Method for producing thermophilic bacterium-derived protein

Abstract

The present invention provides a method for producing a thermophilic bacterium-derived protein, the method comprising a step in which a host that expresses a thermophilic bacterium-derived protein and in which a cell wall has been destroyed is heated at a temperature below 64°C and at which the host cell-derived protein is denatured.

Description

Method for producing thermophilic protein

[0001] The present invention broadly relates to a method for producing a protein derived from a thermophilic bacterium.

[0002] Proteins derived from thermophiles are widely used in various technical fields, such as DNA polymerases used in amplification reactions such as PCR. To mass-produce such proteins, various hosts, such as Escherichia coli, transformed to express the desired protein are used. However, to recover the expressed protein from the host, various steps, such as disruption of the host and purification of the protein, are required.

[0003] For example, proteins derived from thermophilic bacteria generally do not denature even at temperatures above 55°C, whereas proteins derived from the host denature at such high temperatures. Therefore, it is common to utilize this difference in heat resistance to purify the desired protein by subjecting it to a heat treatment step.

[0004] When the host is Escherichia coli and the thermophilic protein is a ligase, such a heat treatment step has conventionally been carried out at 64°C or higher (Non-Patent Document 1).

[0005] Jianying Luo et al., Nucleic Acids Research, 1996, Vol. 24, No. 15

[0006] Conventional production methods do not provide sufficient yields of proteins derived from thermophilic bacteria, and there is thought to be room for improvement in the processes after protein expression in the host.

[0007] The present inventors have investigated various steps such as disruption of the host and denaturation of host-derived proteins, and have found conditions that improve the yield.

[0008] That is, the present application encompasses the following inventions: (1) A method for producing a protein derived from a thermophilic bacterium, comprising a step of heating a host expressing a protein derived from a thermophilic bacterium, the host having its cell wall disrupted, at a temperature below 64°C at which the protein derived from the host cells is denatured. (2) The method according to (1), wherein the host cells are disrupted by a mechanical method or a non-mechanical method, and when disrupted by a non-mechanical method, the treatment temperature is not room temperature. (3) The method according to (1) or (2), wherein the heating temperature is 50°C to 60°C. (4) The method according to any one of (1) to (3), wherein the heating time is less than 20 minutes. (5) The method according to any one of (1) to (4), wherein the amount of the protein derived from a thermophilic bacterium produced from the host is greater than when the host is heated at a temperature of 65°C or higher. (6) The method according to any one of (1) to (5), further comprising a step of cooling the host and / or the protein produced from the host after the heating step. (7) The method according to any one of (1) to (6), wherein the protein is an enzyme. (8) The method according to (7), wherein the enzyme is a ligase or a polymerase. (9) The method according to any one of (1) to (8), wherein the host is Escherichia coli. (10) The method according to any one of (1) to (9), wherein the thermophilic bacterium is a bacterium belonging to the genus Thermus. (11) The method according to (10), wherein the bacterium belonging to the genus Thermus is one or more of Thermus brockianus, Thermus thermophilus, Thermus species, and / or Thermus aquaticus.

[0009] According to the production method of the present invention, the yield of thermophilic bacterium-derived protein can be significantly increased compared to conventional methods.

[0010]

[0046] Figure 1 shows the results of the yield of Tbr LigA protein purified from E. coli lysates lysed at different temperatures. Figure 1 shows the results of SDS-PAGE of the proteins in Figure 1. Figure 1 shows the results of measuring the enzymatic activity of the proteins in Figure 1. Figure 1 shows the results of the yield of Tth LigA protein purified from E. coli lysates lysed at different temperatures. Figure 4 shows the results of SDS-PAGE of the proteins in Figure 4. Figure 4 shows the results of measuring the enzymatic activity of the proteins in Figure 4. Figure 1 shows the results of the yield of Tsp LigA protein purified from E. coli lysates lysed at different temperatures. Figure 7 shows the results of SDS-PAGE of the proteins in Figure 7. Figure 7 shows the results of measuring the enzymatic activity of the proteins in Figure 7. Figure 10 shows the results of the yield of Taq DNA polymerase mutant proteins purified from E. coli lysates lysed at different temperatures. Figure 11 shows the results of SDS-PAGE of the proteins in Figure 10. 11 shows the results of measuring the enzyme activity of the protein in Figure 10. 12 shows the results of measuring the enzyme activity of the protein in Figure 10 again.

[0011] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0012] (Method for Producing Proteins) In a first embodiment, there is provided a method for producing a protein derived from a thermophilic bacterium, the method comprising the step of heating a host that expresses a protein derived from a thermophilic bacterium, the host having a destroyed cell wall, at a temperature below 64°C, at which the protein derived from the host cell is denatured.

[0013] As used herein, the term "thermophilic bacteria" generally refers to microorganisms that can grow in high-temperature environments. Examples of such microorganisms include bacteria, fungi, archaea, and viruses. There is no particular lower limit to the high temperature as long as the bacteria can grow, but the optimal growth temperature for thermophilic bacteria is, for example, 40°C or higher.

[0014] Thermophiles are sometimes classified according to the difference in growth temperature, and are generally defined as moderate thermophiles that can grow at 65° C. or higher, extreme thermophiles that can grow at 75° C. or higher, and hyperthermophiles that can grow at 80° C. or higher. The type of thermophile is not particularly limited, but extreme thermophiles or hyperthermophiles are preferred, with extreme thermophiles being more preferred.

[0015] Examples of thermophilic bacteria include bacteria belonging to the genera Thermus, Thermotoga, Thermoactinomyces, Thermobacillus, Thermobifida, Aquifex, Geobacillus, and Bacillus. Among these, bacteria belonging to the genus Thermus are preferred.

[0016] In one embodiment, the bacterium belonging to the genus Thermus is Thermus thermophilus (Tth), Thermus brockianus (Tbr), Thermus species (Tsp), Thermus aquaticus (Taq), Thermus oshimai (Tos), or Thermus filiformis (Tfi).

[0017] Examples of thermophilic fungi include fungi belonging to the genera Rasamsonia, Thermomyces, Byssochlamys, Eupenicillium, and Neosartorya.

[0018] Examples of thermophilic archaea include those belonging to the genera Thermococcus, Pyrococcus, Thermoproteus, Thermoplasma, Methanothermus, Archaeoglobus, Sulfolobus, Aeropyrum, Pyrolobus, Pyrodictium, and Pyrobaculum. Among these, archaea belonging to the genus Thermococcus are preferred.

[0019] Thermophilic viruses include, for example, phages that infect prokaryotes.

[0020] The notations of bacteria, fungi, and archaea in this specification may be shown in the notation of the new classification or the notation of the old classification.

[0021] The type of thermophile-derived protein is not particularly limited as long as it can be expressed in a host, and may be, for example, an enzyme derived from a thermophile. Proteins derived from thermophiles are not intended to be limited to those naturally present in thermophiles, but may also be artificially modified versions of such naturally occurring proteins. Proteins derived from thermophiles may be, for example, proteins derived from thermophiles that have been modified to have mutations. Proteins derived from thermophiles preferably have heat resistance and other resistances, such as resistance to protease, acid, alkali, denaturants, etc. Examples of enzymes include ligase, polymerase, endonuclease, and exonuclease. Among enzymes, ligase and polymerase are preferred.

[0022] As used herein, the term "host" refers to an organism, such as a bacterium, fungus, or plant cell, that has been transformed to express a desired protein derived from a thermophilic bacterium. The host bacterium may be Escherichia coli, Bacillus subtilis, actinomycete, or the like, and the host fungus may be a yeast or filamentous fungus, or the like.

[0023] In one embodiment, the host is E. coli.

[0024] When the host is a prokaryote, the protein expressed in the host may be a protein derived from a thermophilic prokaryote or a protein derived from a thermophilic eukaryote. Furthermore, when the host is a eukaryote, the protein expressed in the host may be a protein derived from a thermophilic eukaryote or a protein derived from a thermophilic prokaryote. For example, when the host is Escherichia coli, the protein expressed in the host may be a protein derived from any thermophilic bacterium, thermophilic fungus, or thermophilic archaea.

[0025] Transformation of host cells may be carried out by any method known to those skilled in the art, such as electroporation or heat shock.

[0026] To transform a host, an exogenous expression vector encoding a protein of interest is prepared. The expression vector may encode multiple proteins of interest. The expression vector preferably encodes a selectable marker, such as an antibiotic resistance gene, used to select transformants.

[0027] To increase the transformation efficiency, the host is preferably subjected to heat treatment or electrical treatment before transformation, which increases the efficiency of expression vector uptake in the resulting competent cells.

[0028] The transformed host is also referred to as a transformant. Selection of the transformant, growth of the transformant, induction of expression of the target protein, and recovery of the host expressing the target protein may be carried out by any method known to those skilled in the art.

[0029] For example, a mixture of an expression vector and host competent cells is cultured in a medium containing an antibiotic corresponding to the selection marker encoded by the expression vector, and the resulting colonies are selected to select transformants. While the type of medium varies depending on the host cell, LB medium is an example of a medium for E. coli. Plate media are preferred for the selection of transformants. Culture conditions are appropriately adjusted, for example, at a culture temperature of approximately 30°C to 37°C and for a culture time of approximately 12 hours to overnight.

[0030] The selected transformant is grown by culturing. Pre-culturing may be performed before the main culturing. The growth of the transformant can also be carried out under the culture conditions used for selecting the transformant, but the medium is preferably a liquid medium. The culture temperature and culture time are appropriately adjusted depending on the desired level of growth of the transformant, and are not intended to be limiting, but are appropriately adjusted between about 16°C and 37°C and about 12 hours to overnight.

[0031] The conditions for inducing expression of a target protein vary depending on the expression system used. While not intended to be limiting, the pET system is a preferred expression system when Escherichia coli is used as a host. The pET system is a protein expression system in which T7 RNA polymerase transcribes the target gene under the control of the lacUV5 promoter and expression induction occurs in the presence of allolactose or the lactose analog IPTG (isopropyl-β-thiogalactopyranoside).

[0032] In the case of an expression system using the pET system, expression of the target protein can be induced by adding IPTG to the medium and culturing under specified conditions. The culture temperature and culture time are appropriately adjusted depending on the desired level of expression, and are not intended to be limiting, but are appropriately adjusted between about 1 hour and overnight, and between about 16°C and 37°C.

[0033] In order to recover the expressed protein from the host, the host cells are subjected to a disruption process. As used herein, cell disruption refers to the destruction of the cell wall. The disruption method is not particularly limited, but can be broadly divided into mechanical and non-mechanical methods. Examples of mechanical methods include ultrasonic disruption, disruption using known devices such as homogenizers and blenders. Non-mechanical methods are methods not included in the mechanical methods, and examples include chemical methods using chemical agents such as surfactants or enzymes such as lysozyme. When disrupting host cells using chemical agents such as surfactants or enzymes such as lysozyme, protease inhibitors, DNase, etc. may be added as needed in addition to the surfactants, lysozyme, etc. Non-mechanical methods are not limited to chemical methods and may also utilize osmotic pressure differences.

[0034] Various conditions, such as temperature conditions, used in the process of disrupting host cells can be determined appropriately by those skilled in the art. For example, mechanical methods are generally performed on ice because heat is generated during the disruption process. Non-mechanical chemical methods include the lysis reagent method, in which the host cell wall is disrupted by suspending the host in a buffer containing a surfactant or lysozyme and treating at room temperature. However, when a non-mechanical method is used to disrupt host cells in the present invention, it is preferable that the treatment temperature is not room temperature. Room temperature may be, for example, 10 to 30°C.

[0035] The conditions for non-mechanical disruption of host cells at a treatment temperature other than room temperature can be determined appropriately by those skilled in the art. For example, when disrupting host cells using a lysis reagent method, among other non-mechanical methods, the treatment temperature is adjusted by mixing the host in a buffer containing necessary reagents, such as a surfactant or lysozyme, on ice. When removing the mixture of the host and buffer containing the added reagent from the ice and suspending it using a known device such as a vortex mixer, it is preferable to suspend the mixture quickly, for example, within 10 seconds, so that the temperature of the mixture does not rise to room temperature. After removing the mixture from the ice and suspending it using a vortex mixer, the mixture may be returned to ice, and then removed from the ice and suspended using a vortex mixer. This procedure may be repeated. When suspending the mixture without using a known device such as a vortex mixer, it is preferable to always perform the suspension operation on ice. Avoiding incubation at room temperature after mixing and suspension allows for rapid transition to the heating step. If the heating step is not to be transitioned to immediately after mixing and suspension, it is preferable to store the mixture on ice, etc.

[0036] After the disruption step, the host cells are subjected to a heating step. The heating step is carried out to denature the host cell-derived proteins, and is carried out at a temperature below 64°C at which the host cell-derived proteins denature. The temperature used in the heating step is also referred to as the "heating temperature" or the "treatment temperature," and these terms are used interchangeably. Denaturation occurs when the helical structure or sheet structure of the protein is disrupted, and the temperature at which the protein denatures can be determined by known methods for analyzing the secondary structure of proteins, such as circular dichroism spectroscopy or fluorescence spectroscopy.

[0037] As used herein, "a temperature below 64°C at which a host cell-derived protein denatures" refers to a temperature equal to or higher than a specific temperature at which the structure of a host cell-derived protein changes and the function of the protein is lost, but lower than 64°C. It also refers to a temperature at which a host cell-derived protein denatures but a thermophilic bacterium-derived protein does not denature.

[0038] The heating temperature can be appropriately determined by those skilled in the art depending on the growth temperature of the thermophilic bacterium. For example, when using thermophilic bacteria with a low growth temperature, such as moderate thermophiles, the heat treatment may be carried out at a temperature lower than the heating temperature used in the heating step for extreme thermophiles.

[0039] In one embodiment, the heating temperature is 50° C. or higher.

[0040] The heating temperature is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, or 63°C. The heating temperature is preferably 50°C to 60°C, more preferably 55°C to 60°C. The heating time varies depending on the heating temperature, but may be any time as long as the host cell-derived protein is denatured.

[0041] In one embodiment, when the protein derived from a thermophilic bacterium is a ligase derived from Thermus broeckianus, the heating temperature is about 53 to 60°C, preferably about 55 to 60°C, more preferably about 54 to 56°C.

[0042] In one embodiment, when the thermophilic bacterium-derived protein is Thermus thermophilus-derived ligase, the heating temperature is about 55 to 62°C, preferably about 55 to 60°C, more preferably about 59 to 61°C.

[0043] In one embodiment, when the thermophilic bacterium-derived protein is a ligase derived from Thermus species, the heating temperature is about 55 to 60°C, preferably about 54 to 56°C.

[0044] In one embodiment, when the thermophilic bacterium-derived protein is a polymerase derived from Thermus aquaticus, the heating temperature is about 50 to 62°C, preferably about 54 to 60°C.

[0045] In one embodiment, when the heating temperature is within the range of 50 to 60°C, the heating step time is, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, or 19 minutes.

[0046] Heating may be performed using any device known to those skilled in the art, for example, an incubator such as a heat block, or, in the case of industrial production, a heating tank.

[0047] The host and / or the protein produced by the host may be cooled after the heating step. Proteins produced in a host are usually separated from other components by steps such as centrifugation, but by cooling the host and / or the protein produced by the host, the insoluble fraction and the soluble fraction can be separated more clearly than when not cooled, thereby increasing the recovery amount of the target protein contained in either fraction. Cooling is preferably performed on ice, but may be performed at a temperature lower than room temperature, for example, at 0 to 10°C. The cooling time may be any time as long as the insoluble fraction and the soluble fraction can be clearly separated, for example, 1 to 30 minutes. The cooling step is preferably performed promptly after the heat treatment.

[0048] After cooling, the protein produced from the host is collected by centrifugation or the like.

[0049] The produced protein may be purified by any method known to those skilled in the art, such as affinity chromatography, ion exchange chromatography, or gel filtration chromatography. The produced protein may have similar properties and physiological activity, e.g., similar purity and activity, as the protein when heated at a temperature of 65°C or higher.

[0050] The yield, purity, activity, etc. of the produced protein can be evaluated by any method known to those skilled in the art. For example, the yield can be evaluated by quantifying the protein concentration using absorptiometry, fluorometry, or the like as the corrected yield, and converting it into protein per bacterial cell weight, preferably per wet bacterial cell weight, of the host used.

[0051] The purity of the produced protein can be evaluated by, for example, electrophoretic analysis such as SDS-PAGE, chromatographic analysis, mass spectrometry, or the like.

[0052] The activity of the produced protein can be appropriately evaluated by those skilled in the art depending on the type of protein obtained. For example, if the obtained protein is an enzyme, the activity can be evaluated by quantitatively analyzing the results of reacting the enzyme with a substance containing its substrate. For example, if the enzyme is a ligase, the activity can be evaluated by reacting the ligase with a solution containing nucleic acid and performing quantitative real-time PCR analysis on the resulting product. Furthermore, for example, if the enzyme is a polymerase, the activity can be evaluated by subjecting the polymerase to a PCR reaction and quantitatively analyzing the resulting PCR amplicon of the desired length.

[0053] (Method for increasing protein yield) In a second embodiment, there is provided a method for increasing the yield of a protein derived from a thermophilic bacterium, the method comprising the step of heating a host expressing a protein derived from a thermophilic bacterium, the host having a destroyed cell wall, at a temperature below 64°C at which the protein derived from the host cell is denatured.

[0054] The heating temperature can be appropriately determined by those skilled in the art depending on the growth temperature of the thermophilic bacterium. For example, when a thermophilic bacterium with a low growth temperature, such as a moderate thermophilic bacterium, is used, the treatment may be carried out at a lower temperature than that used in the heating step for an extreme thermophilic bacterium.

[0055] In one embodiment, the heating temperature is 50° C. or higher.

[0056] The heating temperature is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, or 63°C. The heating temperature is preferably 50°C to 60°C, and more preferably 55°C to 60°C. The heating time varies depending on the heating temperature, but may be any time as long as the host cell-derived protein is denatured. In one embodiment, when the thermophilic bacterium-derived protein is Thermus broeckianus-derived ligase, the heating temperature is about 53 to 60°C, preferably about 55 to 60°C, and more preferably about 54 to 56°C.

[0057] In one embodiment, when the thermophilic bacterium-derived protein is Thermus thermophilus-derived ligase, the heating temperature is about 55 to 62°C, preferably about 55 to 60°C, more preferably about 59 to 61°C.

[0058] In one embodiment, when the thermophilic bacterium-derived protein is a ligase derived from Thermus species, the heating temperature is about 55 to 60°C, preferably about 54 to 56°C.

[0059] In one embodiment, when the thermophilic bacterium-derived protein is a polymerase derived from Thermus aquaticus, the heating temperature is about 50 to 62°C, preferably about 54 to 60°C.

[0060] In one embodiment, when the heating temperature is within the range of 50 to 60°C, the heating step time is, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, or 19 minutes.

[0061] Preferably, the thermophilic protein is a ligase or a polymerase.

[0062] The yield of protein produced by the above method can be significantly increased by adjusting the heating temperature.

[0063] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0064] Example 1 Purification of Ligase (LigA) and DNA Polymerase from Bacteria Belonging to the Genus Thermus <Transformation> Thermus brochianus (Tbr), Thermus thermophilus (Tth), Thermus species (Tsp), and Thermus aquaticus (Taq) were selected as bacteria belonging to the genus Thermus. BL21(DE3) competent cells (Thermo Fisher, Cat: EC0114) were transformed with the Tbr LigA construct (Example 2), the Tth LigA construct (Example 3), the Tsp LigA construct (Example 4), and a pET vector (Example 5) incorporating a Taq DNA polymerase mutant (SEQ ID NO: 1). The transformed E. coli bacteria were plated on an ampicillin-containing LB plate and cultured overnight at 37°C to allow E. coli colonies to form.

[0065]

[0066] <Induction of Enzyme Protein Expression> A single colony was inoculated into a small amount of carbenicillin-containing liquid LB medium and cultured overnight at 37°C with shaking (225 rpm, preculture). The next day, 1 / 40 of the preculture solution was added to fresh carbenicillin-containing liquid LB medium and cultured at 37°C for 4 hours with shaking (225 rpm, main culture). After confirming that the OD600 value of the main culture was between 0.4 and 0.6, IPTG (Fujifilm Wako, Cat: 099-05013) was added to a final concentration of 0.1 mM and cultured at 30°C for 3 hours with shaking (225 rpm, IPTG induction). The IPTG-induced E. coli solution was collected and centrifuged (10,000 x g, 10 minutes, 4°C) to recover the E. coli pellet. The wet weight was measured and the pellet was frozen overnight or longer in a -80°C freezer.

[0067] Lysis of E. coli and collection of soluble fraction: Frozen E. coli was transferred to ice, and 5 mL of BugBuster (Novagen, Cat. No. 70921-4) containing 1,000 U / mL rLysozyme (Novagen, Cat. No. 71110-4) + 1 / 200 Volume Protease Inhibitor Cocktail (Millipore, Cat. No. 539134-1MLCN) was added per gram of wet E. coli. The mixture was quickly vortexed to completely suspend the E. coli. The E. coli suspension was dispensed into 1.5 mL microtubes and transferred to heat blocks set at 50, 55, 60, 65, 70, or 80°C, followed by shaking at 600 rpm for 15 minutes. After the heat treatment, the mixture was immediately transferred onto an aluminum block placed on ice and cooled for 15 minutes to allow the precipitate to mature. After centrifugation (16,000 x g, 20 minutes, 4°C), the soluble fraction was collected and transferred to a new 1.5 mL microtube.

[0068] <His affinity purification> 250 μL of His-Affinity Gel (included in the His-Spin Protein Miniprep kit (ZymoResearch, Cat: P2002)) was transferred to a His-Spin column (included in the His-Spin Protein Miniprep kit) and centrifuged (15,000 × g, 20 seconds, 4°C) to remove the gel dispersion. 300 μL of the soluble fraction was added to the His-Spin column and shaken (1,500 rpm) at 25°C for 5 minutes. After centrifugation (same as above), the flow-through liquid was decanted and discarded. An additional 250 μL of wash buffer (10 mM imidazole (Nacalai Tesque, Cat. No. 08787-22), 0.03% Triton® X-100 (Fujifilm Wako, Cat. No. 160-24751) was added to the column, which was then gently mixed by vortexing and centrifuged (same as above). The flow-through solution was discarded, and the same washing procedure was repeated two more times. The column was placed in a new 1.5 mL microtube, and 250 μL of His-Elution Buffer (included in the His-Spin Protein Miniprep kit) was added to the column. The column was mixed by vortexing for approximately 10 seconds and then allowed to stand on ice for at least 1 minute. The eluate was collected by centrifugation (same as above). The volume of the eluate was accurately recorded.

[0069] <Sample preparation for SDS-PAGE> 4x Laemmli SDS-PAGE sample buffer (containing 40% glycerol (Nacalai Tesque, Cat. No. 17045-94), 8% sodium lauryl sulfate (Nacalai Tesque, Cat. No. 02873-75), 400 mM dithiothreitol (Fujifilm Wako, Cat. No. 042-29222), and bromophenol blue (Nacalai Tesque, Cat. No. 05808-61), 250 mM Tris-HCl (Nacalai Tesque, Cat. No. 35434-05), pH 6.8) and UltraPure DNase / RNase-Free Distilled Water (Thermo Fisher Scientific) were used. The eluate was mixed with the eluate at a ratio of 2:1:1 to prepare a sample for SDS-PAGE. Each SDS-PAGE sample was treated at 100°C for 5 minutes before electrophoresis, and then centrifuged (13,000 x g, 10 minutes, room temperature). The supernatant was then subjected to SDS-PAGE.

[0070] <SDS-PAGE> A polyacrylamide gel was prepared by layering a separating gel (8.0% acryl-bisacrylamide (Nacalai Tesque, Cat. No. 06140-45), 375 mM Tris-HCl, pH 8.8, 0.1% sodium lauryl sulfate, 0.1% ammonium peroxodisulfate (Nacalai Tesque, Cat. No. 06284-04), 0.1 v / v% N,N,N',N'-tetramethylethylenediamine (Nacalai Tesque, Cat. No. 33401-72)) as the lower layer and a stacking gel (4.0% acryl-bisacrylamide, 125 mM Tris-HCl, pH 6.8, 0.1% sodium lauryl sulfate, 0.1% ammonium peroxodisulfate, 0.1 v / v% N,N,N',N'-tetramethylethylenediamine) as the upper layer. The gel was placed in an electrophoresis tank filled with SDS-PAGE running buffer (25 mM Tris-HCl, 0.1% sodium lauryl sulfate, 191 mM glycine (Nacalai Tesque, Cat: 17109-35)), and 200 ng of each SDS-PAGE sample was loaded. A constant current of 30 mA per gel was applied for approximately 30 minutes to concentrate each sample in the concentrating gel, and a constant current of 40 mA was applied for approximately 50 minutes per gel to separate the proteins contained in each sample by molecular weight in the separating gel.

[0071] <Coomassie Brilliant Blue (CBB) Staining> After electrophoresis, the gel was gently washed with ultrapure water and treated three times for 10 minutes each in a fixative (50% methanol (Fujifilm Wako, Cat: 132-06471), 10% acetic acid (Nacalai Tesque, Cat: 10218-15)). Subsequently, the gel was stained for 10 minutes with a quick-CBB kit (Fujifilm Wako, Cat: 299-50101), destained in ultrapure water, and the stained protein bands were imaged.

[0072] <Measurement of protein concentration in eluate> Protein concentration was measured using an absorption spectrometer by measuring the amount of absorbance at 280 nm derived from the side chains of aromatic amino acids (tyrosine, tryptophan) that constitute the protein. During the measurement, His-Elution Buffer was used as a blank solution. The yield was calculated as the corrected yield (µg protein / µg wet weight of bacterial cells) by multiplying the eluate volume and the measured value and dividing the result by the wet weight of E. coli used.

[0073] <Evaluation of Ligase Activity—Ligase Reaction> Each eluate was diluted with LigA diluent (10 mM Tris-HCl, pH 7.4, 100 mM potassium chloride (Nacalai Tesque, Cat: 28538-75), 0.1 mM EDTA (Thermo Fisher Scientific, Cat: 15575-020), 1 mM dithiothreitol, 0.1% Triton (registered trademark) X-100, 50% glycerol) to a protein concentration of 3.4 ng / μL to obtain a diluted eluate. The diluted eluate was added to a reaction solution containing 20 nM BRAF E1(+) template (DNA oligo of SEQ ID NO: 2), 20 nM BRAF E1 bridge oligo (DNA oligo of SEQ ID NO: 3), and 1x HiFi Taq Ligase Buffer (New England Biolabs, Cat: M0647S) at a concentration of 10 v / v% and mixed thoroughly. As a positive control, an equal amount of HiFi Taq DNA Ligase was added. Heat denaturation was performed at 95°C for 5 minutes, followed by ligation at 50°C for 1 hour. After the reaction was completed, the mixture was cooled quickly, and a reaction stop solution (80% formamide (Fujifilm Wako, Cat: 066-02301), 100 mM EDTA) was added in an amount 1.5 times the reaction solution, mixed, and then allowed to stand on ice.

[0074]

[0075] <Ligase activity evaluation - quantitative real-time PCR reaction> The reaction solution after reaction termination treatment was diluted 40,000-fold with UltraPure DNase / RNase-Free Distilled Water, and the diluted solution was mixed with 1X Prime Time PCR Assay (Integrated DNA Technologies, consisting of 20 μM DNA oligos of SEQ ID NOs: 4 and 5 and 10 μM DNA oligo of SEQ ID NO: 6. The DNA oligo of SEQ ID NO: 6 is modified with a fluorescent molecule, FAM, at the 5' end and a quenching molecule, Iowa Black (registered trademark) FQ, at the 3' end), 1X Prime Time Gene Expression Master Mix (Integrated DNA Technologies), and 1X Prime Time Gene Expression Master Mix (Integrated DNA Technologies). 2 μL of the PCR product was added to a quantitative real-time PCR reaction solution consisting of a 95°C heat denaturation reaction for 3 minutes, followed by a 95°C heat denaturation reaction for 15 seconds and a 60°C extension reaction for 30 seconds. A total of 45 PCR cycles were performed. Fluorescent signals were detected once after each cycle. The maximum position of the second derivative (twice differentiated curve) of the measured fluorescent signal amplification curve was recorded as the Ct value.

[0076]

[0077] <Polymerase Activity Evaluation - PCR Reaction> Each eluate was diluted to a Taq DNA polymerase mutant protein concentration of 62.6 ng / μL (molar concentration: 1 μM) to obtain diluted proteins. The reaction mixture contained 200 nM BRAF forward primer (DNA oligo of SEQ ID NO: 7), 200 nM BRAF reverse primer (DNA oligo of SEQ ID NO: 8), 10 ng of genomic DNA purified from colon cancer cell line HCT-116 (ATCC, Cat: CCL-247), 200 μM dNTP (TOYOBO, Ref: NTP-201), 50 mM Tris-HCl, pH 9.2, 16 mM (NH)SO (Nacalai Tesque, Cat: 02633-15), 0.1% Tween-20 (Nacalai Tesque, Cat: 28353-14), and 2.5 mM MgCl (Nacalai Tesque, Cat: 20937-72). The diluted protein was added to a final molar concentration of 200 nM. After an initial heat treatment reaction at 95°C for 2 minutes, 35 PCR cycles were performed, each cycle consisting of 95°C for 15 seconds, 62°C for 30 seconds, and 72°C for 30 seconds. The purified products were separated by electrophoresis on a 3.0% agarose / TAE gel and stained with SYBR Gold staining reagent for 10 minutes to detect PCR amplicons of the desired length.

[0078]

[0079] Example 2 <Result 1> As a result of purifying the Tbr LigA protein, a plot of the lysis treatment temperature and the corrected yield is shown in Figure 1. Compared to the treatment temperatures (64°C or higher) described in Non-Patent Document 1, the yield increased by 1.5 to 5.5 times at 55°C and 60°C.

[0080] LigA protein purified from E. coli lysates lysed at various temperatures was separated by SDS-PAGE and then stained with CBB. The results are shown in Figure 2. No significant differences in the amount or purity of degradation products were observed due to differences in treatment temperature.

[0081] Furthermore, the enzyme activity of the LigA protein purified from the E. coli lysate subjected to lysis treatment at each temperature was measured, and the results are shown in Figure 3. No difference in enzyme activity was observed due to the difference in treatment temperature.

[0082] Example 3 <Result 2> As a result of purifying the Tth LigA protein, a plot of the lysis treatment temperature and corrected yield is shown in Figure 4. Compared to the treatment temperatures (64°C or 70°C) described in Non-Patent Document 1, International Publication No. 2000-026381, and Jenny Tong et al., "Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D. Nucleic Acids Research." 1999, 27(3), 788-794, the yield increased by 1.4 to 2.1 times at 55°C and 60°C.

[0083] LigA protein purified from E. coli lysates lysed at various temperatures was separated by SDS-PAGE and then stained with CBB. The results are shown in Figure 5. No significant differences in the amount or purity of degradation products were observed due to differences in treatment temperature.

[0084] Furthermore, the enzyme activity of the LigA protein purified from the E. coli lysate subjected to lysis treatment at each temperature was measured, and the results are shown in Figure 6. No difference in enzyme activity was observed due to the difference in treatment temperature.

[0085] Example 4 <Result 3> As a result of purifying the Tsp LigA protein, a plot of the lysis treatment temperature and corrected yield is shown in Figure 7. Compared to the treatment temperature (70°C) described in International Publication No. 2000-026381 and Jenny Tong et al., "Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D. Nucleic Acids Research." 1999, 27(3), 788-794, the yield increased 1.8-fold at 55°C.

[0086] LigA protein purified from E. coli lysates lysed at various temperatures was separated by SDS-PAGE and then stained with CBB. The results are shown in Figure 8. No significant differences in the amount or purity of degradation products were observed due to differences in treatment temperature.

[0087] Furthermore, the enzyme activity of the LigA protein purified from the E. coli lysate subjected to lysis treatment at each temperature was measured, and the results are shown in Figure 9. No difference in enzyme activity was observed due to the difference in treatment temperature.

[0088] Example 5 <Result 4> As a result of purifying Taq DNA polymerase mutant proteins, a plot of the lysis treatment temperature and corrected yield is shown in Figure 10. Compared to the treatment temperature (72°C) used in purifying Taq-derived polymerase as described in Christian Gloechner et al. "Evolving a Thermostable DNA Polymerase That Amplifies from Highly Damaged Templates." Angewandte Chemie. 2007, 46(17), 3115-3117, the yield increased approximately two-fold at 55°C to 65°C.

[0089] The Taq DNA polymerase mutant proteins purified from E. coli lysates lysed at various temperatures were separated by SDS-PAGE and stained with CBB. The results are shown in Figure 11. No significant differences in the amount or purity of degradation products were observed due to differences in treatment temperature.

[0090] Furthermore, the enzyme activity of the Taq DNA polymerase mutant protein purified from the E. coli lysate lysed at each temperature was measured, and the results are shown in Figure 12. No polymerase activity was observed at treatment temperatures of 70°C or higher.

[0091] The lack of activity at 70°C was thought to be due to the fact that a large amount of the His-Elution Buffer used for extraction in His affinity purification was carried over into the PCR reaction system. Therefore, two lots were purified again under the heat treatment conditions (72°C) described in Christian Gloechner et al. "Evolving a Thermostable DNA Polymerase That Amplifies from Highly Damaged Templates." Angewandte Chemie. 2007, 46(17), 3115-3117. and 60°C, which gave the highest yield, and the polymerase activity was evaluated. As shown in Figure 13, no difference in polymerase activity was observed between the treatment temperatures of 60°C and 72°C.

Claims

1. A method for producing a protein derived from a thermophilic bacterium, comprising the step of heating a host expressing the protein derived from a thermophilic bacterium, the host having a destroyed cell wall, to a temperature below 64°C at which the protein derived from the host cell is denatured.

2. The method of claim 1, wherein the host cells are disrupted by a non-mechanical method or a mechanical method, and if disrupted by a non-mechanical method, the processing temperature is not room temperature.

3. The method according to claim 1 or 2, wherein the heating temperature is 50°C to 60°C.

4. The method according to claim 1 or 2, wherein the duration of the heating step is less than 20 minutes.

5. The method according to claim 1 or 2, wherein the amount of thermophilic bacterium-derived protein produced from the host is greater than that produced when the host is heated at a temperature of 65°C or higher.

6. The method of claim 1 or 2, further comprising the step of cooling the host and / or the protein produced from the host after the heating step.

7. The method of claim 1 or 2, wherein the protein is an enzyme.

8. The method of claim 7, wherein the enzyme is a ligase or a polymerase.

9. The method of claim 1 or 2, wherein the host is Escherichia coli.

10. The method according to claim 1 or 2, wherein the thermophilic bacterium is a bacterium belonging to the genus Thermus.

11. The method according to claim 10, wherein the bacterium belonging to the genus Thermus is one or more of Thermus brockianus, Thermus thermophilus, Thermus species and / or Thermus aquaticus.

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