反応後の反応液を、12,000rpmで5分間、室温で遠心した。その上清80μLを、96穴フィルタープレート(ミリポア社)に加え、そこに60μLのAバッファー(20mM Tris-HCl pH7.5、300mM NaCl)、および予めAバッファーで平衡化した60μLの50% TALON(登録商標)樹脂(TAKARABIO社)を加えて、10分間振とうした。フィルタープレートを1500rpmで1分間遠心し、抽出液由来の夾雑タンパク質を除いた。ここで、目的のタンパク質はTALON樹脂に結合している。次に、150μLのAバッファーを加えて1500rpmで1分間遠心する洗浄工程を、3回行った。最後に、50μLのBバッファー(20mM Tris-HCl pH7.5、300mM NaCl、300mM イミダゾール)を加えて3分間振とう撹拌した後に1500rpmで1分間遠心して粗精製タンパク質を得る溶出工程を2回行った。 (His tag affinity purification of EGFP protein, UBA protein, Ras protein, and β-galactosidase protein)
The reaction solution after the synthesis reaction was centrifuged at 12,000 rpm for 5 minutes at room temperature. 80 μL of the supernatant was added to a 96-well filter plate (Millipore), and 60 μL of A buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl) and 60 μL of 50% TALON (equilibrated with A buffer in advance) (Registered trademark) resin (TAKARA BIO) was added and shaken for 10 minutes. The filter plate was centrifuged at 1500 rpm for 1 minute to remove contaminant proteins derived from the extract. Here, the protein of interest is bound to the TALON resin. Next, a washing step of adding 150 μL of A buffer and centrifuging at 1500 rpm for 1 minute was performed three times. Finally, 50 μL of B buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 300 mM imidazole) was added, and the mixture was shaken and stirred for 3 minutes, and then centrifuged at 1500 rpm for 1 minute to carry out an elution step twice to obtain a crude protein. .
(EGFPタンパク質、UBAタンパク質、Rasタンパク質、およびβガラクトシダーゼタンパク質の定量)
EGFPタンパク質、UBAタンパク質、Rasタンパク質およびβガラクトシダーゼタンパク質の各粗精製タンパク質の定量は、ウシ血清アルブミン(BSA)を標準物質として、Protein assay試薬(Bio-Rad社)を用いて、ブラッドフォード法で行った。また、粗精製タンパク質についてSDS-ポリアクリルアミドゲル電気泳動を行い、分子量の確認を行った。結果を図2および3に示す。 (Quantification of EGFP protein, UBA protein, Ras protein, and β-galactosidase protein)
Quantification of each crude protein of EGFP protein, UBA protein, Ras protein, and β-galactosidase protein was performed by the Bradford method using Protein assay reagent (Bio-Rad) with bovine serum albumin (BSA) as a standard substance. It was. The crude protein was subjected to SDS-polyacrylamide gel electrophoresis to confirm the molecular weight. The results are shown in FIGS.
EGFPタンパク質、UBAタンパク質、Rasタンパク質およびβガラクトシダーゼタンパク質の各粗精製タンパク質の定量は、ウシ血清アルブミン(BSA)を標準物質として、Protein assay試薬(Bio-Rad社)を用いて、ブラッドフォード法で行った。また、粗精製タンパク質についてSDS-ポリアクリルアミドゲル電気泳動を行い、分子量の確認を行った。結果を図2および3に示す。 (Quantification of EGFP protein, UBA protein, Ras protein, and β-galactosidase protein)
Quantification of each crude protein of EGFP protein, UBA protein, Ras protein, and β-galactosidase protein was performed by the Bradford method using Protein assay reagent (Bio-Rad) with bovine serum albumin (BSA) as a standard substance. It was. The crude protein was subjected to SDS-polyacrylamide gel electrophoresis to confirm the molecular weight. The results are shown in FIGS.
図2は、ブラッドフォード法により定量した各タンパク質の収量を示す図である。図3は、電気泳動の結果を示す図であり、レーンA~Dは、鋳型DNAとしてそれぞれpCR2.1 NHis-EGFP、pCR2.1 N11-UBA、pK7b2 NHis-RasおよびpUC19 N11-β-galactosidaseを用いた合成系由来の粗精製物であり、レーンEは合成系に鋳型DNAを含まないコントロールの合成系由来の粗精製物である。図2に示されるように、リボヌクレオシドを添加した合成系の場合には、NTPを添加した合成系の場合と比較して、タンパク質の収量が約1.2倍~1.4倍となり、より高い収量で得られた。また、図3に示されるように、リボヌクレオシドを添加した合成系において合成されたタンパク質は、NTPを添加した合成系の場合と同じく、目的のタンパク質の分子量を有するタンパク質であった。以上から、ごく小さなタンパク質から100kDaを超える高分子量タンパク質まで様々なタンパク質においても、NTPの代わりにリボヌクレオシドを用いることによって正しく合成が可能であるばかりでなく、NTPを用いた場合よりも収量を増加させることができることが示された。
FIG. 2 is a graph showing the yield of each protein quantified by the Bradford method. FIG. 3 shows the results of electrophoresis. Lanes A to D show pCR2.1 NHis-EGFP, pCR2.1 N11-UBA, pK7b2 NHis-Ras and pUC19 N11-β-galactosidase as template DNAs, respectively. Lane E is a crude product derived from a control synthesis system that does not contain template DNA in the synthesis system. As shown in FIG. 2, in the case of the synthetic system to which ribonucleoside was added, the protein yield was about 1.2 to 1.4 times that of the synthetic system to which NTP was added. A high yield was obtained. Further, as shown in FIG. 3, the protein synthesized in the synthesis system to which ribonucleoside was added was a protein having the molecular weight of the target protein, as in the case of the synthesis system to which NTP was added. From the above, various proteins ranging from very small proteins to high molecular weight proteins exceeding 100 kDa can be synthesized correctly by using ribonucleosides instead of NTP, and also increase the yield compared to using NTP. It was shown that can be made.
〔実施例3:添加するリボヌクレオシドの種類による評価〕
添加するリボヌクレオシドの種類および量を変化させた場合のタンパク質の合成量について比較を行った。 [Example 3: Evaluation by type of ribonucleoside to be added]
A comparison was made of the amount of protein synthesized when the type and amount of ribonucleoside to be added were changed.
添加するリボヌクレオシドの種類および量を変化させた場合のタンパク質の合成量について比較を行った。 [Example 3: Evaluation by type of ribonucleoside to be added]
A comparison was made of the amount of protein synthesized when the type and amount of ribonucleoside to be added were changed.
(合成反応条件)
鋳型DNAとしてpCR2.1 NHis GFP-S1を用い、4種類のリボヌクレオシドのうち、いずれか1種類、2種類または3種類を、各0.1~1.6mM添加した以外は、実施例1と同じである。プラスミドpCR2.1 NHis GFP-S1は、GFP-S1タンパク質をコードする配列を有しており、この配列の上流にT7プロモーターおよびリボソーム結合配列を有しており、下流に、T7ターミネーターを有している(非特許文献:Seki E, Matsuda N, Yokoyama S, Kigawa T.(2008)Anal Biochem. 377(2):156-161.参照)。 (Synthetic reaction conditions)
Example 1 except that pCR2.1 NHis GFP-S1 was used as the template DNA and any one, two or three of the four ribonucleosides were added at 0.1 to 1.6 mM each. The same. The plasmid pCR2.1 NHis GFP-S1 has a sequence encoding the GFP-S1 protein, has a T7 promoter and a ribosome binding sequence upstream of this sequence, and has a T7 terminator downstream. (Non-patent literature: See Seki E, Matsuda N, Yokoyama S, Kigawa T. (2008) Anal Biochem. 377 (2): 156-161.).
鋳型DNAとしてpCR2.1 NHis GFP-S1を用い、4種類のリボヌクレオシドのうち、いずれか1種類、2種類または3種類を、各0.1~1.6mM添加した以外は、実施例1と同じである。プラスミドpCR2.1 NHis GFP-S1は、GFP-S1タンパク質をコードする配列を有しており、この配列の上流にT7プロモーターおよびリボソーム結合配列を有しており、下流に、T7ターミネーターを有している(非特許文献:Seki E, Matsuda N, Yokoyama S, Kigawa T.(2008)Anal Biochem. 377(2):156-161.参照)。 (Synthetic reaction conditions)
Example 1 except that pCR2.1 NHis GFP-S1 was used as the template DNA and any one, two or three of the four ribonucleosides were added at 0.1 to 1.6 mM each. The same. The plasmid pCR2.1 NHis GFP-S1 has a sequence encoding the GFP-S1 protein, has a T7 promoter and a ribosome binding sequence upstream of this sequence, and has a T7 terminator downstream. (Non-patent literature: See Seki E, Matsuda N, Yokoyama S, Kigawa T. (2008) Anal Biochem. 377 (2): 156-161.).
反応は、30℃で3時間行った。
The reaction was performed at 30 ° C. for 3 hours.
(GFP-S1タンパク質の合成量の評価)
反応終了後、10μLの反応液に90μLのPBSを加えて、485nm(励起光)/535nm(発光)の蛍光強度を測定することにより、GFP-S1タンパク質の合成量を評価した。結果を図4に示す。 (Evaluation of synthesis amount of GFP-S1 protein)
After completion of the reaction, 90 μL of PBS was added to 10 μL of the reaction solution, and the fluorescence intensity at 485 nm (excitation light) / 535 nm (luminescence) was measured to evaluate the amount of GFP-S1 protein synthesized. The results are shown in FIG.
反応終了後、10μLの反応液に90μLのPBSを加えて、485nm(励起光)/535nm(発光)の蛍光強度を測定することにより、GFP-S1タンパク質の合成量を評価した。結果を図4に示す。 (Evaluation of synthesis amount of GFP-S1 protein)
After completion of the reaction, 90 μL of PBS was added to 10 μL of the reaction solution, and the fluorescence intensity at 485 nm (excitation light) / 535 nm (luminescence) was measured to evaluate the amount of GFP-S1 protein synthesized. The results are shown in FIG.
図4は各合成系におけるGFP-S1タンパク質由来の蛍光強度を示す図であり、(a)は、リボヌクレオシドとして少なくともアデノシンおよびグアノシンが添加されている合成系における結果を示しており、(b)は、リボヌクレオシドとして少なくともアデノシンが添加されている合成系における結果を示しており、(c)は、リボヌクレオシドとして少なくともグアノシンが添加されている合成系における結果を示しており、(d)は、リボヌクレオシドとしてアデノシンおよびグアノシンのいずれもが添加されていない合成系における結果を示している。
FIG. 4 is a diagram showing the fluorescence intensity derived from the GFP-S1 protein in each synthetic system, and (a) shows the results in the synthetic system to which at least adenosine and guanosine are added as ribonucleosides, (b) Shows the result in a synthetic system in which at least adenosine is added as a ribonucleoside, (c) shows the result in a synthetic system in which at least guanosine is added as a ribonucleoside, (d) The results are shown in a synthetic system in which neither adenosine nor guanosine is added as a ribonucleoside.
図4(a)に示されるように、4種類のリボヌクレオシドを添加した場合には、0.1~1.6mMの範囲で安定した蛍光値が得られた。このことから、仮に、リボヌクレオシドの濃度が多少ずれたとしても、タンパク質合成は安定していることが示された。また、リボヌクレオシドは必ずしも4種類加える必要はなく、例えば、グアノシンおよびシチジンの2種類のみを添加した場合にも蛍光値の上昇が検出され、0.4mM以上のグアノシンおよびシチジンのみを添加した場合には、4種類すべてのリボヌクレオシドを添加した場合と同程度の蛍光値が得られた(図4(c)参照)。これは、グアノシンまたはシチジンから、ATPおよびUTPが合成されたものと推定される。
As shown in FIG. 4 (a), when four types of ribonucleosides were added, stable fluorescence values were obtained in the range of 0.1 to 1.6 mM. This indicates that the protein synthesis is stable even if the ribonucleoside concentration is slightly deviated. In addition, it is not always necessary to add four types of ribonucleosides. For example, when only two types of guanosine and cytidine are added, an increase in fluorescence value is detected, and when only guanosine and cytidine of 0.4 mM or more are added. Obtained fluorescence values similar to those obtained when all four types of ribonucleosides were added (see FIG. 4C). This is presumed that ATP and UTP were synthesized from guanosine or cytidine.
さらに、グアノシンのみを添加した場合にも蛍光値の上昇が検出され、1.2mM以上のグアノシンを添加した場合には、NTPを添加した場合と同程度の蛍光値が得られた(図4(c)参照)。このことから、リボヌクレオシドを添加して合成を行う系において、グアノシンを添加することにより、合成量をより向上させることができることが示された。
Furthermore, when only guanosine was added, an increase in fluorescence value was detected, and when guanosine of 1.2 mM or more was added, a fluorescence value comparable to that obtained when NTP was added was obtained (FIG. 4 ( c)). From this, it was shown that the amount of synthesis can be further improved by adding guanosine in a system in which ribonucleoside is added for synthesis.
〔実施例4:透析法によるCATタンパク質の合成〕
上述の実施例1では、バッチ法によりCATタンパク質の合成を行ったが、透析法によってもCATタンパク質の合成を試みた。 [Example 4: Synthesis of CAT protein by dialysis]
In Example 1 described above, CAT protein was synthesized by a batch method, but synthesis of CAT protein was also attempted by a dialysis method.
上述の実施例1では、バッチ法によりCATタンパク質の合成を行ったが、透析法によってもCATタンパク質の合成を試みた。 [Example 4: Synthesis of CAT protein by dialysis]
In Example 1 described above, CAT protein was synthesized by a batch method, but synthesis of CAT protein was also attempted by a dialysis method.
(合成反応条件)
透析法による無細胞合成系の内液組成は、次の通りである:60mM HEPES-KOH(pH7.5)、230mM D-グルタミン酸カリウム、4% PEG8000、3mM DTT、0.05% NaN3、アデノシン、グアノシン、シチジン、ウリジン、36μg/mL フォリン酸、80mM クレアチンリン酸、175μg/mL E.coli tRNA、10mM 酢酸マグネシウム、各1.5mM タンパク質構成アミノ酸、100μg/mL クレアチンキナーゼ、60μg/mL T7RNAポリメラーゼ、1μg/mL 鋳型DNA、および30% 大腸菌S30抽出液。また、外液組成は、次の通りである:60mM HEPES-KOH(pH7.5)、230mM D-グルタミン酸カリウム、4% PEG8000、3mM DTT、0.05% NaN3、アデノシン、グアノシン、シチジン、ウリジン、36μg/mL フォリン酸、80mM クレアチンリン酸、10mM 酢酸マグネシウム、各1.5mM タンパク質構成アミノ酸、30% 大腸菌S30バッファー。 (Synthetic reaction conditions)
The internal solution composition of the cell-free synthesis system by dialysis is as follows: 60 mM HEPES-KOH (pH 7.5), 230 mM potassium D-glutamate, 4% PEG8000, 3 mM DTT, 0.05% NaN 3 , adenosine Guanosine, cytidine, uridine, 36 μg / mL folinic acid, 80 mM creatine phosphate, 175 μg / mL E. coli tRNA, 10 mM magnesium acetate, each 1.5 mM protein constituent amino acid, 100 μg / mL creatine kinase, 60 μg / mL T7 RNA polymerase, 1 μg / mL template DNA and 30% E. coli S30 extract. The composition of the external solution is as follows: 60 mM HEPES-KOH (pH 7.5), 230 mM potassium D-glutamate, 4% PEG8000, 3 mM DTT, 0.05% NaN 3 , adenosine, guanosine, cytidine,uridine 36 μg / mL folinic acid, 80 mM creatine phosphate, 10 mM magnesium acetate, 1.5 mM protein constituent amino acids, 30% E. coli S30 buffer.
透析法による無細胞合成系の内液組成は、次の通りである:60mM HEPES-KOH(pH7.5)、230mM D-グルタミン酸カリウム、4% PEG8000、3mM DTT、0.05% NaN3、アデノシン、グアノシン、シチジン、ウリジン、36μg/mL フォリン酸、80mM クレアチンリン酸、175μg/mL E.coli tRNA、10mM 酢酸マグネシウム、各1.5mM タンパク質構成アミノ酸、100μg/mL クレアチンキナーゼ、60μg/mL T7RNAポリメラーゼ、1μg/mL 鋳型DNA、および30% 大腸菌S30抽出液。また、外液組成は、次の通りである:60mM HEPES-KOH(pH7.5)、230mM D-グルタミン酸カリウム、4% PEG8000、3mM DTT、0.05% NaN3、アデノシン、グアノシン、シチジン、ウリジン、36μg/mL フォリン酸、80mM クレアチンリン酸、10mM 酢酸マグネシウム、各1.5mM タンパク質構成アミノ酸、30% 大腸菌S30バッファー。 (Synthetic reaction conditions)
The internal solution composition of the cell-free synthesis system by dialysis is as follows: 60 mM HEPES-KOH (pH 7.5), 230 mM potassium D-glutamate, 4% PEG8000, 3 mM DTT, 0.05% NaN 3 , adenosine Guanosine, cytidine, uridine, 36 μg / mL folinic acid, 80 mM creatine phosphate, 175 μg / mL E. coli tRNA, 10 mM magnesium acetate, each 1.5 mM protein constituent amino acid, 100 μg / mL creatine kinase, 60 μg / mL T7 RNA polymerase, 1 μg / mL template DNA and 30% E. coli S30 extract. The composition of the external solution is as follows: 60 mM HEPES-KOH (pH 7.5), 230 mM potassium D-glutamate, 4% PEG8000, 3 mM DTT, 0.05% NaN 3 , adenosine, guanosine, cytidine,
アデノシン、グアノシン、シチジンおよびウリジンの濃度は、内液および外液ともに、0.2mM、0.4mM、0.8mM、1.2mMまたは1.6mMである。なお、NTPを用いた対照としては、内液および外液ともに、アデノシン、グアノシン、シチジン、およびウリジンの代わりに、1.3mM ATP、0.9mM GTP、0.9mM CTP、および0.9mM UTPが添加されている。
The concentrations of adenosine, guanosine, cytidine, and uridine are 0.2 mM, 0.4 mM, 0.8 mM, 1.2 mM, or 1.6 mM for both the internal solution and the external solution. In addition, as a control using NTP, 1.3 mM ATP, 0.9 mM GTP, 0.9 mM CTP, and 0.9 mM UTP were used instead of adenosine, guanosine, cytidine, and uridine in both the inner and outer solutions. It has been added.
鋳型DNAには、pK7-CATを用いた。大腸菌S30抽出液ならびにシチジン、ウリジン、アデノシンおよびグアノシンは実施例1と同様にして調製した。
PK7-CAT was used as the template DNA. E. coli S30 extract and cytidine, uridine, adenosine and guanosine were prepared in the same manner as in Example 1.
反応は、30℃で14時間行った。
The reaction was carried out at 30 ° C. for 14 hours.
(CATタンパク質の定量)
CATタンパク質の定量方法は、実施例1と同様である。結果を図5に示す。 (Quantification of CAT protein)
The CAT protein quantification method is the same as in Example 1. The results are shown in FIG.
CATタンパク質の定量方法は、実施例1と同様である。結果を図5に示す。 (Quantification of CAT protein)
The CAT protein quantification method is the same as in Example 1. The results are shown in FIG.
図5は、CATタンパク質の合成量を比較した図である。図5に示されるように、0.8~1.6mMのリボヌクレオシドを添加した場合には、NTPを添加した場合の80~103%のCATタンパク質が合成されていた。すなわち、ミリグラム単位の大量調製に用いられる透析法においても、リボヌクレオシドを用いて、従来と同程度のタンパク質を合成できることが示された。
FIG. 5 is a diagram comparing the amount of CAT protein synthesis. As shown in FIG. 5, when 0.8 to 1.6 mM ribonucleoside was added, 80 to 103% of CAT protein was synthesized when NTP was added. That is, it was shown that the same degree of protein can be synthesized using ribonucleosides even in the dialysis method used for mass preparation in milligram units.
透析法を行う場合、通常、内液および外液の両方にNTPを加える必要があり、外液には内液の10倍の量を用いることが一般的である。すなわち、透析法ではより多くのNTPが必要となるため、NTPを安価なリボヌクレオシドに入れ替えることによるコストの削減効果は、透析法において顕著である(図6参照)。
When performing dialysis, it is usually necessary to add NTP to both the internal and external liquids, and it is common to use 10 times the amount of the internal liquid as the external liquid. That is, since more NTP is required in the dialysis method, the cost reduction effect by replacing NTP with an inexpensive ribonucleoside is remarkable in the dialysis method (see FIG. 6).
〔実施例5:NTP合成の確認〕
上記実施例1の反応において、鋳型DNAおよびT7 RNA polymeraseの何れをも添加していない反応液を調製し、反応開始前のサンプル、および反応開始から30分後のサンプルを各30μl回収し、5%TCAを等量加えて氷冷した。これを12,000rpmで5分間遠心を行い、得られた上清39μLに内部標準として2mM NADを1μL加えた。この溶液20μLに含まれる各ヌクレオシドおよび各NTPの検出を、C18カラムをつないだ高速液体クロマトグラフィー(HPLC)により行った。結果を図7に示す。 [Example 5: Confirmation of NTP synthesis]
In the reaction of Example 1 above, a reaction solution to which neither template DNA nor T7 RNA polymerase was added was prepared, and 30 μl each of a sample before the start of the reaction and a sample after 30 minutes from the start of the reaction were collected. An equal amount of% TCA was added and ice-cooled. This was centrifuged at 12,000 rpm for 5 minutes, and 1 μL of 2 mM NAD was added as an internal standard to 39 μL of the obtained supernatant. Each nucleoside and each NTP contained in 20 μL of this solution were detected by high performance liquid chromatography (HPLC) connected to a C18 column. The results are shown in FIG.
上記実施例1の反応において、鋳型DNAおよびT7 RNA polymeraseの何れをも添加していない反応液を調製し、反応開始前のサンプル、および反応開始から30分後のサンプルを各30μl回収し、5%TCAを等量加えて氷冷した。これを12,000rpmで5分間遠心を行い、得られた上清39μLに内部標準として2mM NADを1μL加えた。この溶液20μLに含まれる各ヌクレオシドおよび各NTPの検出を、C18カラムをつないだ高速液体クロマトグラフィー(HPLC)により行った。結果を図7に示す。 [Example 5: Confirmation of NTP synthesis]
In the reaction of Example 1 above, a reaction solution to which neither template DNA nor T7 RNA polymerase was added was prepared, and 30 μl each of a sample before the start of the reaction and a sample after 30 minutes from the start of the reaction were collected. An equal amount of% TCA was added and ice-cooled. This was centrifuged at 12,000 rpm for 5 minutes, and 1 μL of 2 mM NAD was added as an internal standard to 39 μL of the obtained supernatant. Each nucleoside and each NTP contained in 20 μL of this solution were detected by high performance liquid chromatography (HPLC) connected to a C18 column. The results are shown in FIG.
図7(a)は、反応開始前のサンプルにおける結果を示しており、図7(b)は、反応開始から30分後のサンプルにおける結果を示している。図7に示されるように、反応開始直後に検出されていたアデノシン、グアノシン、シチジンおよびウリジンのピークは、反応開始から30分後には大幅に低減しており、一方で、ATP、GTP、CTPおよびUTPのピークが増大していた。このことから、合成系内において、アデノシン、グアノシン、シチジンおよびウリジンから、ATP、GTP、CTPおよびUTPが合成されていることが確認できた。
FIG. 7 (a) shows the result in the sample before the start of the reaction, and FIG. 7 (b) shows the result in the sample 30 minutes after the start of the reaction. As shown in FIG. 7, the peaks of adenosine, guanosine, cytidine and uridine detected immediately after the start of the reaction are greatly reduced 30 minutes after the start of the reaction, while ATP, GTP, CTP and The UTP peak increased. This confirmed that ATP, GTP, CTP and UTP were synthesized from adenosine, guanosine, cytidine and uridine in the synthesis system.
〔実施例6:EGFPタンパク質の合成〕
エネルギー再生系の成分として、(i)クレアチンリン酸(CP)およびクレアチンキナーゼ(CK)、(ii)30mM PEP、0.33mM NAD、0.26mM CoAおよび2.7mM シュウ酸ナトリウム、または(iii)30mM グルコース、0.33mM NAD、0.26mM CoAおよび10mM K2HPO4を用い、かつ、pK7-CATの代わりに、pCR2.1 NHis-EGFPを用いたこと以外は、実施例1と同様にしてタンパク質の合成を行った。なお、ピルビン酸キナーゼは大腸菌S30抽出液中に含まれているため、ピルビン酸キナーゼのさらなる添加は行っていない。反応は、30℃で3時間行った。何れの場合にもさらなる対照として、リボヌクレオシドの代わりに、1.3mM AMP、0.9mM GMP、0.9mM CMP、および0.9mM UMPを添加して、あるいはリボヌクレオシドおよびその代替物を添加せずに、タンパク質の合成を行った。反応終了後、実施例3と同様にして、485nm(励起光)/535nm(発光)の蛍光強度を測定することにより、EGFPタンパク質の合成量を評価した。結果を図8に示す。 [Example 6: Synthesis of EGFP protein]
Components of the energy regeneration system include (i) creatine phosphate (CP) and creatine kinase (CK), (ii) 30 mM PEP, 0.33 mM NAD, 0.26 mM CoA and 2.7 mM sodium oxalate, or (iii) Example 1 except that 30 mM glucose, 0.33 mM NAD, 0.26 mM CoA and 10 mM K 2 HPO 4 were used, and pCR2.1 NHis-EGFP was used instead of pK7-CAT. Protein synthesis was performed. Since pyruvate kinase is contained in the E. coli S30 extract, no further addition of pyruvate kinase is performed. The reaction was carried out at 30 ° C. for 3 hours. As an additional control in each case, add 1.3 mM AMP, 0.9 mM GMP, 0.9 mM CMP, and 0.9 mM UMP instead of ribonucleoside, or add ribonucleoside and its substitutes. Without synthesis, protein synthesis was performed. After completion of the reaction, the amount of EGFP protein synthesized was evaluated by measuring the fluorescence intensity at 485 nm (excitation light) / 535 nm (emission) in the same manner as in Example 3. The results are shown in FIG.
エネルギー再生系の成分として、(i)クレアチンリン酸(CP)およびクレアチンキナーゼ(CK)、(ii)30mM PEP、0.33mM NAD、0.26mM CoAおよび2.7mM シュウ酸ナトリウム、または(iii)30mM グルコース、0.33mM NAD、0.26mM CoAおよび10mM K2HPO4を用い、かつ、pK7-CATの代わりに、pCR2.1 NHis-EGFPを用いたこと以外は、実施例1と同様にしてタンパク質の合成を行った。なお、ピルビン酸キナーゼは大腸菌S30抽出液中に含まれているため、ピルビン酸キナーゼのさらなる添加は行っていない。反応は、30℃で3時間行った。何れの場合にもさらなる対照として、リボヌクレオシドの代わりに、1.3mM AMP、0.9mM GMP、0.9mM CMP、および0.9mM UMPを添加して、あるいはリボヌクレオシドおよびその代替物を添加せずに、タンパク質の合成を行った。反応終了後、実施例3と同様にして、485nm(励起光)/535nm(発光)の蛍光強度を測定することにより、EGFPタンパク質の合成量を評価した。結果を図8に示す。 [Example 6: Synthesis of EGFP protein]
Components of the energy regeneration system include (i) creatine phosphate (CP) and creatine kinase (CK), (ii) 30 mM PEP, 0.33 mM NAD, 0.26 mM CoA and 2.7 mM sodium oxalate, or (iii) Example 1 except that 30 mM glucose, 0.33 mM NAD, 0.26 mM CoA and 10 mM K 2 HPO 4 were used, and pCR2.1 NHis-EGFP was used instead of pK7-CAT. Protein synthesis was performed. Since pyruvate kinase is contained in the E. coli S30 extract, no further addition of pyruvate kinase is performed. The reaction was carried out at 30 ° C. for 3 hours. As an additional control in each case, add 1.3 mM AMP, 0.9 mM GMP, 0.9 mM CMP, and 0.9 mM UMP instead of ribonucleoside, or add ribonucleoside and its substitutes. Without synthesis, protein synthesis was performed. After completion of the reaction, the amount of EGFP protein synthesized was evaluated by measuring the fluorescence intensity at 485 nm (excitation light) / 535 nm (emission) in the same manner as in Example 3. The results are shown in FIG.
図8に示されるように、PEPを用いた場合であっても、リボヌクレオシドを添加することにより、添加しない場合と比較し、タンパク質の合成量が増加した。
As shown in FIG. 8, even when PEP was used, the amount of protein synthesis increased by adding ribonucleoside compared to the case where PEP was not added.
また、CPおよびCKを含む系においてNMPを用いた場合、得られた蛍光値はCPおよびCKを含む系においてNTPを用いた場合の約9割程度であったのに対し、CPおよびCKを含む系においてリボヌクレオシドを用いた場合、得られた蛍光値はCPおよびCKを含む系においてNTPを用い場合の約1.2倍であった。
In addition, when NMP was used in a system containing CP and CK, the obtained fluorescence value was about 90% of that when NTP was used in a system containing CP and CK, whereas CP and CK were included. When ribonucleoside was used in the system, the fluorescence value obtained was about 1.2 times that obtained when NTP was used in the system containing CP and CK.
本発明は、タンパク質の合成、機能の解析、および構造の解析を必要とする、製薬分野および保健医学分野をはじめ、生命科学分野の産業に広く利用することができる。
The present invention can be widely applied to industries in the life science field including the pharmaceutical field and the health medicine field, which require protein synthesis, functional analysis, and structural analysis.
Claims (11)
- 無細胞タンパク質合成系によるタンパク質の合成方法であって、
タンパク質をコードする鋳型DNAと、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドとを、タンパク質合成用の細胞抽出液を含む反応液に添加することを特徴とするタンパク質の合成方法。 A protein synthesis method using a cell-free protein synthesis system,
A protein DNA characterized by adding a template DNA encoding a protein and at least one ribonucleoside of adenosine, guanosine, uridine and cytidine to a reaction solution containing a cell extract for protein synthesis. Synthesis method. - 少なくともグアノシンを添加することを特徴とする請求項1に記載のタンパク質の合成方法。 The protein synthesis method according to claim 1, wherein at least guanosine is added.
- アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか2種のリボヌクレオシドを添加することを特徴とする請求項1に記載のタンパク質の合成方法。 The method for synthesizing a protein according to claim 1, wherein at least any two ribonucleosides of adenosine, guanosine, uridine and cytidine are added.
- 少なくともグアノシンおよびシチジンを添加することを特徴とする請求項1~3のいずれか1項に記載のタンパク質の合成方法。 The method for synthesizing a protein according to any one of claims 1 to 3, wherein at least guanosine and cytidine are added.
- 上記細胞抽出液は細菌細胞から調製した抽出液であることを特徴とする請求項1~4の何れか1項に記載のタンパク質の合成方法。 The method for synthesizing a protein according to any one of claims 1 to 4, wherein the cell extract is an extract prepared from bacterial cells.
- 上記細胞抽出液は大腸菌から調製した抽出液であることを特徴とする請求項5に記載のタンパク質の合成方法。 6. The protein synthesis method according to claim 5, wherein the cell extract is an extract prepared from E. coli.
- ATP再生系の成分を上記反応液に添加することを特徴とする請求項1~6の何れか1項に記載のタンパク質の合成方法。 The method for synthesizing a protein according to any one of claims 1 to 6, wherein a component of the ATP regeneration system is added to the reaction solution.
- 上記ATP再生系の成分として、クレアチンキナーゼおよびクレアチンリン酸を上記反応液に添加することを特徴とする請求項7に記載のタンパク質の合成方法。 The method for synthesizing a protein according to claim 7, wherein creatine kinase and creatine phosphate are added to the reaction solution as components of the ATP regeneration system.
- 透析法による合成方法であることを特徴とする請求項1~8の何れか1項に記載のタンパク質の合成方法。 The protein synthesis method according to any one of claims 1 to 8, wherein the protein synthesis method is a dialysis method.
- バッチ法による合成方法であることを特徴とする請求項1~8の何れか1項に記載のタンパク質の合成方法。 The protein synthesis method according to any one of claims 1 to 8, wherein the protein synthesis method is a batch method.
- 請求項1~10の何れか1項に記載のタンパク質の合成方法に使用されるタンパク質の合成キットであって、アデノシン、グアノシン、ウリジンおよびシチジンのうちの少なくとも何れか1種のリボヌクレオシドと、細胞抽出液とを含むことを特徴とするタンパク質の合成キット。 A protein synthesis kit used in the protein synthesis method according to any one of claims 1 to 10, comprising at least one ribonucleoside of adenosine, guanosine, uridine, and cytidine, and a cell A protein synthesis kit comprising an extract.
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CALHOUN K.A. ET AL.: "An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates.", BIOTECHNOL. PROG., vol. 21, no. 4, 2005, pages 1146 - 1153 * |
KIM H.C. ET AL.: "Methods for energizing cell- free protein synthesis.", J. BIOSCI. BIOENG., vol. 108, no. 1, 2009, pages 1 - 4 * |
YOKOYAMA J. ET AL.: "An economical method for producing stable-isotope labeled proteins by the E.coli cell-free system.", J. BIOMOL. NMR, vol. 48, no. 4, 2010, pages 193 - 201 * |
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