JPWO2006109751A1 - Protein production method using cell-free protein synthesis system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a protein under a high temperature condition in a cell-free protein translation system containing a cell extract derived from a hyperthermophilic bacterium or a cell-free protein transcription / translation system. In a cell-free protein translation system containing a cell extract derived from a hyperthermophilic bacterium, translation of mRNA encoding the target protein is carried out under high temperature conditions to produce the protein. In a cell-free protein transcription / translation system containing a cell extract derived from a hyperthermophilic bacterium, transcription and translation of a plasmid encoding the target protein is performed under high temperature conditions to produce the protein. [Selection figure] None

Description

  The present invention relates to a method for producing a protein using a cell-free protein translation system or a cell-free protein transcription / translation system under high temperature conditions.

  The cell-free protein synthesis system is a system that synthesizes proteins using cell extracts. Compared to systems that use living cells such as E. coli, protein synthesis and purification procedures are much simpler. Demand is increasing. By using this system, the system can be easily modified, so that not only industrially useful cell-derived proteins can be obtained, but, for example, proteins highly toxic to cells, There is an advantage that a protein introduced with a natural amino acid can also be obtained.

  As the cell extract, those derived from Escherichia coli, wheat embryo and rabbit reticulocyte are mainly used. Conventionally, it has been pointed out that the cell-free protein synthesis system using these cell extracts has a low protein synthesis amount. To solve such problems, for example, a concentrated cell extract is used. (Patent Document 1), and a technique of adding a linear nucleic acid that does not serve as a template for protein synthesis to a synthesis reaction solution at a specific concentration (Patent Document 2) is disclosed.

  The cell-free protein synthesis system described above synthesizes proteins at room temperature. For example, Pseudomonas microorganisms that can be grown at low temperatures to synthesize proteins that are difficult to synthesize at room temperature, such as nucleases that degrade template DNA. A cell-free protein synthesis system that synthesizes proteins under low temperature conditions using an extract or a Rhodococcus microorganism extract is disclosed (Patent Document 3).

JP 2000-175695 A JP 2001-136971 A JP 2004-290181 A

  As described above, a cell-free protein synthesis system at room temperature using cell extracts derived from Escherichia coli, wheat embryos and rabbit reticulocytes, and a cell-free protein synthesis system at low temperatures using cell extracts derived from the related species of E. coli Has been developed. However, no cell-free protein synthesis system under high temperature conditions has been disclosed so far.

  If a cell-free protein synthesis system can be established under high temperature conditions, the protein production rate will be improved compared to conventional cell-free protein synthesis systems, and the reaction will be less likely to occur at room temperature. Even if it is mixed, it can be expected to produce such effects that the effect is small, that it is possible to synthesize a protein resistant to high temperature, and that it is possible to screen a protein resistant to high temperature by introducing a mutant DNA library.

  Therefore, in order to establish a cell-free protein synthesis system under high-temperature conditions, the present inventors have focused on Thermococcus kodakaraensis KOD1 strain, which is a kind of hyperthermophilic bacterium, and a reaction solution containing a cell extract derived from the strain. As a result of intensive investigations on reaction conditions suitable for protein synthesis using the present invention, the present invention has been completed.

That is, the gist of the present invention is as follows.
[1] A method for producing a protein using a cell-free protein translation system, wherein the mRNA encoding the target protein is translated under high temperature conditions in a cell-free protein translation system containing a cell extract derived from a hyperthermophilic bacterium. A method for producing proteins by
[2] The method according to [1], wherein the cell-free protein translation system contains mRNA, Mg ²⁺ and an amino acid encoding the target protein,
[3] The method according to [2], further comprising at least one component of NH ₄ ⁺ , K ⁺ , polyethylene glycol, ATP regeneration system and energy source,
[4] The method according to any one of [1] to [3], wherein the cell extract is derived from Thermococcus kodakaraensis KOD1 strain or a strain derived from the KOD1 strain,
[5] The method according to any one of [1] to [4], wherein the mRNA encoding the target protein has the ribosome binding sequence shown in SEQ ID NO: 4 upstream of the base sequence encoding the target protein;
[6] A kit for producing a protein by a cell-free protein translation system, which contains a cell extract derived from a hyperthermophilic bacterium, mRNA encoding the target protein, Mg ²⁺ and amino acids as a cell-free protein translation system A kit for producing a protein by translating the mRNA under high temperature conditions,
[7] The kit according to [6], wherein the cell-free protein translation system further contains at least one component of NH ₄ ⁺ , K ⁺ , polyethylene glycol, ATP regeneration system and energy source. ,
[8] The kit according to [6] or [7], wherein the cell extract is derived from Thermococcus kodakaraensis KOD1 strain or a strain derived from the KOD1 strain,
[9] The kit according to any one of [6] to [8], wherein the mRNA encoding the target protein has the ribosome binding sequence shown in SEQ ID NO: 4 upstream of the base sequence encoding the target protein;
[10] A method for producing a protein using a cell-free protein transcription / translation system, wherein a plasmid encoding a target protein is transferred in a cell-free protein transcription / translation system containing a cell extract derived from a hyperthermophilic bacterium. A method for producing a protein by performing translation under high temperature conditions;
[11] The method according to [10], wherein the cell-free protein transcription / translation system contains a plasmid encoding the target protein, RNA polymerase, Mg ²⁺ and an amino acid,
[12] The method according to [11], wherein the RNA polymerase has an optimum temperature under high temperature conditions,
[13] The method according to any one of [10] to [12], wherein the cell extract is derived from Thermococcus kodakaraensis KOD1 strain or a strain derived from the KOD1 strain,
[14] The method according to any one of [10] to [13], wherein the plasmid encoding the target protein comprises a promoter, a ribosome binding sequence shown in SEQ ID NO: 4 and a base sequence encoding the target protein in order from upstream.
[15] In performing transcription and translation of a plasmid encoding the target protein under high temperature conditions, the transcription temperature is set to the optimum temperature of the RNA polymerase, and the translation temperature is any one of [1] to [9] The method according to any one of [11] to [14], wherein the temperature is set to an optimum temperature of the cell-free protein translation system according to
[16] A kit for producing a protein by a cell-free protein transcription / translation system, wherein the cell-free protein transcription / translation system includes a cell extract derived from a hyperthermophilic bacterium, a plasmid encoding a target protein, an RNA polymerase A kit for producing a protein containing Mg ²⁺ and an amino acid, wherein transcription and translation of the plasmid are performed under high temperature conditions;
[17] The kit according to [16], wherein the RNA polymerase has an optimum temperature under high temperature conditions,
[18] The kit according to [16] or [17], wherein the cell extract is derived from Thermococcus kodakaraensis KOD1 strain or a strain derived from the KOD1 strain,
[19] The kit according to any one of [16] to [18], wherein the plasmid encoding the target protein includes a promoter, a ribosome binding sequence shown in SEQ ID NO: 4 and a base sequence encoding the target protein in order from upstream.

  According to the present invention, a protein can be produced under a high temperature condition in a cell-free protein translation system containing a cell extract derived from a hyperthermophilic bacterium. Moreover, according to the present invention, a protein can be produced under high temperature conditions in a cell-free protein transcription / translation system containing a cell extract derived from a hyperthermophilic bacterium.

FIG. 2 is a schematic diagram of pTRC1 and an outline of a method for preparing ChiAΔ4 mRNA. It is the result of performing a synthesis reaction of ChiAΔ4 using the sample shown in Table 2, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. The results are shown in Table 3. Samples 1, 3, 4 and 9 in Table 3 were used for ChiAΔ4 synthesis, subjected to polyacrylamide gel electrophoresis and analyzed by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 and 5 to 9 in Table 3, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 and 2 in Table 4, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 and 3 to 5 in Table 4, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 2 and 6 to 8 in Table 4, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 9 in Table 5, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is a figure which shows the synthetic | combination amount of ChiA (DELTA) 4 after the synthesis reaction was performed using the samples 2-9 of Table 5, and the synthesis reaction was complete | finished. It is a figure which shows the time change of the synthesis amount of ChiAΔ4 when the synthesis reaction of ChiAΔ4 was performed using samples 5, 7 and 9 in Table 5. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 3 in Table 6, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 4 to 6 in Table 6, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. The results are shown in Table 7. Samples 1-3 and 9 in Table 7 were used for ChiAΔ4 synthesis reaction, subjected to polyacrylamide gel electrophoresis and analyzed by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 4 to 9 in Table 7, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. The results are shown in Table 8. Samples 1 and 9 in Table 8 were used for ChiAΔ4 synthesis reaction, subjected to polyacrylamide gel electrophoresis and analyzed by Western blotting. The results are shown in Table 8. Samples 2, 3 and 9 in Table 8 were used for ChiAΔ4 synthesis reaction, subjected to polyacrylamide gel electrophoresis and analyzed by Western blotting. The results are shown in Table 8. Samples 4, 5 and 9 in Table 8 were used for ChiAΔ4 synthesis reaction, subjected to polyacrylamide gel electrophoresis and analyzed by Western blotting. The results are shown in Table 8. Samples 6 and 9 in Table 8 were used for ChiAΔ4 synthesis reaction, subjected to polyacrylamide gel electrophoresis and analyzed by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 7 in Table 9, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. The results are shown in Table 10. Samples 8 and 9 in Table 10 were used for ChiAΔ4 synthesis reaction, subjected to polyacrylamide gel electrophoresis and analyzed by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using Sample 8 in Table 11, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. The results of ChiAΔ4 synthesis reaction using sample 8 in Table 12, polyacrylamide gel electrophoresis, and analysis by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 4 in Table 13, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. Among the procedures described in “2. Preparation of cell extract”, the protocol is a protocol summarizing each procedure after culturing the KOD1 strain. It is the result of performing a synthetic reaction of ChiAΔ4 using samples 1 to 5 in Table 14, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 4 in Table 15, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 4 in Table 18, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 and 2 in Table 20, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. This is a protocol for the preparation of S30FP-1G. It is the result of performing a synthetic reaction of ChiAΔ4 using samples 1 to 5 in Table 21, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 6 to 10 in Table 22, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 5 in Table 23, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 4 in Table 24, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 6 in Table 25, polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 4 in Table 27, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the figure which showed the relationship between the PEP density | concentration in a reaction liquid, and the synthesis | combination ChiA (DELTA) 4 density | concentration. It is the result of performing a synthetic reaction of ChiAΔ4 using samples 1 to 11 in Table 28, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the figure which showed the relationship between Mg2 ⁺ density | concentration in a reaction liquid, and the density | concentration of ChiA (DELTA) 4 synthesize | combined. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 6 in Table 29, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the figure which showed the relationship between NH4 ⁺ density | concentration in a reaction liquid and the density | concentration of ChiA (DELTA) ₄ synthesize | combined. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 8 in Table 30, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the figure which showed the relationship between the amino acid density | concentration in a reaction liquid, and the synthesis | combination ChiA (DELTA) 4 density | concentration. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 7 in Table 31, performing polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the figure which showed the relationship between the polyethyleneglycol density | concentration in a reaction liquid, and the synthesis | combination ChiA (DELTA) 4 density | concentration. It is the result of performing a synthesis reaction of ChiAΔ4 using samples 1 to 11 in Table 32, polyacrylamide gel electrophoresis, and analyzing by Western blotting. It is the figure which showed the relationship between K ⁺ density | concentration in a reaction liquid, and the density | concentration of ChiA (DELTA) 4 synthesized. It is a figure which performs the synthetic reaction of ChiA (DELTA) 4 using the samples 1-5 of Table 34, and shows the synthetic | combination amount of ChiA (DELTA) 4 after completion | finish of a synthetic reaction.

  Hereinafter, the present invention will be described in detail. The present invention relates to a method for producing a protein using a cell-free protein translation system, and in a cell-free protein translation system containing a cell extract derived from a hyperthermophilic bacterium, translation of mRNA encoding the target protein is performed under high temperature conditions. To produce a protein. The present invention also relates to a method for producing a protein using a cell-free protein transcription / translation system, which comprises a DNA encoding a target protein in a cell-free protein transcription / translation system containing a cell extract derived from a hyperthermophilic bacterium. This is a method for producing a protein by performing transcription and translation of (plasmid) under high temperature conditions.

  In the present invention, the “high temperature condition” means a temperature higher than the optimum growth temperature of a normal microorganism, and 40 to 85 ° C., preferably 55 to 80 ° C. when producing a protein by a cell-free protein translation system. More preferably, it refers to a temperature range of 57-78 ° C. When a protein is produced by a cell-free protein transcription / translation system, it is 40-85 ° C, preferably 40-80 ° C, more preferably 40-75 ° C. Refers to the temperature range.

  The “cell-free protein translation system” means a coexistence of a cell extract derived from a living cell and an mRNA encoding the target protein (the mRNA is not derived from the cell extract but separately prepared) A system that synthesizes the protein through translation of the mRNA. “Cell-free protein transcription / translation system” refers to the coexistence of a cell extract derived from a living cell and a DNA encoding the target protein (the DNA is not derived from the cell extract but prepared separately). A system for synthesizing the protein through transcription and translation using the DNA as a template. In the following description, a cell-free protein translation system and a cell-free protein transcription / translation system are collectively referred to as a “cell-free protein synthesis system” unless otherwise distinguished.

  The term “hyperthermophilic bacterium” used in the present invention refers to a thermophilic bacterium or thermophilic progenitor that grows at 60 ° C. or higher, preferably 90 ° C. or higher. The hyperthermophilic bacterium applicable to the present invention is not particularly limited as long as it meets the above definition. Currently, more than 100 types of hyperthermophilic bacteria have been isolated and identified, and any of these can be applied to the present invention. Such hyperthermophilic bacteria include, for example, Aquifex pyrophilus, Geothermobacterium ferrireducens, Thermotoga maritima, Thermotoga neopolitana, Thermotoga petrophila, Thermotoga naphtophila, Acidianus infernus, Aeropyrum pernix, Archaeoglous lucidus, Archaeoglousus mucosus, Ferroglobus placidus, Geoglobus ahangari, Hyperthermus butylicus, Ignicoccus islandicus, Ignicoccus pacificus, Methanococcus jannaschii, Methanococcus fervens, Methanococcus igneus, Methanococcus infernus, Methanopyrus kandleri, Methanothermus fervidus, Methanothermus sociabilis, Palaeococcus ferrophilus, Pyrobaculum aerophilum, Pyrobaculum calidifontis, Pyrobaculum islandicum, Pyrobaculum oguniense, Pyrococcus furiosus, Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus woesei, Pyrodictium abyssi, Pyrodictium brockii, Pyrodictium occultum, Pyrolobus fumarii, Staph ylothermus marinus, Stetteria hydrogenophila, Sulfolobus solfataricus, Sulfolobus shibatae, Sulfolobus tokodaii, Sulfophobococcus zilligii, Sulfurisphaera ohwakuensis, Thermococcus kodakaraensis, Thermococcus celer, Thermococcus litoralis, Thermodiscus maritimus, Thermofilum pendens, Thermoproteus tenax, Thermoproteus neutrophilus, Thermosphaera aggregans, Vulcanisaeta distributa, Vulcanisaeta souniana , Acidianus ambivalens, Acidianus brierleyi, Acidianus convivator, Acidianus manzaensis, Acidianus pozzuoliensis, Acidianus tengchongenses, Metallosphaera hakonensis, Metallosphaera prunae, Metallosphaera sedula, Stygiolobus azoricus, Sulfolobus acidocaldarius, Sulfolobus islandicus, Sulfolobus metallicus, Sulfolobus neozealandicus, Sulfolobus tengchongensis, Sulfolobus thuringiensis, Sulfolobus yangmingensis, Sulfurisphaera ohwakuensis, Caldisphaera lagunensis, Acidilobus aceticus, Caldivirga maquilingensis, Thermocoladium modestius, Methanothermobac ter thermautotrophicus, Methanothermobacter defluvii, Methanothermobacter marburgensis, Methanothermobacter thermoflexus, Methanothermobacter thermophilus, Methanothermobacter wolfeii, Methanothermococcus okinawensis, Methanothermococcus thermolithotrophicus, Picrophilus oshimae, Picrophilus torridus, Thermoplasma acidophilum, Thermoplasma volcanium, Halobacterium salinarium, Chlorobium tepidum, Chloroflexus aurantiacus, Heliothrix oregonensis, Thermomicrobium roseum , Ammonifex degensii, Anaerobranca gottschalkii, Bacillus thermooleovorans, Bacillus caldevelox, Bacillus thermoglucosidasius, Bacillus thermantarcticus, Bacillus thermoalkalophilus, Bacillus thermoamyloliquefaciens, Bacillus thermoamylovorans, Bacillus thermocloaceae, Bacillus thermoproteolyticus, Bacillus thermoterrestris, Bacillus thermozeamaize, Caldicellulosiruptor saccharolyticus, Carboxydothermus hydrogenoformans, Clostridium acetobutylicum, Clostridium caminithermale, Clostridium ce llulose, Clostridium isatidis, Clostridium stercorarium, Clostridium straminisolvens, Clostridium thermoalcaliphilum, Clostridium thermobutyricum, Clostridium thermocellum, Clostridium thermopalmarium, Clostridium thermosuccinogenes, Coprothermobacter proteolyticus, Geobacillus kaustophilus, Geobacillus thermocatenulatus, Geobacillus thermodenitrificans, Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Geobacillus thermoleovorans, Heliobacterium modesticaldum, Moorella thermoacetica, Thermoanaerobacter acetoethylicus, Thermoanaerobacter brockii, Thermoanaerobacter ethanolicus, Thermoanaerobacter italicus, Thermoanaerobacter keratinophilus, Thermoanaerobacter kivui, Thermoanaerobacter mathranii, Thermoanaerobacter siderophilus, Thermoanaerobacter subterraneus, Thermoanaerobacter sulfurigignens, Thermoanaerobacter sulfurophilus, Thermoanaerobacter tengcongensis, Thermoanaerobacter thermocopriae, Thermoanaerobacter thermohydrosulfuricus, The rmoanaerobacter wiegelii, Thermoanaerobacter yonseiensis, Thermoanaerobacterium aotearoense, Thermoanaerobacterium bryantii, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium zeae, Thermovenabulum ferriorganovorum, Methylococcus capsulatus, Symbiobacterium thermophilum, Acidothermus cellulolyticus, Rubrobacter xylanophilus, Rubrobacter radiotolerans, Thermobifida fusca , Thermosynechococcus elongatus, Thermus antranikianii, Thermus aquaticus, Thermus brockianus, Thermus caldophilus, Thermus filiformis, Thermus igniterrae, Thermus kawarayensis, Thermus nonproteolyticus, Thermus oshimai, Themusus reoshir, Thermus reoshir thermophilum 、 Thermodesulfobacterium commune Such Thermodesulfovibrio yellowstonii the like (e.g., Atomi HareMiyukira, Biochemistry, Chapter 75, pp. No. 7, see pp.561-575,2003).

  In the present invention, among the hyperthermophilic bacteria described above, Thermococcus kodakaraensis KOD1 strain (hereinafter referred to as “KOD1 strain” as necessary) and Aeropyrum pernix K1 strain are used in the Examples. KOD1 stock has been deposited at the RIKEN BioResource Center, and its deposit number is JCM 12380. Aeropyrum pernix K1 strain is also deposited at the RIKEN BioResource Center, and its deposit number is JCM 9820.

  The protein that can be produced in the present invention is a protein derived from a hyperthermophilic bacterium, a protein introduced with an unnatural amino acid, a protein introduced with a target mutation, or a peptide having an arbitrary molecular weight composed of a plurality of amino acid residues. Including. Proteins derived from hyperthermophilic bacteria generally have high heat resistance. For example, from the viewpoint of industrial use, heat-resistant enzymes derived from hyperthermophilic bacteria are suitable for the purpose of the present invention.

  The cell extract refers to a hyperthermophile extract containing components necessary for protein synthesis such as ribosome and tRNA. This cell extract can be prepared by the method of Pratt et al. (Pratt, Transcription and Translation-a practical approach, Henes, BD and Higgins, SJ ed., Pp.179-209, IRL. Press, Oxford., Pp.179-209 (1984)). Specifically, it can be prepared by crushing using a French press or crushing using glass beads.

  For cell-free protein translation systems, in addition to the cell extract described above, mRNA encoding the target protein, ATP regeneration system, ATP, GTP, CTP, UTP and other energy sources, buffers, salts, amino acids, RNase inhibition Agents, tRNA, dithiothreitol (DTT), spermine, spermidine, stabilizers such as polyethylene glycol (eg, PEG8000), antibacterial agents and the like. The cell-free protein transcription / translation system includes a plasmid encoding the target protein instead of the mRNA encoding the target protein based on the above-described components, and can further include an RNA polymerase. Here, in the present invention, the mRNA encoding the target protein and the plasmid encoding the target protein are not derived from the cell extract but separately prepared.

  The ATP regeneration system is not particularly limited, and a known combination of a phosphate donor and a kinase can be used. Examples of this combination include phosphoenolpyruvate (PEP) and pyruvate kinase (PK), creatine phosphate (CP) and creatine kinase (CK), acetyl phosphate (AP) and acetate. A combination of kinase (AK) and the like can be mentioned. An ATP regeneration system comprising these combinations may be added to the cell-free protein synthesis system. In addition, when the above kinase is endogenous to the cell extract, only the above phosphate donor can be added to the cell-free protein synthesis system.

  As the buffer, a buffer known in the art may be used, and examples thereof include Tris-acetate buffer, Tris-hydrochloric acid buffer, Hepes-KOH, and the like. As salts, salts known in the art may be used, and examples thereof include acetates (magnesium acetate, ammonium acetate, potassium acetate, etc.), glutamates and the like. Examples of the antibacterial agent include sodium azide and ampicillin.

As the plasmid encoding the target protein, a plasmid containing a promoter, a ribosome binding sequence (RBS) and a base sequence encoding the target protein in order from the upstream is preferable. In addition, an appropriate terminator may be introduced downstream of the base sequence encoding the target protein in order to terminate the transcription reaction. When a terminator is not introduced, a fragment obtained by treating with a suitable restriction enzyme in advance may be used (run off method). The plasmid that can be used in the present invention is not particularly limited, and known plasmids can be used, and appropriate promoters, RBS, and other terminators can be appropriately introduced and used by known genetic engineering techniques. .

  As the promoter, an endogenous promoter possessed by the hyperthermophilic bacterium used in the preparation of the cell extract described above may be used, or an exogenous promoter may be used. The endogenous promoter is not particularly limited as long as a strong promoter capable of mass-producing the gene product encoded by the chromosomal DNA of the hyperthermophilic bacterium to be used is appropriately selected. When the KOD1 strain is used as a hyperthermophilic bacterium, for example, a GDH promoter that controls transcription of a thermostable glutamate dehydrogenase (GDH) gene can be used (RNZARahman et al., Mol Gen Genet. 257. pp.338). -449, (1998), JP 2000-41680). In addition, when an exogenous promoter is used, it is necessary to be able to act under the high temperature conditions described above. As a promoter satisfying such conditions, for example, the T7 promoter can be mentioned.

As RBS, endogenous RBS possessed by the hyperthermophilic bacterium used in the preparation of the cell extract described above can be appropriately selected and used. The inventors of the present invention have conventionally isolated and functionally analyzed many genes derived from the KOD1 strain, and as a result of comparing many upstream sequences of the start codon of the gene, the consensus sequence of the RBS of the KOD1 strain is , 5′-GG (T / A) G (A / G) (T / G) -3 ′ (SEQ ID NO: 4). Therefore, when using the KOD1 strain as a hyperthermophilic bacterium, RBS can be appropriately selected based on the base sequence of SEQ ID NO: 4.
In Examples described later, RBS (5′-GGTGGT-3 ′, SEQ ID NO: 5) of the above-mentioned thermostable glutamate dehydrogenase (GDH) gene is used.

  The target protein is not particularly limited as described above, but in order to know the behavior of the cell-free protein synthesis system in the present invention, a reporter gene encoding a protein whose characteristics are understood is expressed as a target gene, It is necessary to evaluate the efficiency of the system. Accordingly, as described in the Examples described later, the present inventors focused on chitinase derived from the KOD1 strain (SEQ ID NO: 1) and reported the gene encoding the partially deleted fragment (ChiAΔ4, SEQ ID NO: 2) as a reporter. I decided to use it as a gene. The inventors of the present invention have conventionally isolated a large number of proteins derived from the KOD1 strain and analyzed their functions, of which chitinase has also been isolated and analyzed (Japanese Patent Laid-Open No. 11-313688). According to the study by the present inventors, the 910th Gly of SEQ ID NO: 1 is substituted with Met, and this is used as the N-terminus, and a partial deletion fragment consisting of the 911-1115th amino acid residues downstream thereof ( ChiAΔ4, SEQ ID NO: 2) has extremely high thermal stability, such as a residual activity of 70% or more even after heat treatment at 100 ° C. for 3 hours. Therefore, in the present invention, in consideration of performing protein synthesis under the high-temperature conditions described above, a gene encoding ChiAΔ4 is selected as a reporter gene, and the efficiency of the cell-free protein synthesis system is analyzed through analysis of the synthesized ChiAΔ4. Decided to consider.

  As the terminator, an endogenous terminator possessed by the hyperthermophilic bacterium used in the preparation of the cell extract described above may be used, or an exogenous known terminator may be used. When an exogenous terminator is used, it can be appropriately selected in consideration of the combination with the promoter used. For example, when a T7 promoter is used as an exogenous promoter, a T7 terminator can be selected as an exogenous terminator.

  As the mRNA encoding the target protein, mRNA having a ribosome binding sequence (RBS) upstream of the base sequence encoding the target protein is suitable. Here, the base sequence encoding the target protein and the ribosome binding sequence can be appropriately selected according to the same criteria as the plasmid encoding the target protein described above. The mRNA used in the present invention can be easily prepared, for example, by first preparing the plasmid described above and then transcribe the plasmid.

  As the RNA polymerase, those having an optimum temperature under high temperature conditions are suitable. Here, “high temperature condition” means 40 ° C. or higher, preferably 50 ° C. or higher, more preferably 60 ° C. or higher. As the RNA polymerase exhibiting the above characteristics, an RNA polymerase derived from a hyperthermophile used in the preparation of the cell extract described above may be used, or an exogenous RNA polymerase may be used. The RNA polymerase may be appropriately selected in consideration of the combination with the promoter described above. For example, when a T7 promoter is used as the promoter, T7 RNA polymerase (for example, Thermo T7 RNA Polymerase, Toyobo Co., Ltd.) is used as the RNA polymerase. You can choose.

When a protein is produced by a cell-free protein translation system, a reaction solution can be prepared by adding various components as described above in addition to a cell extract derived from a hyperthermophilic bacterium. If the cell extract, mRNA encoding the target protein, Mg ²⁺ and 20 kinds of amino acids are contained in the reaction solution, the target protein can be produced. In addition to the above components, if the reaction solution contains at least one of NH ₄ ⁺ , K ⁺ , polyethylene glycol, ATP regeneration system and energy source, the expression level of the target protein is increased. Can be made. The concentration of each component can be appropriately set according to the cell extract to be used, reaction temperature, reaction time, reaction scale, and the like.

  Moreover, in order to increase the expression level of the target protein using a cell-free protein translation system, it is preferable to prepare a cell extract suitable for it. Here, the cell extract derived from hyperthermophilic bacteria is displayed in the step of culturing the cells, the step of suspending the cultured cells in artificial seawater, the step of crushing the cell suspension, and the lysate after crushing. Including the step of adding the pre-incubation mix according to 1 (hereinafter referred to as “preincubation”) and the subsequent dialysis step, etc., and substantially the same as the cell extract derived from E. coli. Can be prepared by the method. In the present invention, among the above steps, when a French press is used as a means for crushing bacterial cells, as long as the bacterial cells are efficiently disrupted, the pressure during pressing is minimized and the number of times of pressing is minimized. It is preferable to use a hypotonic solution having a osmotic pressure as low as possible as a suspension of bacterial cells. Moreover, it is preferable not to preincubate or dialyze the lysate after crushing. In the present invention, a cell extract suitable for increasing the expression level of the target protein can be prepared by adopting one or more of the preferred steps.

  Specifically, for example, when a French press is used as a means for disrupting cells after culturing in preparing a cell extract derived from the KOD1 strain, the pressure during pressing is preferably 7,000 to 8,000 p.si, The number of presses is preferably 1, and the cell suspension is preferably the S30 buffer described later in “2. Preparation of cell extract”, and the pre-incubation and dialysis are preferably not performed on the lysate after disruption. When the cell extract derived from the KOD1 strain is prepared in the preferred embodiment, the target protein can be produced in a batch of 6 μg / ml or more.

Furthermore, in the present invention, the expression level of the target protein can be further increased by using a reaction solution in which one to a plurality of component concentrations are optimized among the various additive components described above. Specifically, a suitable concentration of Mg ²⁺ is 0.5 to 6 mM, and more preferably 2 to 4 mM. A suitable concentration of K ⁺ is 100 to 450 mM, more preferably 170 to 350 mM. A suitable concentration of NH ⁴⁺ is 50 to 120 mM, more preferably 65 to 100 mM. The preferred concentration of the 20 amino acids is 1-7 mM, more preferably 2-7 mM. The preferred concentration of phosphoenolpyruvate is 2.5-25 mM, more preferably 5-20 mM. The preferred concentration of polyethylene glycol is 0-2.5% (w / v). In addition, the density | concentration of each said component is a final density | concentration in a reaction liquid.

  In the present invention, as described above, when a cell extract derived from the KOD1 strain is prepared in a preferred embodiment for increasing the expression level of the target protein, and the above components are set to suitable concentrations, the target protein 60 μg / ml or more can be produced.

When a protein is produced by a cell-free protein transcription / translation system, a reaction solution can be prepared by adding various components as described above in addition to a cell extract derived from a hyperthermophilic bacterium. If the cell extract, plasmid encoding the target protein, RNA polymerase, Mg ²⁺ and 20 kinds of amino acids are contained in the reaction solution, the target protein can be produced. In addition to the above components, if the reaction solution contains at least one of NH ₄ ⁺ , K ⁺ , polyethylene glycol, ATP regeneration system and energy source, the expression level of the target protein is increased. Can be made. The concentration of each component can be appropriately set according to the cell extract to be used, reaction temperature, reaction time, reaction scale, and the like.

  The cell-free protein transcription / translation system is a reaction that combines transcription and translation. Therefore, in order to efficiently express the target protein, consider the reaction temperature separately for the transcription reaction and the translation reaction. A temperature suitable for the reaction can be set (hereinafter referred to as “first temperature setting standard”). The temperature setting of the transcription reaction can be set according to the optimum temperature of the RNA polymerase in the reaction solution. In addition, the temperature of the translation reaction is set so that the protein is efficiently expressed by using the mRNA encoding the target protein by the above-described cell-free protein translation system. (Hereinafter, this temperature is referred to as “the optimum temperature for translation reaction”). Here, the optimum temperature for each of the transcription reaction and the translation reaction is usually about 5 to 15 ° C., including the temperature at which the target protein can be expressed most efficiently (hereinafter referred to as “optimum temperature”) Temperature range. Therefore, when the optimum temperatures of the transcription reaction and the translation reaction partially overlap, the reaction temperature is set to be the same between the transcription reaction and the translation reaction (that is, set to the overlap temperature). Proteins can also be expressed.

  In addition, the target protein can be expressed by setting the reaction temperature in the transcription reaction and the translation reaction to be the same without considering the reaction temperature separately in the transcription reaction and the translation reaction (hereinafter referred to as “second temperature setting”). Standard ”). That is, even when there is an optimum temperature suitable for the reaction in each reaction and the optimum temperature of each reaction does not overlap, if the intermediate temperature of the optimum temperature of each reaction is set, the transcription reaction can be performed at that temperature. The target protein can be expressed by performing a translation reaction.

  The optimum temperature of thermostable RNA polymerase available at present is 45 to 50 ° C., and the optimum temperature of the cell-free protein translation system when using a cell extract derived from a hyperthermophilic bacterium (for example, KOD1 strain). Is 60-70 ° C. Therefore, in order to efficiently promote protein synthesis in the cell-free protein transcription / translation system in the present invention, the transcription temperature is set to 45 to 50 ° C. and the translation temperature is set to 60 based on the first temperature setting standard. What is necessary is just to set to -70 degreeC. Further, based on the second temperature setting standard, both the transfer temperature and the translation temperature may be set to about 50 ° C.

  In addition, the synthesis | combination of the protein by a cell-free protein synthesis system can use the well-known batch method performed by mixing the reaction liquid mentioned above in one container. Further, in order to increase the expression level of the protein, a known method such as allowing the cell-free protein synthesis system to carry out the reaction by continuously supplying reaction substrates such as amino acids, ATP, and GTP through an ultrafiltration membrane, etc. The continuous method may be used, or a concentrated cell extract may be used.

The present invention also includes a kit for producing a target protein by the above-described method of the present invention. The kit includes a kit for producing a protein by a cell-free protein translation system and a kit for producing a protein by a cell-free protein transcription / translation system. The former kit contains at least a cell extract derived from a hyperthermophilic bacterium, mRNA encoding the target protein, Mg ²⁺ and an amino acid. The latter kit contains at least a cell extract derived from hyperthermophilic bacteria, a plasmid encoding a target protein, RNA polymerase, Mg ²⁺ and amino acids. Furthermore, these reagents may be provided in an independent form, or may be provided in a combined form of two or more.

  Purification of the protein produced by the method of the present invention can be performed relatively easily because the amount and type of contaminants mixed in is significantly smaller than that of separation from living cells. As the purification method, for example, conventionally known methods such as gel filtration, ion exchange chromatography, affinity chromatography, HPLC, and electrophoresis can be used alone or in appropriate combination.

  Hereinafter, as an example of the present invention, in the super thermostable chitinase (SEQ ID NO: 1) derived from the KOD1 strain, the 910th Gly is substituted with Met, and this is used as the N-terminal, and the 911 to 1115th amino acids downstream thereof The results of studying the method of the present invention using a partially deleted fragment consisting of residues (ChiAΔ4, SEQ ID NO: 2) as the target protein will be described in detail, but the present invention is not limited to these examples. .

1. Construction of plasmid and mRNA encoding ChiAΔ4 In order to synthesize ChiAΔ4 using a cell-free protein transcription / translation system, the T7 promoter and glutamate dehydrogenase (GDH) ribosome binding sequence (RBS) are incorporated upstream of the gene encoding ChiAΔ4. A plasmid (pTRC1) was prepared. In addition, in order to synthesize ChiAΔ4 using a cell-free protein translation system, the cell-free protein transcription / translation plasmid (pTRC1) obtained above was transcribed to produce a cell-free protein translation mRNA (ChiAΔ4 mRNA).

1.1 Preparation of plasmid incorporating T7 promoter The restriction enzyme recognition sequence XbaI present in the plasmid pUC118 was removed. Specifically, pUC118 was first treated with XbaI, and then the XbaI-cleaved fragment was blunted using a Blunting High kit (Toyobo Co., Ltd.) according to the instruction manual, and then the blunt ends obtained were ligated.

  Next, plasmid pET-21a (Novagen) was double-digested with BglII and EcoRI to excise a DNA fragment containing the T7 promoter (SEQ ID NO: 3). Then, the pUC118 from which the XbaI recognition sequence was removed was double-digested with BamHI and EcoRI and ligated with the DNA fragment containing the T7 promoter. Hereinafter, the obtained plasmid is referred to as “pT”.

1.2 Preparation of DNA fragment containing RBS To prepare a DNA fragment containing the ribosome binding sequence (RBS) (SEQ ID NO: 5) of glutamate dehydrogenase (GDH), the following was performed. That is, oligo DNA containing the recognition sequence for XbaI (5'-AAAA TCTAGA CGCAGATTACCGAAATGAGGT- 3 ', SEQ ID NO: 6) and oligo DNA comprising a recognition sequence for NdeI (5'-AAAA CATATG TACCACCTCATTTCGGTAATCTGCG- 3', SEQ ID NO: 7) Each oligo DNA was synthesized and annealed to prepare a DNA fragment (SEQ ID NO: 8) containing the RBS. Thereafter, this DNA fragment was amplified by PCR, and the obtained amplified fragment was double-digested with XbaI and NdeI. Hereinafter, the obtained cleaved fragment (SEQ ID NO: 9) is referred to as “RBS fragment”.

1.3 Preparation of DNA fragment containing ChiAΔ4
A DNA fragment containing ChiAΔ4 was prepared by amplifying the ChiAΔ4 gene by PCR using the chromosomal DNA of the KOD1 strain as a template and using two kinds of primers described later. Below, the preparation method of the chromosomal DNA of KOD1 strain is demonstrated first.

(Preparation of chromosomal DNA of KOD1 strain)
Artificial seawater (1.6% NaCl, 0.24% MgCl ₂ .6H _{2 0,} 0.48% MgSO ₄ .7H _{2 0,} 0.48% (NH ₄ ) ₂ SO ₄ , 0.08% NaHCO ₃ , 0.016% CaCl ₂ · 2H _{2 0,} 0.04% KCl, 0.0336% KH ₂ PO ₄ , 0.004% NaBr, 0.0016% SrCl ₂ · 6H _{2 0,} 0.0008% Fe (NH ₄ ) ₂ H (C ₆ H ₅ 0 ₇ ) ₂ ), a medium consisting of 0.5% yeast extract and 0.5% tryptone is placed in a 2 liter culture bottle and autoclaved (121 ° C., 2 atoms, 20 minutes) And sterilized. 0.1% sulfur was added to the sterilized medium, and 100 ml of this medium was inoculated with the KOD1 strain and cultured. The culture was anaerobically performed at 85 ° C. for about 16 hours. After completion of the culture, the cells were collected by centrifuging the culture at 8,000 rpm for 10 minutes. After removing the supernatant from the collected cells, the cells were suspended in the artificial seawater and centrifuged at 8,000 rpm for 10 minutes twice to wash the cells.

5 g of the washed cells are suspended in 1.6 ml of TE buffer A (100 mM Tris-HCl (pH 8.0), 10 mM EDTA), and 20 μl of 10% sodium dodecyl sulfate and 20 μl of proteinase K are added to the suspension. , Reacted at 55 ° C. for 1 hour. Next, 1 ml of neutral phenol was added to the suspension and mixed for 10 minutes. The obtained mixed solution was centrifuged at 20,000 × g for 10 minutes, and then the aqueous layer portion in the upper layer was taken out. An equivalent amount (about 1.5 ml) of PCI (phenol / chloroform / isopropyl alcohol) (50: 48: 2% (v / v)) was added to the extracted aqueous layer and mixed for 10 minutes, and the resulting mixture was obtained. Was centrifuged at 20,000 × g for 10 minutes, and then the aqueous layer portion in the upper layer was taken out. About 3 ml of ethanol and 150 μl of 3M sodium acetate solution were added to the removed aqueous layer, and the mixture was allowed to stand at −20 ° C. for 30 minutes. Then, the mixture after standing was centrifuged at 20,000 × g for 10 minutes to obtain a nucleic acid precipitate.

  To this precipitate, 5 ml of 70% ethanol was added, and the nucleic acid was rinsed by centrifuging at 20,000 × g for 10 minutes. After removing the supernatant, the precipitate was dried, and 400 μl of TE buffer B (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) was added to the dried precipitate to dissolve the nucleic acid. Next, 4 μl of ribonuclease (1 mg / ml) was added to 400 μl of the obtained nucleic acid solution and reacted at 37 ° C. for 1 hour. 400 μl of the PCI was added to the nucleic acid solution after completion of the reaction and mixed for 5 minutes. This mixed solution was centrifuged at 20,000 × g for 10 minutes, and the aqueous layer portion was taken out. Then, the addition of PCI, centrifugation, and the operation of taking out the aqueous layer portion were performed again. About 3 ml of ethanol and 40 μl of 3M sodium acetate solution were added to the removed aqueous layer (about 400 μl), and the mixture was allowed to stand at −20 ° C. for 30 minutes.

  The solution subjected to the stationary treatment was centrifuged at 20,000 × g for 10 minutes to obtain a DNA precipitate. To this precipitate, 0.5 ml of 70% ethanol was added, followed by centrifugation at 20,000 × g for 10 minutes. After removing the supernatant, the precipitate was dried, and chromosomal DNA was obtained by adding 200 μl of TE buffer B (10 mM Tris-HCl (pH 8.0), 1 mM EDTA).

(Amplification of ChiAΔ4 gene)
Primer ChiA-Nd (5′-AAAA CATATG CTTCCCGAGCACTTCTTCGCCC-3 ′, SEQ ID NO: 10) containing the recognition sequence of NdeI and primer ChiA-T1 (5′-AAAA GAATTC TCCAATTTCATTATGGAC-3 ′, SEQ ID NO: 11 containing the recognition sequence of EcoRI ), PCR was performed using the chromosomal DNA obtained above as a template to amplify the DNA fragment of the ChiAΔ4 gene. PCR was first reacted at 94 ° C. for 180 seconds in a 50 μl reaction solution containing the chromosomal DNA (400 ng) prepared above, 20 pmol of each primer and KOD-plus-DNA polymerase (manufactured by Toyobo Co., Ltd.) at 94 ° C. The cycle of 15 seconds, 63 ° C. for 30 seconds and 72 ° C. for 60 seconds was repeated 25 times, and finally the reaction was performed at 72 ° C. for 300 seconds. Subsequently, the obtained amplified fragment was double-digested with NdeI and EcoRI. Hereinafter, the obtained cleaved fragment is referred to as “ChiAΔ4 fragment”.

1.4 Preparation of plasmid (pTRC1) encoding ChiAΔ4 The plasmid pT obtained above was double-digested with XbaI and EcoRI. Then, the obtained cleaved fragment, the above-described RBS fragment and ChiAΔ4 fragment were mixed, and the respective DNA fragments were combined to prepare plasmid pTRC1 encoding ChiAΔ4. A schematic diagram of the obtained pTRC1 is shown in FIG.

The pTRC1 that produced the above-mentioned mRNA (ChiAΔ4mRNA) encoding 1.5 ChiAderuta4 was treated with EcoRI, T7 RNA synthesis kit obtained EcoRI-digested fragment as a template ^{(T7 RiboMAX TM Express Large Scale RNA} Production System (Promega Corp.)) and In use, ChiAΔ4 mRNA, a transcription product of pTRC1, was prepared according to the attached protocol. An outline of the preparation method of ChiAΔmRNA is shown in FIG.

2. Preparation of cell extract The cell extract was prepared by culturing the KOD1 strain, collecting and washing the resulting cells, and adding Pre-incubation Mix. Specifically, a medium in which 0.5% sodium pyruvate was added to the artificial seawater was placed in a 3 liter culture bottle and sterilized by autoclaving (121 ° C., 2 atoms, 20 minutes). The sterilized medium was inoculated with KOD1 strain and cultured. The culture was anaerobically performed at 85 ° C. for about 16 hours.

  After completion of the culture, the cells were collected by centrifuging the obtained culture at 4 ° C. and 6,000 × g for 10 minutes. Next, the collected cells were suspended in the artificial seawater described above, and the cells were washed by centrifuging at 4 ° C. and 6,000 × g for 5 minutes. The washed cells are resuspended in artificial seawater, transferred to a centrifuge tube whose weight has been measured in advance, centrifuged at 4 ° C., 6,000 × g for 10 minutes, and the supernatant is removed. The weight of the microbial cells was measured (the amount of microbial cells 4.78 g). All subsequent operations were performed under RNase-free conditions. The obtained cells were suspended in 1.27 ml / g artificial seawater and gently mixed with vortex. The suspension was transferred to a French press and pressed under 10,000 p.s.i, 3 conditions. The obtained lysate was transferred to a centrifuge tube, 100 ml of 0.1 M DTT was added per 10 ml of the lysate, and then centrifuged at 30,000 × g for 30 minutes at 4 ° C. The supernatant was collected, and the collected supernatant was centrifuged at 4 ° C. and 30,000 × g for 30 minutes. Then, 80% of the supernatant was collected, and 0.3 ml of Pre-incubation Mix shown in Table 1 was added per 1 ml of the collected supernatant. This mixture was incubated at 37 ° C. for 80 minutes to terminate ongoing mRNA translation. After incubation, the mixture was dialyzed against S30 buffer (10 mM Tris-acetic acid (pH 7.4), 1.0 mM DTT, 14 mM magnesium acetate, 60 mM potassium acetate) (3 times for 45 minutes). The mixture after dialysis was centrifuged at 4,000 × g for 10 minutes, and the supernatant was collected to obtain a cell extract used in the present invention (protein concentration: 34 mg / ml).

3. Cell-free protein synthesis reaction and its analysis The cell-free protein synthesis reaction described later is prepared by mixing the above cell extract and various reagents described later on ice to maintain a constant reaction temperature using a PCR device. It was done by doing. Then, after a predetermined time, the reaction solution was allowed to stand on ice to complete the synthesis reaction, and the reaction result was analyzed by the Western blot method shown below. First, the solution after completion of the cell-free protein synthesis reaction was diluted 30-fold with milliQ water. The diluted solution was subjected to SDS-polyacrylamide gel electrophoresis according to a conventional method (acrylamide concentration: 12.5%). After completion of the electrophoresis, the sample in the gel was transferred to a PVDF membrane (Hybond-P, Amersham Biosciences) using horizon blot (Ato). Next, an anti-rabbit ChiAΔ4 antibody was added as a primary antibody to the PVDF membrane and incubated at room temperature for 1 hour. Subsequently, HRP-rec-ProteinG (Zymed) was added as a secondary antibody and incubated at room temperature for 1 hour. Then, the PVDF membrane in which the secondary antibody was incubated was subjected to chemiluminescence according to the reaction conditions described in the instruction manual using ECL Advance Western Blot Detection Kit (Amersham Biosciences). The target band was detected using Hyperfilm ECL (Amersham Biosciences). The anti-rabbit ChiAΔ4 antibody is a polyclonal antibody prepared according to a conventional method using purified ChiAΔ4 obtained according to the method described in JP-A-11-313688 as an antigen and rabbit as an immunized animal.

4). Cell-free protein translation system
4.1 Effect of ChiAΔ4 mRNA
The protein translation reaction using ChiAΔ4 mRNA was carried out at 48 ° C. for 90 minutes using a reagent, composition and final concentration sample shown in Table 2 in a total volume of 30 μl. As for T. kodakaraensis tRNA in Table 2, the KOD1 strain was first cultured by the method described above, and the cells recovered from the obtained culture broth were used with Nucleobond AX (Macherey-Nagel) and the attached protocol. According to the above. The same T.kodakaraensis tRNA was used in subsequent experiments. The results are shown in FIG. In FIG. 2, lanes 1 to 4 and C (control sample) correspond to the respective sample numbers in Table 2. The band of 33.8 kDa indicated by the arrow in FIG. 2 is ChiAΔ4, and the band of ChiAΔ4 was confirmed in all of lanes 1 to 4, but the band of ChiAΔ4 was not observed in lane C. From this, it was found that ChiAΔ4 mRNA is an essential component for synthesizing ChiAΔ4. From comparison of lanes 2, 3 and 4, it was found that the amount of ChiAΔ4 synthesized increased by increasing the concentration of ChiAΔ4 mRNA.

4.2 Effect of reaction temperature
In order to investigate the effect of reaction temperature in the synthesis reaction of ChiAΔ4, the reaction was performed at 48 ° C. or 68 ° C. for 90 minutes with the reagent, composition and final concentration shown in Table 3, samples and the final concentration of 30 μl. A cellular protein translation reaction was performed. Sample 9 is a control sample to which no ChiAΔ4 mRNA was added. The results are shown in FIG. In FIG. 3, lanes 1, 3, 4 and lane 9 correspond to the respective sample numbers in Table 3, and lane C shows ChiAΔ4 derived from Escherichia coli as a molecular weight marker. As described in JP-A-11-313688, this E. coli-derived ChiAΔ4 is obtained by culturing, collecting, crushing, and heat-treating Escherichia coli having a ChiAΔ4 expression vector, Next, the crude enzyme solution was applied to an anion exchange column and a gel filtration column and purified until it was observed as a single band by SDS-PAGE. The same ChiAΔ4 derived from E. coli was used in subsequent experiments. In FIG. 3, a band of ChiAΔ4 was confirmed in all of lanes 1, 3 and 4. From comparison of each lane, the synthetic amount of ChiAΔ4 increased more specifically at 68 ° C. than 48 ° C. as the reaction temperature increased. I found out that In addition, since the synthesis of ChiAΔ4 was confirmed in lane 1, and the amount of ChiAΔ4 band in lane 3 and lane 4 was equivalent, tRNA (T.kodakaraensis tRNA in this experiment) was used in the translation reaction of ChiAΔ4. It can be said that it has no effect.

4.3 Influence of magnesium ions
In order to examine the influence of magnesium ion concentration in the synthesis reaction of ChiAΔ4, the total amount of the reaction solution was 30 μl with the reagents, compositions and final concentrations shown in Samples 1 and 5 to 8 in Table 3, and cell-free protein at 48 ° C. for 90 minutes. A translation reaction was performed. Sample 9 is a control sample to which no ChiAΔ4 mRNA was added. The results are shown in FIG. In FIG. 4, lanes 1, 5 to 8 correspond to the sample numbers in Table 3, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. In FIG. 4, a band of ChiAΔ4 was confirmed in lanes 1 and 5 to 7, but a band of ChiAΔ4 was not confirmed in lane 8. From this, it was found that as the magnesium ion concentration increased, the amount of ChiAΔ4 synthesized increased, and the preferred concentration was 5 to 10 mM. However, when it exceeded 25 mM, the translation reaction was inhibited and ChiAΔ4 was not synthesized.

4.4 Effects of polyethylene glycol 1
In order to examine the influence of polyethylene glycol in the synthesis reaction of ChiAΔ4, the cell-free protein translation reaction was carried out at 68 ° C. for 90 minutes with the total amount of the reaction solution being 30 μl with the reagents, composition and final concentration shown in samples 1 and 2 of Table 4. I did it. The results are shown in FIG. In FIG. 5, lanes 1 and 2 correspond to the respective sample numbers in Table 4, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. In FIG. 5, since the amount of ChiAΔ4 synthesized is increased in Lane 2 (PEG8000: 2%) compared to Lane 1 (PEG8000: 0%), the amount of ChiAΔ4 synthesized increases when polyethylene glycol is added. I understood.

4.5 Effects of spermine and spermidine
In order to examine the effects of spermine and spermidine in the synthesis reaction of ChiAΔ4, the total volume of the reaction solution was 30 μl with the reagents, compositions and final concentrations shown in Samples 1 and 3 to 5 in Table 4, and a cell-free protein at 68 ° C. for 90 minutes. A translation reaction was performed. The results are shown in FIG. In FIG. 6, lanes 1 and 3 to 5 correspond to the sample numbers in Table 4, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. In FIG. 6, since no significant difference was observed in the band of ChiAΔ4 in lanes 3 to 5, it was found that spermine or spermidine did not affect the translation reaction.

4.6 Effects of sodium ions
In order to examine the influence of sodium ions in the synthesis reaction of ChiAΔ4, cell-free protein translation was performed at 68 ° C. for 90 minutes with the total volume of the reaction solution being 30 μl with the reagents, compositions and final concentrations shown in Samples 2 and 6-8 in Table 4. Reaction was performed. The results are shown in FIG. In FIG. 7, lanes 2 and 6 to 8 correspond to the respective sample numbers in Table 4, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. In FIG. 7, the amount of ChiAΔ4 synthesized is smaller in Lane 6 (Na ⁺ : 100 mM), Lane 7 (Na ⁺ : 300 mM), Lane 8 (Na ⁺ : 500 mM) than in Lane 2 (Na ⁺ : 0 mM). From the results, it was found that sodium ion had no positive effect on the translation reaction.

4.7 Examination of optimization of reaction temperature
In order to examine the influence of reaction temperature in the synthesis reaction of ChiAΔ4, cell-free protein translation was performed at 30-80 ° C. for 90 minutes, with the total amount of the reaction solution being 30 μl, using the reagents, compositions and final concentrations shown in samples 1 to 9 of Table 5 Reaction was performed. Sample 1 is a control sample to which no ChiAΔ4 mRNA was added. The results are shown in FIG. In FIG. 8, lanes 1 to 9 correspond to the sample numbers in Table 5, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. In FIG. 8, a band of ChiAΔ4 was clearly confirmed in lanes 3 to 8 (reaction temperature: 40 to 75 ° C.), but in lane 2 (reaction temperature: 30 ° C.) and lane 9 (reaction temperature: 80 ° C.) The band of was not confirmed.

In order to examine the above results in detail, the amount of ChiAΔ4 at the end of synthesis after 90 minutes of reaction at each of the above temperatures was quantified by activity measurement. A method for measuring the activity of ChiAΔ4 is described below. Reaction of 500 μl of fluorescent substrate GlcNAc _2-4 -methylumbelliferone (0.02 mM) dissolved in 0.1 M acetate buffer (pH 5.0), 480 μl of purified water, and 20 μl of the reaction solution at 80 ° C. for 10 to 30 minutes. It was. After completion of the reaction, 500 μl of the reaction solution was added to 500 μl of ice-cold 100 mM glycine-NaOH (pH 11) to stop the enzyme reaction. The excitation light at 350 nm and the fluorescence at 440 nm of this sample were measured using a spectrofluorometer, and the amount of ChiAΔ4 synthesized by the translation reaction was calculated using the specific activity value of ChiAΔ4. The results are shown in FIG. From FIG. 9, it was found that ChiAΔ4 was synthesized for all the samples 2 to 9 in Table 5, and the synthesis amount was maximum at 60 to 70 ° C. From this, it can be said that 60-70 degreeC is an optimal temperature in this experiment system. Therefore, in the subsequent experiments, the reaction temperature was set to 60 ° C., which is in the optimum temperature range, and the influence of various components was examined.

  In addition, each of samples 5, 7 and 9 in Table 5 was sampled at 5, 15, 30, 60, 90, and 120 minutes after the reaction, analyzed by the same method as above, and the expression level of ChiAΔ4 was time-dependent. The sex was examined. The results are shown in FIG. FIG. 10 shows that at 70 ° C., the synthesized amount of ChiAΔ4 is almost saturated in about 30 minutes after the start of the reaction, and the synthetic amount of ChiAΔ4 reaches 0.8 μg / ml at the maximum.

4.8 Effects of potassium ions
In order to examine the influence of potassium ions in the synthesis reaction of ChiAΔ4 under the optimum temperature conditions, the reaction solution was prepared in 30 μl for 90 minutes at 60 ° C. with the total amount of the reaction solution being 30 μl with the reagents, compositions and final concentrations shown in Samples 1 to 3 in Table 6. A protein translation reaction was performed. The results are shown in FIG. In FIG. 11, lanes 1 to 3 correspond to the sample numbers in Table 6, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. In FIG. 11, the band of ChiAΔ4 becomes smaller from lane 1 (K ⁺ : 100 mM) to lane 3 (K ⁺ : 700 mM). From this, the amount of potassium ion is increased to increase the amount of ChiAΔ4 synthesized. It can be said that reducing the concentration is effective.

4.9 Effects of polyethylene glycol 2
In order to examine the influence of polyethylene glycol in the synthesis reaction of ChiAΔ4 under the optimum temperature condition, the total volume of the reaction solution was 30 μl with the reagents, compositions and final concentrations shown in Samples 4 to 6 in Table 6, and cell-free at 60 ° C. for 90 minutes. A protein translation reaction was performed. The results are shown in FIG. In FIG. 12, lanes 4 to 6 correspond to the sample numbers in Table 6, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. In FIG. 12, the band of ChiAΔ4 increases from lane 4 (PEG 8000: 1%) to lane 6 (PEG 8000: 5%). From this, the concentration of polyethylene glycol is increased to increase the amount of ChiAΔ4 synthesized. It can be said that raising the value is effective.

4.10 Effects of various inorganic ions
In order to examine the influence of magnesium ion, ammonium ion and potassium ion on the synthesis of ChiAΔ4 under optimum temperature conditions, the total volume of the reaction solution was set to 30 μl with the reagents, compositions and final concentrations shown in samples 1 to 3 and 9 in Table 7. The cell-free protein translation reaction was performed at 60 ° C. for 90 minutes. The results are shown in FIG. In FIG. 13, lanes 1 to 3 and 9 correspond to the sample numbers in Table 7, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. In FIG. 13, a ChiAΔ4 band was not confirmed in Lane 1 (Mg ²⁺ : 0 mM), whereas a ChiAΔ4 band was confirmed in Lane 9 (Mg ²⁺ : 7.5 mM). From this, it can be said that magnesium ion is an essential component for synthesizing ChiAΔ4.

In FIG. 13, it was confirmed that the band of ChiAΔ4 was larger in lane 9 (NH ₄ ⁺ : 80 mM) than in lane 2 (NH ₄ ⁺ : 0 mM). From this, it can be said that the ammonium ion is not an essential component for synthesizing ChiAΔ4, but is a component necessary for increasing the synthesis amount of ChiAΔ4. Further, it was confirmed that the band of ChiAΔ4 was larger in lane 9 (K ⁺ : 100 mM) than in lane 3 (K ⁺ : 0 mM). From this, it can be said that potassium ion is not an essential component for synthesizing ChiAΔ4, but is a component necessary to some extent to increase the amount of ChiAΔ4 synthesized.

4.11 Effects of phosphoenolpyruvate
In order to examine the influence of phosphoenolpyruvate (PEP) in the synthesis reaction of ChiAΔ4 under the optimum temperature condition, the total amount of the reaction solution was set to 30 μl at 60 ° C. with the reagents, compositions and final concentrations shown in Samples 4 to 9 of Table 7. The cell-free protein translation reaction was performed for 90 minutes. The results are shown in FIG. In FIG. 14, lanes 4 to 9 correspond to the sample numbers in Table 7, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. In FIG. 14, it was confirmed that the band of ChiAΔ4 was larger in lane 9 (PEP: 33 mM) than in lane 6 (PEP: 0 mM). Here, the reaction solution contains pyruvate kinase (PK) derived from Pre-incubation Mix as described above. It has also been found that PK exists in the KOD1 strain itself. Considering these matters together, it can be said that PEP acts as a substrate for the ATP regeneration system and is a necessary component for increasing the amount of ChiAΔ4 synthesized. In addition, comparison of lanes 4 to 8 confirms that ATP is regenerated using PEP as a substrate for PK, and it can be said that the amount of ChiAΔ4 synthesized tends to decrease if the concentration of ATP is too high. The relationship between various energy sources and PEP will be examined again later in 4.16.

4.12 Effect of Tris-acetate buffer
In order to examine the influence of the Tris-acetate buffer in the synthesis reaction of ChiAΔ4 under the optimum temperature condition, the total amount of the reaction solution was set to 30 μl at 90 ° C. at 60 ° C. with the reagents, compositions and final concentrations shown in Samples 1 and 9 of Table 8. A cell-free protein translation reaction was performed for a minute. The results are shown in FIG. In FIG. 15, lanes 1 and 9 correspond to the sample numbers in Table 8, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. In FIG. 15, lane 1 (Tris acetic acid: 0 mM) and lane 9 (Tris acetic acid: 56.2 mM) showed bands of almost the same size. From this, it was found that the Tris-acetate buffer had no effect on the ChiAΔ4 synthesis reaction.

4.13 Effects of amino acids
In order to examine the effect of 20 amino acids in the synthesis reaction of ChiAΔ4 under optimum temperature conditions, the total volume of the reaction solution was 30 μl at 60 ° C. with the reagents, compositions and final concentrations shown in samples 2, 3 and 9 of Table 8. Cell-free protein translation reaction was performed for 90 minutes. The results are shown in FIG. In FIG. 16, lanes 2, 3, and 9 correspond to the respective sample numbers in Table 8, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. In FIG. 16, a band of ChiAΔ4 was not confirmed in lane 2 (each amino acid: 0 mM), whereas a band of ChiAΔ4 was confirmed in lanes 3 (each amino acid: 4 mM) and 9 (each amino acid: 2 mM). . From this, it can be said that 20 kinds of amino acids are essential components for synthesizing ChiAΔ4.

4.14 Effect of spermidine 2
In order to examine the influence of spermidine in the synthesis reaction of ChiAΔ4 under the optimum temperature conditions, the total amount of the reaction solution was 30 μl at 60 ° C. for 90 minutes with the reagents, compositions and final concentrations shown in Samples 4, 5 and 9 in Table 8. A cellular protein translation reaction was performed. The results are shown in FIG. In FIG. 17, lanes 4, 5 and 9 correspond to the respective sample numbers in Table 8, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. In FIG. 17, spermidine has an influence on the translation reaction since no significant difference is observed in the bands of ChiAΔ4 in lanes 4 (spermidine: 0.5 mM), 5 (spermidine: 1 mM) and 9 (spermidine: 0 mM). I found out that it would not reach.

4.15 Effects of GTP, CTP and UTP
In order to examine the influence of GTP, CTP and UTP (hereinafter referred to as “GCU mixture”) in the synthesis reaction of ChiAΔ4 under the optimum temperature conditions, the reaction solution was used with the reagents, compositions and final concentrations shown in Samples 6 and 9 in Table 8. Cell-free protein translation reaction was performed at 60 ° C. for 90 minutes. The results are shown in FIG. In FIG. 18, lane 6 and lane 9 correspond to the respective sample numbers in Table 8, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. In FIG. 18, there is no significant difference in the band of ChiAΔ4 in lane 6 (GCU mixture: 0 mM) and lane 9 (GCU mixture: 0.85 mM each), so that the GCU mixture as an energy source affects the translation reaction. It was found that it does not affect.

4.16 Relationship between various energy sources and PEP
In order to examine the relationship between the GCU mixture / ATP and PEP in the synthesis reaction of ChiAΔ4 under optimum temperature conditions, the total amount of the reaction solution was set to 30 μl at 60 ° C. with the reagents, compositions and final concentrations shown in Samples 1 to 7 of Table 9. The cell-free protein translation reaction was performed for 90 minutes. The results are shown in FIG. In FIG. 19, lanes 1 to 7 correspond to the sample numbers in Table 9, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. First, a ChiAΔ4 band was also confirmed in Lane 4 (only PEP) in which neither ATP nor GCU mixture was present. Since the energy substances required for the translation reaction are ATP and GTP, these substances are present in the cell extract, or their precursors ADP and GDP are present and regenerated and used by PEP. Recognize. In lane 5 (ATP only) and lane 6 (GCU mixture only), where no PEP is present, the ChiAΔ4 band is not seen, while in lane 1 (ATP and GCU mixture) where both are mixed, a band can be confirmed. It was. Since ATP and GTP can be interconverted by intracellular enzymes (nucleoside-diphosphate kinase), the synergistic effect in this case is the result of simply increasing the amount of energy substance in the absence of PEP as an energy regeneration substance. it is conceivable that. In addition, when ATP (lane 3) or GCU mixture (lane 2) was added in the presence of PEP, the amount of ChiAΔ4 synthesized increased synergistically, so if either ATP or GCU mixture and PEP were present It can be said that the energy required for the synthesis of ChiAΔ4 can be obtained efficiently.

5. Cell-free protein transcription / translation system
5.1 Effect of reaction temperature 1
The protein synthesis reaction using pTRC1 was carried out at 40 ° C. or 60 ° C. for 90 minutes using the reagents, compositions and final concentrations shown in Samples 8 and 9 in Table 10 with a total reaction volume of 30 μl. The reaction solution used in this test was the same as the reaction solution in the translation reaction system described above except that pTRC1 was used instead of ChiAΔ4 mRNA in the translation reaction system described above, and T7 RNA polymerase (Toyobo Co., Ltd.) was newly added. Basically the same. In addition, pTRC1 used was a fragment that had been treated with EcoRI in advance and cleaved the EcoRI recognition site downstream of the ChiAΔ4 gene. The T7 RNA polymerase (trade name: Thermo T7 RNA Polymerase) has a maximum activity temperature of 50 ° C. and has high thermal stability. The results are shown in FIG. In FIG. 20, lane 8 and lane 9 correspond to the respective sample numbers in Table 10, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. In FIG. 20, no ChiAΔ4 band was observed in either lane 8 (reaction temperature: 40 ° C.) or lane 9 (reaction temperature: 60 ° C.). Therefore, in this experimental system, even if the transcription / translation reaction is performed at the optimal temperature for either the transcription reaction or the translation reaction, the reaction does not proceed efficiently in either the transcription reaction or the translation reaction. It turns out that the effect is small.

5.2 Effect of reaction temperature 2
In order to examine the influence of the reaction temperature in the synthesis reaction of ChiAΔ4, the reaction was carried out at 40 ° C. for 60 minutes with the reagent, composition and final concentration shown in Sample 8 of Table 11 as 30 μl, and then reacted at 60 ° C. The reaction was performed for 90 minutes. The results are shown in FIG. In FIG. 21, lane 8 corresponds to the sample number in Table 11, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. In FIG. 21, a band of ChiAΔ4 was confirmed in lane 8 although it was a trace amount. From this, it can be said that it is effective to set the temperature to the optimum temperature in each reaction during the transcription reaction and the translation reaction in order to efficiently perform the transcription / translation reaction in this experimental system.

5.3 Effects of RNase inhibitors
In order to examine the influence of RNase in the synthesis reaction of ChiAΔ4, the reaction was carried out at 40 ° C. for 60 minutes with the total amount of the reaction solution being 30 μl with the reagents, composition and final concentration shown in Sample 8 of Table 12, and then at 60 ° C. The reaction was performed for 90 minutes. The results are shown in FIG. In FIG. 22, lane 8 corresponds to the sample number in Table 12, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. In Lane 8, a band of ChiAΔ4 was clearly confirmed as compared with the experimental system in which the RNase inhibitor was not added (see FIG. 21). From this, it can be said that it is effective to add an RNase inhibitor in order to efficiently perform the transcription / translation reaction in this experimental system.

5.4 Effects of RNA polymerase
In order to examine the influence of the addition amount of RNA polymerase in the synthesis reaction of ChiAΔ4, the reaction was carried out at 40 ° C. for 60 minutes with the total amount of the reaction solution being 30 μl with the reagents, compositions and final concentrations shown in samples 1 to 4 of Table 13. Then, it was made to react at 60 degreeC for 90 minutes. The results are shown in FIG. In FIG. 23, lanes 1 to 4 correspond to the sample numbers in Table 13, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. In FIG. 23, the band of ChiAΔ4 increases from lane 3 to lane 1. From this, it can be said that in this experimental system, if the concentration of T7 RNA polymerase is too high, the transcription reaction is inhibited. In addition, from the comparison between lane 3 and lane 4, it was reconfirmed that the RNase inhibitor was functioning in this experimental system.

6). Cell-free protein translation system 2 (Study on a suitable method for preparing cell extract for the purpose of increasing the amount of target protein synthesis)
6.1 Influence of the number of times of French press FIG. 24 is a protocol summarizing the numbered procedures after the cultivation of the KOD1 strain among the procedures described in “2. Preparation of cell extract”. In step 9 of FIG. 24, the number of French presses is set to 3. However, if the number of French presses is increased, elements necessary for protein transcription / translation (for example, RNA polymerase) may be destroyed. Conceivable. Therefore, the effect of reducing the number of French presses on the amount of chiAΔ4 synthesized was investigated. Specifically, among the procedures in FIG. 24, a cell extract was prepared in the same procedure except that the number of French presses in procedure 9 was once, twice, or three times, and samples 1 to 5 in Table 14 were prepared. The cell-free protein translation reaction was carried out at 60 ° C. for 90 minutes with the reagent, composition and final concentration shown in the above and the total volume of the reaction solution being 30 μl. In Table 14, the S30 fraction indicates the cell extract used in “4. Cell-free protein translation system” and “5. Cell-free protein transcription / translation system”, and S30-FP1, S30-FP2 and S30- FP3 is a cell extract obtained by performing French press once, twice and three times, respectively. Note that the protein concentration of each cell extract is shown in parentheses after each cell extract. In FIG. 25, each lane corresponds to each sample number in Table 14, lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker, and lane 1 is a control sample to which no ChiAΔ4 mRNA was added. Among the lanes 2 to 5, when comparing the band of ChiAΔ4, the band of lane 3 (one French press) was the largest. From this, it was found that reducing the number of French presses increased the amount of ChiAΔ4 synthesized, and that one French press was sufficient.

6.2 Effect of French Press Pressure In order to examine how the French press pressure affects the amount of target protein synthesized, among the steps in FIG. A cell extract was prepared in the same procedure except that the pressure was lowered to 8,400 psi (this cell extract is referred to as “S30FP-1A”), and the reaction was carried out with the reagents, compositions and final concentrations shown in Samples 1 to 4 in Table 15. Cell-free protein translation reaction was performed at 65 ° C. for 90 minutes with a total volume of 30 μl. Samples 1 and 2 in Table 15 are samples using S30FP-1 prepared in “6.1 Effect of French Press” as a cell extract. FIG. 26 shows the results of Western blot analysis. In FIG. 26, each lane corresponds to each sample number in Table 15, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. Table 15 shows the amount of ChiAΔ4 synthesized for the samples corresponding to the lanes in FIG. 26 using a chemiluminescence detector (Lumi Vision PRO 400EX, Aisin Seiki). ChiAΔ4 synthesis for sample 3 (pressure: 8,400 psi) and sample 1 (pressure: 10,000 psi) (or sample 4 (pressure: 8,400 psi) and sample 2 (pressure: 10,000 psi)) , Sample 3 (or sample 4) showed a value about 5 times higher. From this, it was found that setting the pressure of the French press lower than the conventional 10,000 psi is effective for increasing the amount of ChiAΔ4 synthesized.

6.3 Effect of hypotonic solution When comparing the osmotic pressure of artificial seawater and S30 buffer described above, S30 buffer is clearly lower. Therefore, it was decided to examine how the synthesis amount of the target protein is affected when S30 buffer is used as a dispersion medium instead of artificial seawater during cell disruption. Specifically, among the procedures in FIG. 24, the artificial seawater in step 7 is replaced with the S30 buffer, and in step 9, the number of French presses is set to one and the pressure is 2,500 psi, 5,000 psi or A cell extract was prepared by setting the temperature to 7,500 psi and incubating the procedure 16 at 60 ° C. for 40 minutes. And the effect at the time of using a hypotonic solution at the time of cell disruption was evaluated by comparing the protein concentration of the obtained cell extract. The results are shown in Table 16. In Table 16, S30FP-1A is the cell extract prepared in “6.2 Effect of French Press Pressure”. From the protein concentration of each cell extract shown in Table 16, the amount of protein obtained was sufficient even when the pressure of the French press was low by allowing the cells to permeate the hypotonic solution for a sufficient time.

6.4 Effect of pre-incubation We examined how the amount of target protein synthesized increased or decreased depending on the presence or absence of pre-incubation. Specifically, among the procedures in FIG. 24, the artificial seawater in step 7 is replaced with the S30 buffer, and in step 9, the number of French presses is set to 1 and the pressure is set to 7,500 psi. Three cell extracts were prepared by performing 15 and 16 under the conditions shown in Table 17. Next, using the obtained cell extract (S30FP-1E, S30FP-1F, S30FP-1G), the total amount of the reaction solution was 30 μl with the reagents, compositions and final concentrations shown in Samples 2, 3 and 4 of Table 18. As a result, a cell-free protein translation reaction was performed at 60 ° C. for 90 minutes. In Table 18, Sample 1 is a reference sample using S30FP-1A prepared in “6.2 Effect of French Press Pressure” as a cell extract. Note that the protein concentration of each cell extract is shown in parentheses after each cell extract. FIG. 27 shows the results of Western blot analysis. In FIG. 27, each lane corresponds to each sample number in Table 18. Of the lanes 2 to 4 in FIG. 27, when comparing the ChiAΔ4 band, the lane 4 showed a larger band than the lanes 2 and 3. From this, it was found that in order to increase the synthesis amount of ChiAΔ4, it is preferable not to add Pre-Incubation and not perform preincubation.

6.5 Effects of dialysis We examined how the amount of target protein synthesized increased or decreased depending on the presence or absence of dialysis. Specifically, among the procedures in FIG. 24, the artificial seawater in step 7 is replaced with the S30 buffer, and in step 9, the number of French presses is set to 1 and the pressure is set to 7,500 psi. Cell extracts were prepared with or without 17 dialysis (see Table 19). Next, using the obtained cell extract (S30FP-1H, S30FP-1I), the total amount of the reaction solution was set to 30 μl at 90 ° C. at 60 ° C. with the reagents, compositions and final concentrations shown in Samples 1 and 2 of Table 20. A cell-free protein translation reaction was performed for a minute. FIG. 28 shows the results of analysis by Western blotting. When comparing the band of ChiAΔ4 between lanes 1 and 2, the band of lane 2 was slightly larger. From this, it can be said that it is preferable not to dialyze in order to increase the synthesis amount of ChiAΔ4.

7). Cell-free protein translation system 3 (concentration of mRNA and cell extract suitable for increasing the amount of target protein synthesized)
In “6. Cell-free protein translation system 2”, a method for preparing a cell extract suitable for increasing the synthesis amount of the target protein was examined. Next, in carrying out a cell-free protein translation reaction using the cell extract, it was decided to study suitable concentrations of mRNA and cell extract in the reaction solution in order to increase the amount of ChiAΔ4 synthesized. In the following experiment, S30FP-1G (protein concentration: 35.3 μg / μl) prepared in “6.4 Effect of Preincubation” was used as the cell extract. FIG. 29 shows the preparation protocol for S30FP-1G.

7.1 Preferred concentration of mRNA
In order to study the preferred concentration of ChiAΔ4 mRNA in the synthesis reaction of ChiAΔ4, the cell-free protein translation reaction was carried out at 65 ° C. for 90 minutes with the total volume of the reaction solution being 30 μl with the reagents, composition and final concentration shown in samples 1 to 5 of Table 21 I did it. The ChiAΔ4 mRNA concentration in each sample in Table 21 is 0 mg / ml (sample 1), 0.33 mg / ml (sample 2), 0.42 mg / ml (sample 3), 0.50 mg / ml (sample 4). 0.58 mg / ml (Sample 5). FIG. 30 shows the result of analysis by Western blotting. In FIG. 30, each lane corresponds to each sample number in Table 21, and lane C shows E. coli-derived ChiAΔ4 as a molecular weight marker. FIG. 30 shows that the band of ChiAΔ4 tends to increase from lane 2 (ChiAΔ4 mRNA: 0.33 mg / ml) to lane 5 (ChiAΔ4 mRNA: 0.58 mg / ml). From the above results, the preferred concentration of ChiAΔ4 mRNA is 0.40 to 0.60 mg / ml, and the vicinity of 0.50 mg / ml is most preferred.

7.2 Preferred concentration of cell extract
In order to study the preferred concentration of S30FP-1G in the synthesis reaction of ChiAΔ4, cell-free protein translation was carried out at 65 ° C. for 90 minutes with the reagent, composition and final concentration shown in Samples 6 to 10 in Table 22 and the total volume of the reaction solution being 30 μl. Reaction was performed. The S30FP-1G concentration in each sample in Table 22 is 10.0 mg / ml (sample 6), 12.0 mg / ml (sample 7), 14.0 mg / ml (sample 8), 16.0 mg / ml. (Sample 9) and 18.0 mg / ml (Sample 10). FIG. 31 shows the results of Western blot analysis. In FIG. 31, each lane corresponds to each sample number in Table 22, and lane C shows ChiAΔ4 derived from E. coli as a molecular weight marker. From FIG. 31, it was found that the preferred concentration of S30FP-1G is 12.0 to 16.0 mg / ml.

7.3 Confirmation of ChiAΔ4 synthesis amount Based on the results of “7.1 preferred concentration of mRNA” and “7.2 preferred concentration of cell extract”, ChiAΔ4 mRNA was set to 0.48 mg / ml and S30FP-1G was set to 13.0 mg / ml. Then, cell-free protein translation reaction was performed. The amount of ChiAΔ4 synthesized was quantified by activity measurement as described in “4.7 Examination of Optimization of Reaction Temperature”. As a result, it was confirmed that the synthetic amount of ChiAΔ4 was 6.2 μg / ml, and the synthetic amount was 6 times or more compared to the maximum synthetic amount of 1.0 μg / ml (see FIG. 9) quantified by the above method. was gotten.

8). Cell-free protein translation system 4 (Examination of suitable concentrations of each component of the reaction solution suitable for increasing the amount of target protein synthesis)
“6. Cell-free protein translation system 2 (preferred method for preparing a cell extract for the purpose of increasing the amount of target protein synthesis)” and “7. Cell-free protein translation system 3 (for mRNA and cell extract, Based on the results obtained in “Examination of Concentration Suitable for Increasing Protein Synthesis Amount”), in order to further increase the synthesis amount of ChiAΔ4, the optimum concentration of each component of the reaction solution was examined.

8.1 Preferred concentration of magnesium ion
In order to examine a suitable concentration of magnesium ions in the synthesis reaction of ChiAΔ4, a cell-free protein translation reaction was carried out at 65 ° C. for 90 minutes using the reagents, composition and final concentration shown in Table 23 with a total reaction volume of 30 μl. The Mg ²⁺ concentration in each sample in Table 23 is 1.87 mM (sample 1), 3.73 mM (sample 2), 5.60 mM (sample 3), 7.47 mM (sample 4), 9.33 mM ( Sample 5). FIG. 32 shows the results of analysis by Western blotting. In FIG. 32, each lane corresponds to each sample number in Table 23. From FIG. 32, it was found that the preferred concentration of magnesium ions is 1.87 to 3.73 mM.

8.2 Preferred concentrations of phosphoenolpyruvate
In order to investigate the preferred concentration of phosphoenolpyruvate in the synthesis reaction of ChiAΔ4, a cell-free protein translation reaction was carried out at 65 ° C. for 90 minutes with the reagents, composition and final concentration shown in Table 24 and the total volume of the reaction solution being 30 μl. . The PEP concentration in each sample in Table 24 is 8.33 mM (sample 1), 16.7 mM (sample 2), 25.1 mM (sample 3), and 33.3 mM (sample 4). FIG. 33 shows the results of analysis by Western blotting. In FIG. 33, each lane corresponds to each sample number in Table 24. From FIG. 33, it was found that the preferred concentration of PEP was 8.33 to 25.1 mM.

8.3 Preferred concentrations of amino acids
In order to examine suitable concentrations of 20 amino acids in the synthesis reaction of ChiAΔ4, a cell-free protein translation reaction was carried out at 65 ° C. for 90 minutes with the reagents, composition and final concentrations shown in Table 25 and a total reaction volume of 30 μl. . The amino acid concentration in each sample in Table 25 is 2.4 mM (sample 1), 2.0 mM (sample 2), 1.6 mM (sample 3), 1.2 mM (sample 4), It is 0.8 mM (sample 5) and each 0.4 mM (sample 6). FIG. 34 shows the results of analysis by Western blotting. In FIG. 34, each lane corresponds to each sample number in Table 25. From FIG. 34, it was found that the preferred concentration of amino acid is about 2.4 mM each because the band of lane 1 is particularly large compared to the other lanes.

9. Cell-free protein translation system 5 (Study on suitable concentrations of each component of the reaction solution for the purpose of increasing the synthesis amount of the target protein)
“6. Cell-free protein translation system 2 (preferred method for preparing a cell extract for the purpose of increasing the amount of target protein synthesis)”, “7. Cell-free protein translation system 3 (for mRNA and cell extract, The results obtained in “Examination of Concentration Suitable for Increasing Protein Synthesis” and “8. Cell-free Protein Translation System 4 (Examination of Optimum Concentration of Each Component of Reaction Solution Suitable for Increasing Synthesis of Target Protein)” In view of this, in order to further increase the amount of ChiAΔ4 synthesized, the preferred concentration of each component of the reaction solution was examined. Specifically, based on the reaction solution composition shown in Table 26, cell-free protein translation reaction was performed by changing the concentration of each component, Western blot analysis was performed, and then synthesized ChiAΔ4 was analyzed using Lumi Vision Analyzer ( A suitable concentration of each component was examined by quantification using Aisin Seiki.

9.1 Preferred concentration of phosphoenolpyruvate
In order to investigate the preferred concentration of phosphoenolpyruvate in the synthesis reaction of ChiAΔ4, a cell-free protein translation reaction was carried out at 65 ° C. for 90 minutes with the reagents, composition and final concentration shown in Table 27 and the total volume of the reaction solution being 30 μl. . The PEP concentrations in each sample in Table 27 are 0 mM (sample 1), 10 mM (sample 2), 20 mM (sample 3), and 30 mM (sample 4). FIG. 35 shows the results of analysis by Western blotting. In FIG. 35, each lane corresponds to each sample number in Table 27. FIG. 36 shows the relationship between the PEP concentration in the reaction solution and the synthesized ChiAΔ4 concentration. From FIG. 36, it was found that the preferable concentration of PEP is 2.5 to 25 mM, more preferably 5 to 20 mM, and the vicinity of 10 mM is optimal.

9.2 Suitable magnesium ion concentration
In order to examine a suitable concentration of magnesium ions in the synthesis reaction of ChiAΔ4, a cell-free protein translation reaction was carried out at 65 ° C. for 90 minutes using the reagents, composition and final concentration shown in Table 28 with a total reaction volume of 30 μl. The Mg ²⁺ concentration in each sample in Table 28 is 0 mM (sample 1), 1.0 mM (sample 2), 2.0 mM (sample 3), 3.0 mM (sample 4), 4.0 mM (sample 5). ), 5.0 mM (sample 6), 6.0 mM (sample 7), 7.0 mM (sample 8), 8.0 mM (sample 9), 9.0 mM (sample 10), 10.0 mM (sample 11). is there. FIG. 37 shows the analysis result by Western blotting. In FIG. 37, each lane corresponds to each sample number in Table 28. FIG. 38 shows the relationship between the Mg ²⁺ concentration in the reaction solution and the synthesized ChiAΔ4 concentration. From FIG. 38, it was found that the preferred concentration of Mg ²⁺ is 0.5 to 6 mM, more preferably 2 to 4 mM, and the vicinity of 3 mM is optimal.

9.3 Preferred concentration of ammonium ion
In order to examine the preferred concentration of ammonium ions in the synthesis reaction of ChiAΔ4, a cell-free protein translation reaction was carried out at 65 ° C. for 90 minutes with the reagents, composition and final concentration shown in Table 29 and the total volume of the reaction solution being 30 μl. Incidentally, _NH ^{4 +} concentration in each sample in Table 29, 0 mM (Sample 1), 25 mM (Sample 2), 50 mM (Sample 3), 75 mM (Sample 4), 100 mM (Sample 5), 125 mM (Sample 6) It is. FIG. 39 shows the results of analysis by Western blotting. In FIG. 39, each lane corresponds to each sample number in Table 29. FIG. 40 is a graph showing the relationship between the NH ₄ ⁺ concentration in the reaction solution and the synthesized ChiAΔ4 concentration. From FIG. 40, it was found that the preferable concentration of NH ₄ ⁺ is 50 to 120 mM, more preferably 65 to 100 mM, and the vicinity of 75 mM is optimal.

9.4 Preferred concentration of amino acids
In order to examine suitable concentrations of 20 amino acids in the synthesis reaction of ChiAΔ4, a cell-free protein translation reaction was performed at 65 ° C. for 90 minutes with the reagents, composition and final concentrations shown in Table 30 and the total volume of the reaction solution being 30 μl. . In addition, the amino acid concentration in each sample of Table 30 is 0 mM (sample 1), 1.0 mM (sample 2), 2.0 mM (sample 3), 3.0 mM (sample 4), 4.0 mM (sample 5). , 5.0 mM (sample 6), 6.0 mM (sample 7), 7.0 mM (sample 8). FIG. 41 shows the results of Western blot analysis. In FIG. 41, each lane corresponds to each sample number in Table 30. FIG. 42 shows the relationship between the amino acid concentration in the reaction solution and the synthesized ChiAΔ4 concentration. From FIG. 42, it was found that the preferred concentration of amino acid is 1 to 7 mM, and more preferably 2 to 7 mM.

9.5 Preferred concentration of polyethylene glycol
In order to examine a suitable concentration of polyethylene glycol in the synthesis reaction of ChiAΔ4, a cell-free protein translation reaction was performed at 65 ° C. for 90 minutes with the reagent, composition and final concentration shown in Table 31 and a total reaction volume of 30 μl. The polyethylene glycol concentration in each sample in Table 31 is 0% (w / v) (sample 1), 1.0% (w / v) (sample 2), 2.0% (w / v) ( Sample 3), 3.0% (w / v) (Sample 4), 4.0% (w / v) (Sample 5), 5.0% (w / v) (Sample 6), 6.0% (W / v) (Sample 7). FIG. 43 shows the results of Western blot analysis. In FIG. 43, each lane corresponds to each sample number in Table 31. FIG. 44 is a graph showing the relationship between the polyethylene glycol concentration in the reaction solution and the synthesized ChiAΔ4 concentration. From FIG. 44, it was found that the preferred concentration of polyethylene glycol is 0 to 2.5% (w / v), and the optimum concentration is around 2% (w / v).

9.6 Preferred concentration of potassium ion
In order to examine the preferred concentration of potassium ions in the synthesis reaction of ChiAΔ4, a cell-free protein translation reaction was carried out at 65 ° C. for 90 minutes with the reagents, composition and final concentration shown in Table 32 and the total volume of the reaction solution being 30 μl. The K ⁺ concentrations in Table 32 are 0 mM (sample 1), 50 mM (sample 2), 100 mM (sample 3), 150 mM (sample 4), 200 mM (sample 5), 250 mM (sample 6), 300 mM (sample 7). ), 350 mM (sample 8), 400 mM (sample 9), 450 mM (sample 10), and 500 mM (sample 11). FIG. 45 shows the results of analysis by Western blotting. In FIG. 45, each lane corresponds to each sample number in Table 32. FIG. 46 shows the relationship between the K ⁺ concentration in the reaction solution and the synthesized ChiAΔ4 concentration. From FIG. 46, it was found that the preferable concentration of K ⁺ is 100 to 450 mM, more preferably 170 to 350 mM, particularly preferably 210 to 320 mM, and the vicinity of 250 mM is optimal.

9.7 Maximum Synthesis Amount of ChiAΔ4 Table 33 summarizes the optimum concentration of each component obtained in “9.1 About Phosphoenolpyruvate” to “9.6 About Potassium Ion”. Therefore, cell-free protein translation reaction was carried out at 65 ° C. for 90 minutes with the reagent, composition and final concentration shown in Table 33, and the total amount of the reaction solution was 30 μl, and the maximum synthesis amount of ChiAΔ4 was examined. As a result, the synthesized amount of ChiAΔ4 was 65.5 μg / ml.

10. Cell-free protein translation system using cell extracts derived from A. pernix
10.1 Cultivation of bacterial cells and preparation of cell extract Artificial seawater (2.2% NaCl, 1% MgCl ₂ · 6H ₂ O, 0.14% CaCl ₂ · 2H ₂ O, 0.37% NaSO ₄ , 0.063 _{% KCl, 0.018% NaHCO 3,} 0.009% KBr, 0.0036% NaB 4 O 7 · 10H 2 O, 0.0013% SrCl 2, 0.0003% NaF, 0.0001% LiCl, 0. 000007% KI, 0.00000008% MnCl ₂ .4H ₂ O, 0.00000002% CoCl ₂ .6H ₂ O, 0.0000008% AlCl ₃ .6H ₂ O, 0.0000005% FeCl ₃ .6H ₂ O, 0. _{00000002% Na 2 WO4 · 2H 2} O, 0.000002% (NH 4) 6Mo 7 O 24 · 4H 2 O), 0.2% yeast extract, 0. After preparing the medium 300ml consisting of 4% tryptone and sodium thiosulfate pentahydrate _{(Na 2 S 2 O 3 ·} 5H 2 0) to pH 7.0, with a silicone stopper placed in Sakaguchi flask 500 ml, autoclaved (121 ° C. (2 atom, 20 minutes). The medium was inoculated with A.pernix K1 strain, and the Sakaguchi flask was subjected to shaking culture at 90 ° C. for about 38 hours (shaking speed 150 times / min). The absorbance (660 nm) of the culture solution after completion of the culture was 0.656. The subsequent cell extract preparation was performed according to the method of FIG. The protein concentration of the obtained cell extract was 13.5 mg / ml.

10.2 Cell-free protein translation reaction
Using a cell extract derived from A. pernix, a cell-free protein translation reaction was performed at 60 to 80 ° C. for 60 minutes with the reagent, composition and final concentration shown in Samples 1 to 5 in Table 34 as the total volume of the reaction solution being 30 μl. I did it. After completion of the reaction, the amount of ChiΔ4 produced was quantified by activity measurement using a fluorescent substrate. The result is shown in FIG. From FIG. 47, it was found that ChiAΔ4 was synthesized for all of samples 1 to 5 in Table 34, and the synthesis amount reached the maximum at 60 to 65 ° C. (synthesis amount at reaction temperature of 65 ° C .: 1.52 mg / ml). . From the above, even when a hyperthermophilic bacterium different from T. kodakaraensis is used, a cell-free protein translation system at a high temperature can be established by preparing a cell extract according to the same protocol as in FIG. found.

  INDUSTRIAL APPLICABILITY The present invention can be widely used as a protein production method under high temperature conditions in a cell-free protein synthesis system containing a cell extract derived from a hyperthermophilic bacterium.

Claims (19)

  A method for producing a protein using a cell-free protein translation system, wherein the mRNA encoding the target protein is translated under high temperature conditions in a cell-free protein translation system containing a cell extract derived from a hyperthermophilic bacterium. A method for producing a protein. The method according to claim 1, wherein the cell-free protein translation system contains mRNA, Mg ²⁺ and an amino acid encoding the target protein. The method according to claim 2, further comprising at least one component selected from NH ₄ ⁺ , K ⁺ , polyethylene glycol, ATP regeneration system and energy source.   The method according to any one of claims 1 to 3, wherein the cell extract is derived from Thermococcus kodakaraensis KOD1 strain or a strain derived from the KOD1 strain.   The method according to any one of claims 1 to 4, wherein the mRNA encoding the target protein has the ribosome binding sequence shown in SEQ ID NO: 4 upstream of the base sequence encoding the target protein. A kit for producing a protein using a cell-free protein translation system, which contains a cell extract derived from a hyperthermophilic bacterium, mRNA encoding the target protein, Mg ²⁺ and an amino acid. A kit for producing a protein by translating the mRNA under high temperature conditions. The kit according to claim 6, wherein the cell-free protein translation system further contains at least one component selected from NH ₄ ⁺ , K ⁺ , polyethylene glycol, ATP regeneration system and energy source.   The kit according to claim 6 or 7, wherein the cell extract is derived from Thermococcus kodakaraensis KOD1 strain or a strain derived from the KOD1 strain.   The kit according to any one of claims 6 to 8, wherein the mRNA encoding the target protein has the ribosome binding sequence shown in SEQ ID NO: 4 upstream of the base sequence encoding the target protein.   This is a method for producing proteins using a cell-free protein transcription / translation system. In a cell-free protein transcription / translation system containing cell extracts derived from hyperthermophilic bacteria, transcription and translation of the plasmid encoding the target protein is carried out at a high temperature. A method for producing a protein by performing under conditions. The method according to claim 10, wherein the cell-free protein transcription / translation system contains a plasmid encoding the target protein, RNA polymerase, Mg ²⁺ and an amino acid.   The method according to claim 11, wherein the RNA polymerase has an optimum temperature under high temperature conditions.   The method according to any one of claims 10 to 12, wherein the cell extract is derived from Thermococcus kodakaraensis KOD1 strain or a strain derived from the KOD1 strain.   The method according to any one of claims 10 to 13, wherein the plasmid encoding the target protein comprises a promoter, a ribosome binding sequence shown in SEQ ID NO: 4 and a base sequence encoding the target protein in order from the upstream.   The cell-free cell culture according to any one of claims 1 to 9, in which a transcription temperature is set to an optimum temperature of the RNA polymerase when transcription and translation of a plasmid encoding a target protein is performed under high temperature conditions. The method according to any one of claims 11 to 14, wherein the temperature is set to an optimum temperature of the protein translation system. A kit for producing a protein using a cell-free protein transcription / translation system. As a cell-free protein transcription / translation system, a cell extract derived from a hyperthermophilic bacterium, a plasmid encoding a target protein, RNA polymerase, Mg ²⁺ And a kit for producing a protein containing the amino acid and allowing the plasmid to be transcribed and translated under high temperature conditions.   The kit according to claim 16, wherein the RNA polymerase has an optimum temperature under a high temperature condition.   The kit according to claim 16 or 17, wherein the cell extract is derived from Thermococcus kodakaraensis KOD1 strain or a strain derived from the KOD1 strain.   The kit according to any one of claims 16 to 18, wherein the plasmid encoding the target protein comprises a promoter, a ribosome binding sequence shown in SEQ ID NO: 4 and a base sequence encoding the target protein in order from the upstream.