JP2009039073A - Method for producing protein by continuous fermentation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a protein by continuous fermentation by a simple operational process so as to maintain its high productivity stably for a long period of time. <P>SOLUTION: The method for producing a protein such as canine interferon-γ by continuous fermentation includes the following process: Using porous membranes such as of polyvinylidene fluoride-based resin as separation membranes for a fermented culture liquid, with both high permeability and cell-blocking ability and high plugging resistance, a filtration treatment is conducted at low intermembrane differential pressure, thus enabling microorganisms to be proliferated to high concentrations, resulting in improving the production level of the objective protein. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention filters a fermentation broth of a microorganism having the ability to produce proteins through a porous membrane that is unlikely to be clogged as a separation membrane, and holds or refluxs the unfiltrated liquid in the fermentation broth, and the culture medium of the microorganism Is added to the fermentation broth and the protein is recovered from the microbial cells, and the present invention relates to a method for producing a protein by continuous fermentation.

  Fermentation methods, which are substance production methods that involve the cultivation of microorganisms, can be broadly classified into (1) batch fermentation methods (Batch fermentation methods) and fed-batch fermentation methods (Fed-Batch fermentation methods), and (2) continuous fermentation methods. Can do.

  The batch fermentation method and fed-batch fermentation method of the above (1) are simple in terms of equipment, and have the advantage that the culture is completed in a short time and damage caused by contamination with bacteria is small. However, nutrients in the fermentation broth decrease with the passage of time, and bacterial cell growth and productivity decrease due to the influence of osmotic pressure or product inhibition. Therefore, it is difficult to stably maintain the growth and high productivity of bacterial cells over a long period of time.

  On the other hand, the continuous fermentation method (2) is characterized in that high productivity can be maintained for a long time by avoiding accumulation of the target substance at a high concentration in the fermentation reaction tank. However, since the microorganisms in the fermentation broth are diluted to continuously supply the medium to the fermentation broth and extract the fermentation broth containing the microorganisms, the improvement in production efficiency is limited. Become.

  Next, the technical background regarding the protein and its production method will be described. An enzyme can be mentioned as protein produced industrially. Many industrially important enzymes, such as amylolytic and proteolytic enzymes, are microorganisms of the genus Bacillus, such as B. pylori. B. subtilis, B. subtilis B. amyloliquefaciens, B. et al. B. licheniformis, B. et al. Stearothermophilus, and B. stearothermophilus. Produced from B. coagulans. Moreover, as an example of mass production, there is an example in which an enzyme nitrile hydratase is produced and an amide compound is produced therefrom (see Patent Document 1). Production of aldehyde oxidase, which is useful as an enzyme for measuring aldehydes and an enzyme for degrading and removing harmful environmental pollutant aldehydes such as formaldehyde, has been studied (see Patent Document 2). Studies on an enzyme that produces docosahexaenoic acid, for which various physiological effects such as cholesterol lowering action, anticoagulant action and learning function improving action have been reported, have also been conducted (see Patent Document 3).

  These proteins are originally produced by a fermentation method in which a microorganism capable of producing a desired protein or a recombinant microorganism into which a gene encoding the desired protein is introduced is cultured. As the fermentation method, a batch fermentation method is used. Specifically, production of α-amylase (see Non-Patent Document 1), production of β-amylase (see Non-Patent Documents 2 and 3), production of dihydrofolate reductase (see Non-Patent Documents 1 and 2) that require efficient productivity. Non-patent document 4), and interferon and insulin production (see non-patent document 5) both use batch fermentation. However, it is difficult to improve the production efficiency by the batch fermentation method.

As described above, in protein production, various studies have been conducted to improve production performance for the purpose of efficient protein production, and further protein production technology innovation has been desired.
JP 2007-061035 A JP 2001-299351 A JP 2001-169780 A Palva, Gene (1982) 19: 81-87; Shinomiya et al. Agric. Biol. Chem. (1981) 45; 1733-1735; Gray and Chang J. Bacteril (1981) 145: 422-428; Williams et al. Gene (1981) 16: 199-206 Palva, Gene (1983) 22: 229-235

  An object of the present invention is to provide a protein production method by a continuous fermentation method that maintains a high productivity stably for a long time with a simple operation method, that is, in a continuous fermentation method, Filtration through a separation membrane, maintaining or refluxing the unfiltered liquid in the fermentation broth, improving the microorganism concentration in the fermentation broth, and maintaining the substance high by maintaining it high, producing a protein by continuous fermentation It is to provide a method.

  As a result of diligent research, the present inventors have significantly suppressed clogging of the membrane when the fermentation culture solution is filtered under the condition that the microorganism has less penetration into the separation membrane and the membrane has a low transmembrane pressure difference. It was discovered that by performing continuous fermentation using a separation membrane, it was possible to grow the microorganisms that were the subject to a high concentration, and the protein production amount was dramatically improved. completed.

  In the method for producing a protein of the present invention, a fermentation broth of a microorganism having protein production ability is filtered through a separation membrane, an unfiltered liquid is retained or refluxed in the fermentation broth, and the microorganism medium is used as the fermentation medium. A method for producing a protein by continuous fermentation which is added to a culture solution and recovers a protein from microbial cells, and has a pore having an average pore diameter of 0.01 μm or more and less than 1 μm as the separation membrane The protein is produced by continuous fermentation, characterized in that the membrane is subjected to filtration treatment with a transmembrane differential pressure in the range of 0.1 to 20 kPa.

According to a preferred aspect of the protein production method of the present invention, the porous membrane has a pure water permeability coefficient of 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ³ / m ^2. / S / pa or less.

  According to a preferred embodiment of the protein production method of the present invention, the average pore diameter of the porous membrane is 0.01 μm or more and less than 0.2 μm, and the standard deviation of the average pore diameter is 0.1 μm or less. is there.

  According to a preferred embodiment of the protein production method of the present invention, the membrane surface roughness of the porous membrane is 0.1 μm or less.

  According to a preferred embodiment of the protein production method of the present invention, the porous membrane is a porous membrane including a porous resin layer.

  According to a preferred embodiment of the protein production method of the present invention, the material of the porous membrane is a polyvinylidene fluoride resin.

  According to a preferred embodiment of the method for producing a protein of the present invention, the fermentation raw material of the microorganism includes a saccharide.

  According to a preferred embodiment of the protein production method of the present invention, the gene encoding the protein is linked downstream of the phosphate promoter, and the concentration of inorganic phosphate contained in the medium is 1 mM or less.

  According to a preferred embodiment of the protein production method of the present invention, the protein is canine interferon γ.

  According to a preferred embodiment of the protein production method of the present invention, the microorganism is Escherichia coli.

  According to the present invention, by using a porous membrane that is less likely to be clogged under simple operating conditions, the microorganism concentration in the fermentation broth is improved and maintained high without suppressing the growth of microorganisms. By doing so, it becomes possible to produce a protein that maintains high substance productivity.

  In the present invention, a fermentation broth of a microorganism capable of producing a protein is filtered through a separation membrane, an unfiltered liquid is retained or refluxed in the fermentation broth, and the culture medium of the microorganism is added to the fermentation broth. A method for producing a protein by continuous fermentation in which a protein is recovered from a microbial cell, wherein a porous membrane having pores having an average pore diameter of 0.01 μm or more and less than 1 μm is used as the separation membrane. A method for producing a protein by continuous fermentation, wherein the filtration is performed with a differential pressure in the range of 0.1 to 20 kPa.

  It is desirable that the porous membrane used in the present invention is one in which clogging due to microorganisms used for fermentation hardly occurs and the filtration performance continues stably for a long period of time. Therefore, it is important that the porous membrane used in the present invention has an average pore diameter of 0.01 μm or more and less than 1 μm. As will be described later, a porous membrane having a porous resin layer on at least one surface of a porous substrate is preferably used.

  When the porous resin layer is present on both surfaces of the porous membrane, it is sufficient that at least one of the porous resin layers satisfies the above average pore diameter condition. If the average pore diameter is within this range, it is possible to achieve both a high exclusion rate that does not cause leakage of bacterial cells and sludge, and high water permeability, and it is difficult to clog and maintains water permeability for a long time. Can be implemented with higher accuracy and reproducibility. If the average pore diameter is within this range, when using Escherichia coli, yeast, or the like, there is little clogging and stable continuous fermentation can be carried out without leakage into the cell filtrate. When bacteria smaller than E. coli or yeast are used, the average pore size is preferably 0.4 μm or less, and the average pore size is preferably 0.2 μm or less. If the average pore diameter is too small, the water permeation rate may decrease. Therefore, it is usually preferably 0.02 μm or more, and more preferably 0.04 μm or more.

  Here, the average pore diameter can be obtained by measuring and averaging the diameters of all pores that can be observed within a range of 9.2 μm × 10.4 μm in a scanning electron microscope observation at a magnification of 10,000 times. it can. The standard deviation σ of the pore diameter is preferably 0.1 μm or less. The standard deviation σ of the pore diameter is Nk, which is the number of pores that can be observed within the above-mentioned range of 9.2 μm × 10.4 μm, and the measured diameter is Xk, and the average pore diameter is X (ave). It computed from Formula (1) below.

In the porous membrane used in the present invention, the permeability of the fermentation broth is one of the important points, and the pure water permeability coefficient of the porous membrane before use can be used as the permeability index. When the pure water permeability coefficient of the porous membrane was calculated by measuring the water permeability at a head height of 1 m using purified water at a temperature of 25 ° C. by a reverse osmosis membrane, 2 × 10 ⁻⁹ m ³ / m ² / s. / Pa or more is preferable, and if it is 2 × 10 ⁻⁹ or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less, a practically sufficient amount of permeated water can be obtained.

  The surface roughness of the porous membrane used in the present invention is a factor that affects the clogging of the separation membrane. Preferably, when the surface roughness is 0.1 μm or less, the separation coefficient and membrane resistance of the separation membrane The continuous culture can be performed with a lower transmembrane pressure difference. In addition, since the surface roughness is low, it can be expected to reduce the shear force generated on the membrane surface in the filtration of microorganisms, the destruction of microorganisms is suppressed, and the clogging of the porous membrane is also suppressed, It is considered that stable filtration is possible for a long time.

Here, the surface roughness can be measured under the following conditions using the following atomic force microscope (AFM).
・ Atomic force microscope (Nanoscope IIIa manufactured by Digital Instruments)
・ Condition probe SiN cantilever (manufactured by Digital Instruments)
Scan mode Contact mode (in-air measurement)
Underwater tapping mode (underwater measurement)
Scanning range 10μm, 25μm square (measurement in air)
5μm, 10μm square (underwater measurement)
Scanning resolution 512 × 512
Sample preparation For measurement, the membrane sample was immersed in ethanol at room temperature for 15 minutes, immersed in RO water for 24 hours, washed, and then air-dried. The RO water refers to water that has been filtered using a reverse osmosis membrane (RO membrane), which is a type of filtration membrane, and impurities such as ions and salts are excluded. The pore size of the RO membrane is approximately 2 nm or less.

The surface roughness d _rough of the film was calculated from the following (Equation 2) from the height of each point in the Z-axis direction by AFM.

  The porous membrane in the present invention has separation performance and water permeability according to the quality of water to be treated and applications, and in terms of separation performance such as blocking performance, water permeability and dirt resistance, the porous resin layer It is preferable that the film contains a porous film. That is, a preferred porous membrane has a porous resin layer that acts as a separation functional layer on the surface of the porous substrate. The porous substrate supports the porous resin layer and gives strength to the separation membrane.

  When the porous membrane used in the present invention has a porous resin layer on the surface of the porous substrate, the porous substrate is porous even if the porous resin layer penetrates the porous substrate. It does not matter if the porous resin layer does not penetrate, and it is selected according to the application.

  The material of the porous substrate is not particularly limited, and is preferably an organic fiber, such as an organic material and an inorganic material. A preferred porous substrate is a woven fabric or a nonwoven fabric using organic fibers such as cellulose fiber, cellulose triacetate fiber, polyester fiber, polypropylene fiber and polyethylene fiber, and among them, the density control is relatively easy, Nonwoven fabrics that are easy to manufacture and inexpensive are preferably used. The average thickness of the porous substrate is preferably 50 μm or more and 3000 μm or less.

  As described above, the porous resin layer functions as a separation functional layer, and an organic polymer membrane can be preferably used. The organic polymer membrane is a separation membrane basically composed of an organic polymer material. For example, a pore size smaller than the pore size of the organic fiber nonwoven fabric or macropore structure porous organic substrate and the porous organic substrate. In many cases, the porous resin layer has a composite structure.

  Here, as the material of the porous resin layer, polyethylene resin, polypropylene resin, polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin, polyacrylonitrile resin, cellulose resin And cellulose triacetate resin, and a mixture of these resins as a main component may be used. Among them, polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethersulfone resins, and polyacrylonitrile resins that are easy to form in solution and have excellent physical durability and chemical resistance. Is preferably used. In particular, a polyvinylidene fluoride resin or a resin containing the same as the main component is most preferably used.

  Here, as the polyvinylidene fluoride-based resin, a homopolymer of vinylidene fluoride is preferably used. In addition to a homopolymer of vinylidene fluoride, a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride is used. A polymer is also preferably used. Examples of such vinyl monomers include tetrafluoroethylene, hexafluoropropylene, and trichlorofluoroethylene.

  The porous membrane used in the present invention may be a flat membrane or a hollow fiber membrane.

  When the porous membrane is a flat membrane, its thickness is selected according to the application, but is preferably in the range of 20 μm to 5000 μm, more preferably in the range of 50 μm to 2000 μm. As described above, the porous film may be formed of a porous substrate and a porous resin layer. The thickness of the porous substrate is preferably selected in the range of 50 μm or more and 3000 μm or less.

  When the porous membrane is a hollow fiber membrane, the inner diameter is preferably selected in the range of 200 μm to 5000 μm, and the film thickness is preferably selected in the range of 20 μm to 2000 μm. Moreover, the woven fabric and knitting which made the organic fiber or the inorganic fiber the cylinder shape may be included.

  First, an outline of a method for producing a flat film among the porous films will be described.

  A film of a stock solution containing a resin and a solvent is formed on the surface of the porous base material, and the porous base material is impregnated with the stock solution. Thereafter, only the coating-side surface of the porous substrate having a coating is brought into contact with a coagulation bath containing a non-solvent to solidify the resin, and a porous resin layer is formed on the surface of the porous substrate.

  The stock solution is prepared by dissolving the resin in a solvent. The temperature of the stock solution is usually preferably selected within the range of 5 to 120 ° C. from the viewpoint of film forming properties. The solvent dissolves the resin and acts on the resin to encourage them to form a porous resin layer. Examples of the solvent include N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), N-methyl-2-pyrrolidone, methyl ethyl ketone, tetrahydrofuran, Tetramethylurea, trimethyl phosphate, cyclohexanone, isophorone, γ-butyrolactone, methyl isoamyl ketone, dimethyl phthalate, propylene glycol methyl ether, propylene carbonate, diacetone alcohol, glycerol triacetate, acetone and methyl ethyl ketone can be used. Of these, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO), which have high resin solubility, are preferably used. These solvents may be used alone or in combination of two or more. The stock solution can be prepared by dissolving the above-mentioned resin in the above-mentioned solvent at a concentration of preferably 5 wt% or more and 60 wt% or less.

  Further, for example, components other than the solvent such as polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone and glycerin may be added to the solvent. Non-solvents can also be added to the solvent. The non-solvent is a liquid that does not dissolve the resin. The non-solvent acts to control the pore size by controlling the rate of solidification of the resin. As the non-solvent, water and alcohols such as methanol and ethanol can be used. Of these, water or methanol is preferably used as the non-solvent from the viewpoint of cost. Components other than the solvent and the non-solvent may be a mixture.

  A pore opening agent may be added to the stock solution. The pore-opening agent is extracted when immersed in the coagulation bath, and has a function of making the resin layer porous. By adding a pore opening agent, the average pore size can be controlled. The pore-opening agent is preferably one having high solubility in the coagulation bath. As the pore opening agent, for example, an inorganic salt such as calcium chloride or calcium carbonate can be used. As the pore opening agent, polyoxyalkylenes such as polyethylene glycol and polypropylene glycol, water-soluble polymer compounds such as polyvinyl alcohol, polyvinyl butyral and polyacrylic acid, and glycerin can be used.

  Next, an outline of a method for producing a hollow fiber membrane among the porous membranes will be described.

  The hollow fiber membrane discharges a stock solution composed of a resin and a solvent from the outer tube of the double-tube base, and discharges a hollow portion forming fluid from the inner tube of the double-tube base in a cooling bath. It can be produced by cooling and solidifying.

  The stock solution can be prepared by dissolving the resin described in the above-described method for producing a flat film, preferably in a concentration of 20% by weight or more and 60% by weight or less, in the solvent described in the above-described method for forming a flat film. . Moreover, a gas or a liquid can be normally used for the hollow portion forming fluid. In addition, a new porous resin layer can be coated (laminated) on the outer surface of the obtained hollow fiber membrane. Lamination can be performed in order to change the properties of the hollow fiber membrane, for example, hydrophilicity / hydrophobicity or pore diameter, to desired properties. A new porous resin layer to be laminated can be produced by bringing a stock solution obtained by dissolving a resin in a solvent into contact with a coagulation bath containing a non-solvent to coagulate the resin. As the material of the resin, for example, the same material as that of the organic polymer film is preferably used. As a lamination method, the hollow fiber membrane may be immersed in the stock solution, or the stock solution may be applied to the surface of the hollow fiber membrane. After the lamination, a part of the attached stock solution is scraped off or an air knife is used. It is also possible to adjust the stacking amount by blowing it away.

  The porous membrane used in the present invention can be used as a separation membrane element by combining with a support. A separation membrane element having a porous membrane in which a support plate is used as a support and the porous membrane used in the present invention is disposed on at least one surface of the support plate is one of the preferred forms of the membrane element used in the present invention. It is. In this embodiment, since it is difficult to increase the membrane area, it is also a preferable aspect to dispose a porous membrane on both surfaces of the support plate in order to increase the water permeability.

In the present invention, the transmembrane pressure difference at the time of filtering the microorganisms may be any condition as long as the microorganisms and the medium components are not easily clogged, but the transmembrane pressure difference is in the range of 0.1 kPa to 20 kPa. It is important to. In addition, when the pure water permeability coefficient before use was calculated by measuring the water permeability at a head height of 1 m using purified water at a temperature of 25 ° C. by a reverse osmosis membrane, 2 × 10 ⁻⁹ m ³ / m ² / It is preferably in the range of s / pa or more, and more preferably in the range of 2 × 10 ⁻⁹ m ³ / m ² / s / pa to 6 × 10 ⁻⁷ m ³ / m ² / s / pa. . As a driving force for filtration, it is possible to generate a transmembrane differential pressure in the porous membrane by siphon using the liquid level difference (water head difference) of the culture solution and the porous membrane treated water. As a force, a suction pump may be installed on the treated water side of the porous membrane, or a pressure pump may be installed on the fermentation culture solution side of the porous membrane. The transmembrane pressure can be controlled by changing the liquid level difference between the fermentation broth and the porous membrane treated water, and when using a pump, it can be controlled by the suction pressure. The pressure on the liquid side can be controlled by the pressure of the gas or liquid introduced. When these pressure controls are performed, the pressure difference between the pressure on the fermentation broth side and the pressure on the porous membrane treated water side can be used as the transmembrane pressure difference, which can be used to control the transmembrane pressure difference.

  The fermentation raw material used in the present invention is not particularly limited as long as it promotes the growth of microorganisms to be fermented and cultivated and can satisfactorily produce the protein that is the target fermentation product. As a fermentation raw material, for example, a liquid medium or the like appropriately containing a carbon source, a nitrogen source, inorganic salts, and if necessary, organic micronutrients such as amino acids and vitamins are preferably used.

  Examples of the carbon source include glucose, sucrose, fructose, saccharides such as galactose and lactose, starch saccharified solution containing these saccharides, sweet potato molasses, sugar beet molasses, high test molasses, and organic acids such as acetic acid, ethanol, etc. Alcohols and lysine are also used. Here, sugar is the first oxidation product of polyhydric alcohol, which is a carbohydrate that has one aldehyde group or ketone group, categorized as aldose, saccharide with aldehyde group, and ketose as sugar with ketone group. Point to.

  As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysate, casein decomposition products, other amino acids, Vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used.

  Moreover, as said inorganic salt, phosphate, magnesium salt, calcium salt, iron salt, manganese salt, etc. are used.

  In addition, neutral amino acids, acidic amino acids, and basic amino acids are used as amino acids that are functional compounds of both amino groups and carboxyl groups and that are organic compounds that are structural units of biological proteins. Neutral amino acids include aliphatics such as glycine, alanine, branched amino acids (valine, leucine, isoleucine), hydroxy amino acids (serine, threonine), sulfur-containing amino acids (cysteine, methionine), and acid amide amino acids (asparagine, glutamine). Examples include amino acids, imino acids such as proline, and aromatic amino acids such as phenylalanine, tyrosine and tryptophan. Acidic amino acids include aspartic acid and glutamic acid. As basic amino acids, lysine, arginine and histidine are used.

  When the microorganism used in the present invention requires a specific nutrient for growth, the nutrient can be added as a preparation or a natural product containing it. Moreover, an antifoamer can be used as needed.

  In the present invention, the culture solution refers to a solution obtained as a result of the growth of microorganisms on the fermentation raw material. You may change suitably the composition of the fermentation raw material to add from the fermentation raw material composition at the time of a culture | cultivation start so that productivity of the target protein may become high.

  The microorganism capable of producing a protein used in the present invention is a microorganism originally having or having a gene encoding a protein to be produced. Examples of microorganisms that can be suitably used in the present invention include yeasts such as baker's yeast often used in the fermentation industry, eukaryotic microorganisms such as mold, prokaryotic microorganisms such as Escherichia coli, Bacillus subtilis and coryneform bacteria. be able to. The microorganism used may be isolated from the natural environment, or may be partially modified by mutation or genetic recombination. In the present invention, Escherichia coli is particularly preferably used.

  The gene encoding the protein produced by the method for producing a protein of the present invention is preferably used by being linked under the control of a promoter capable of expressing the protein. There are constitutive promoters and inducible promoters as types of promoters, and both can be used in the present invention. Among them, it is desirable to use a promoter with high expression ability. As a constitutive promoter, Bacillus sp. A promoter localized upstream of the open reading frame (ORF) of the D-amino acid aminotransferase derived from the SK-1 strain, a promoter of the cauliflower mosaic virus (CaMV) 35S RNA gene, a promoter of the actin gene, a rubisco promoter derived from Bacillus subtilis, and ADH1 promoter etc. are mentioned.

  Examples of the inducible promoter include T7 promoter, Tac promoter, PL promoter, PR promoter, PSE promoter, tryptophan promoter, and phosphate promoter. Moreover, it can be used not only as one promoter but also as a double or triple promoter in which two or three promoters are linked, and as a hybrid promoter in which different types of promoters are linked.

  Here, Bacillus sp. A promoter localized upstream of the open reading frame (ORF) of the D-amino acid aminotransferase derived from the SK-1 strain, a promoter of cauliflower mosaic virus (CaMV) 35S RNA gene, a promoter of an actin gene, a rubisco promoter derived from Bacillus subtilis, And the ADH1 promoter is a promoter whose expression is constitutively induced. T7 promoter, Tac promoter, lac promoter, PL promoter, PR promoter and PSE promoter are promoters whose expression is induced by isopropylthio-β-D-galactoside (IPTG). The tryptophan promoter is a promoter whose expression is induced when the concentration of tryptophan in the culture medium is lowered, and expression is also induced when indoleacetic acid (IAA) is added. The phosphate promoter is a promoter whose expression is induced by a decrease in the concentration of inorganic phosphate in the fermentation broth.

  In the method for producing a protein of the present invention, a fermentation broth of a microorganism capable of producing a protein is filtered through a separation membrane, an unfiltered liquid is retained or refluxed in the fermentation broth, and the culture medium of the microorganism is used as the fermentation. Add to the culture and collect the protein from the microbial cells. In the present invention, since the medium added to the fermentation broth can be changed to a composition suitable for protein production, protein production with high productivity is realized. That is, in the method for producing a protein of the present invention, the Bacillus sp. Constitutive promoter located upstream of the open reading frame (ORF) of D-amino acid aminotransferase derived from SK-1 strain, promoter of cauliflower mosaic virus (CaMV) 35S RNA gene, promoter of actin gene, derived from Bacillus subtilis A constitutively expressible promoter such as the Rubisco promoter and the ADH1 promoter can be used. In addition, a promoter whose expression can be controlled depending on the chemical concentration in the culture medium or culture medium, such as the aforementioned tryptophan promoter or phosphate promoter, can also be used.

  In the protein production method of the present invention, when a constitutive promoter is used, the microorganism medium is not particularly limited, and the microorganism in the fermentation broth is grown to a high concentration as the microorganism grows. Thus, highly productive protein can be produced. In the case of T7 promoter, Tac promoter, PL promoter, PR promoter, PSE promoter and lac promoter, IPTG can be added to the fermentation broth by containing IPTG in the culture medium of the microorganism. Manufacturing can be performed.

  In the case of a tryptophan promoter, by reducing the concentration of tryptophan contained in the culture medium of the microorganism, the tryptophan concentration in the fermentation broth can be reduced, so that a highly productive protein can be produced, and the fermentation broth A protein with high productivity can be produced by adding IAA therein. In the case of a phosphate promoter, since the phosphate concentration in the fermentation broth can be lowered by lowering the inorganic phosphate concentration contained in the microorganism medium, a highly productive protein can be produced.

  In the method for producing a protein of the invention, the above-mentioned phosphate promoter can be preferably used. Here, the phosphate promoter will be described. The DNA encoding the upstream region of the phosphate binding protein (PhoS) gene is designed based on the obtained genomic DNA by designing primers with the 5 ′ and 3 ′ end sequences of the upstream region of the phosphate binding protein gene added. It can be obtained by performing PCR. Genomic DNA can be produced, for example, as follows. First, genomic DNA is extracted from Escherichia coli K-12, particularly KLF48 / KL159. As a method for extracting genomic DNA from the above-mentioned Escherichia coli, extraction by an alkaline method (Nucleic Acid Research Vol. 7, No. 6, 1513-1523), a non-phenolic reagent and a protein flocculant is used, and based on phase allocation. Thus, an appropriate method can be selected from among aggregating and distributing methods in which an aggregating layer is formed in the middle depending on the difference in specific gravity (Shinsei Kagaku Kogaku Kenkyu 2 Nucleic acid 1 separation and purification: 13-51 (1991)).

  Phosphate-binding protein is related to phosphate uptake. When phosphate is low, phosphate-binding protein is synthesized to try to take it in, bind to phosphate in the medium, and send it into the cytoplasm have. Transcriptional control of these proteins is a promoter that exists in the upstream region of the genes encoding these proteins and contains a consensus region ctgtcataaattctgtcac called pho box. This region is upstream of the gene encoding the phosphate binding protein and is a DNA sequence of about 200-1000 bp. A microorganism into which a gene expression cassette linked with a gene encoding a desired protein is introduced downstream of the thus obtained phosphate promoter can be used.

  The fermentation broth of microorganisms is preferably reduced to a concentration at which the phosphate promoter is activated. The phosphate promoter expresses a protein at an inorganic phosphate concentration of less than 1 mM. Therefore, the inorganic phosphate concentration of the first fermentation broth may be 1 mM or more. The inorganic phosphate concentration of the first fermentation culture solution may be less than 1 mM, but the growth of the cells tends to be slow. Moreover, when the inorganic phosphoric acid concentration is high, dilution takes time, and there is a possibility that the protein cannot be produced efficiently. Preferably, a culture solution containing inorganic phosphate added at a concentration of 2 to 5 mM is used. In addition, it is preferable to use a medium containing inorganic phosphate at a concentration of 1 mM or less as the medium to be added. Since the phosphoric acid in the fermentation broth is efficiently diluted, it is preferable to use a medium containing 0 to 0.5 mM inorganic phosphoric acid.

  Culture of microorganisms having the ability to produce proteins is usually performed at a pH of 3 to 9 and a temperature of 15 to 70 ° C. The pH of the fermentation broth can be adjusted to a predetermined value within the above range with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, or the like.

  In the culture of microorganisms, if it is necessary to increase the oxygen supply rate, oxygen is added to the air to maintain the oxygen concentration at, for example, 21% or higher, the fermentation broth is pressurized, the stirring rate is increased, or the aeration rate is increased. The following means can be used. Conversely, if it is necessary to reduce the oxygen supply rate, it is also possible to supply a gas containing no oxygen such as carbon dioxide, nitrogen and argon mixed with air.

  Continuous fermentation (pulling out) may be started after the microbial concentration is increased by performing Batch culture or Fed-Batch culture at the beginning of the culture of the present invention. You can go. It is possible to supply the medium and extract the culture from an appropriate time. The start timing of the medium supply and the withdrawal of the culture is not necessarily the same. Further, the supply of the culture medium and the withdrawal of the culture may be continuous or intermittent. What is necessary is just to add a nutrient required for a microbial cell growth as shown above to a culture medium so that a microbial cell growth may be performed continuously.

  The concentration of microorganisms in the fermentation broth must be maintained at a high level in order to obtain efficient productivity, so long as the environment of the fermentation broth is inappropriate for the growth of microorganisms and the rate of death does not increase. preferable. Further, the upper limit value of the microorganism concentration is not particularly limited as long as it does not cause inconvenience in operation of the continuous fermentation apparatus or decrease in production efficiency.

  The continuous fermentation operation performed while growing fresh cells having protein production ability is usually preferably performed in a single fermentation reaction tank in terms of culture management. However, the number of fermentation reaction tanks is not limited as long as it is a continuous fermentation method in which a product is produced while growing cells. A plurality of fermentation reaction tanks may be used because the capacity of the fermentation reaction tank is small. In this case, high productivity can be obtained even if continuous fermentation is performed by connecting a plurality of fermentation reaction tanks in parallel or in series by piping.

  In the present invention, it is possible to continuously and efficiently extract the fermentation broth from the fermentation reaction tank while maintaining the microorganism in the fermentation reaction tank. It is also possible to efficiently produce the target protein by changing the composition of the medium after ensuring the above.

  Examples of proteins that are preferably produced by the protein production method of the present invention include proteins produced by humans and mammals in view of industrial value, such as growth hormones, cytokines, growth factors, and cytoskeletal proteins. . In addition, enzymes, various proteins, and peptides produced by microorganisms, plants, insects, and the like are also included in the proteins suitably produced in the present invention. In addition, mutant proteins obtained by modifying the amino acid sequences of these proteins are also included in the protein produced in the present invention. Furthermore, a chimeric protein and a chimeric peptide in which two or more kinds of proteins or peptides are linked can also be produced by the protein production method of the present invention.

  An outline of protein recovery in the present invention will be described.

  First, recovering protein from cells means extracting a fermentation broth, recovering the cells contained therein, and extracting and purifying the protein by the method described later.

  The fermentation culture solution may be extracted at any timing as long as the cells are contained in the fermentation culture solution. For example, a fermentation broth immediately after inoculation may be used. However, in order to obtain efficient productivity, it is preferable to collect when the environment of the fermentation broth is maintained in a high state within a range where the ratio of dying due to inappropriate growth of microorganisms does not increase. If the fermentation broth is extracted at that time, protein can be obtained efficiently and with high production. In addition, the fermentation broth may be appropriately extracted and recovered at a time when the protein is highly expressed or highly active.

  In the present invention, the protein is recovered from the cultured microbial cells. Specifically, the fermentation broth is extracted from the fermentation reaction tank, and the protein is recovered from the cultured microbial cells.

  As a method for recovering the protein, for example, a method of crushing and purifying bacterial cells by a mechanical method such as homogenizer, ultrasonic wave, glass beads, blender and breath can be employed. Purification can be performed by column chromatography, liquid chromatography, ion exchange chromatography, or the like.

  In addition, as a method for recovering the protein, a chemical method such as a surfactant or a denaturant can be used. In this case, since the protein may be denatured, the protein can be obtained after purification by removing the surfactant and denaturant by dialysis or dilution after solubilization.

  One example of a continuous fermentation apparatus used in the method for producing a protein by continuous fermentation according to the present invention is mainly a fermentation reaction tank for holding a microorganism and producing a protein, and a porosity for filtering and separating the microorganism and the medium. It is composed of a separation membrane element including a membrane. The separation membrane element may be installed either inside or outside the fermentation reaction tank.

  In the method for producing a protein by continuous fermentation according to the present invention, a porous membrane having an average pore diameter of 0.01 μm or more and less than 1 μm is used for the separation membrane, and the transmembrane differential pressure, which is a filtration pressure, is set in the range of 0.1 to 20 kPa. It is characterized by filtering. Therefore, it is not necessary to keep the inside of the fermentation reaction tank in a pressurized state, so that no power means for circulating the fermentation broth between the filtration separation device and the fermentation reaction tank is required, and the separation membrane element is placed inside the fermentation reaction tank. It is possible to install and downsize the fermentation apparatus.

  Of the continuous fermentation apparatus used in the method for producing a protein by continuous fermentation of the present invention, a typical example in which a separation membrane element is installed inside a fermentation reaction tank is shown in the schematic diagram of FIG. FIG. 1 is a schematic side view showing one embodiment of a continuous fermentation apparatus used in the method for producing a protein by continuous fermentation according to the present invention.

  In FIG. 1, the continuous fermentation apparatus basically includes a fermentation reaction tank 1 having a separation membrane element 2 therein and a water head difference control device 3. A porous membrane is incorporated in the separation membrane element 2 in the fermentation reaction tank 1. As this porous membrane, for example, a membrane and a membrane element disclosed in International Publication No. 2002/064240 pamphlet can be used. The separation membrane element will be described in detail later.

  Next, the form of continuous fermentation by the continuous fermentation apparatus of FIG. 1 will be described. In FIG. 1, the medium is continuously or intermittently charged into the fermentation reaction tank 1 by the medium supply pump 7. The medium can be subjected to heat sterilization, heat sterilization, or sterilization using a filter, if necessary, before being introduced into the fermentation reaction tank 1. At the time of culture, the fermentation culture liquid in the fermentation reaction tank 1 is agitated with the agitator 5 as necessary, and the necessary gas is supplied with the gas supply device 4 as necessary, and the pH sensor / control is performed as necessary. It is possible to perform highly productive protein production by adjusting the pH of the fermentation broth with the apparatus 9 and the pH adjusting solution supply pump 8, and adjusting the temperature of the fermentation broth with the temperature controller 10 as necessary. it can. Here, the pH and temperature are exemplified for the adjustment of the physicochemical conditions of the fermentation broth by the instrumentation / control device. However, if necessary, the dissolved oxygen and ORP can be controlled, and an analytical device such as an online chemical sensor can be used. The concentration of various substances in the culture solution can be measured, and physicochemical conditions can be controlled using the measured concentrations. In addition, regarding the form of continuous or intermittent addition of the medium, for example, the input amount and speed of the medium can be appropriately adjusted using the measured value of the physicochemical environment of the culture solution by the instrumentation device as an index.

  The fermentation broth is filtered and separated into a fermentation broth and a filtrate by a separation membrane element 2 installed in the fermentation reaction tank 1. The fermentation broth contains microbial cells. Moreover, the microbial cell is not contained in the filtrate filtered and separated by the separation membrane. By using the separation membrane element, wastes such as acetic acid generated during cell growth can be taken out from the device system as a filtrate while maintaining the cells in the device system.

  In addition, the filtered and separated microorganisms remain in the apparatus system, so that the microorganism concentration in the apparatus system can be maintained high, and protein production with high productivity is possible. Here, the filtration / separation by the separation membrane element 2 is performed by the water head differential pressure with the water surface of the fermentation reaction tank 1, and no special power is required. Moreover, the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation broth in the fermentation reaction tank can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3 as necessary. Although the filtration / separation by the separation membrane element 2 is exemplified by the water head differential pressure, the filtration / separation can be performed by suction filtration using a pump or the like or pressurizing the inside of the apparatus system as necessary.

  The time for collecting the protein from the cells may be basically any time. In order to obtain efficient productivity, it is preferable to collect when the environment of the fermentation broth is maintained in a high state within a range where the ratio of dying due to inappropriate growth of microorganisms does not increase. In addition, it is preferable to appropriately extract the fermentation broth and collect it at a time when the protein is highly expressed or highly active.

  For protein recovery, the fermentation broth can be withdrawn from the fermentation reaction tank, and the protein can be recovered from the microbial cells contained in the fermentation broth by the means described above.

  Next, among the continuous fermentation apparatuses used in the method for producing a protein by continuous fermentation of the present invention, a typical example in which the separation membrane element is installed outside the fermentation reaction tank is shown in the schematic diagram of FIG. FIG. 2 is a schematic side view for explaining one embodiment of another continuous fermentation apparatus used in the present invention.

  In FIG. 2, the continuous fermentation apparatus basically includes a fermentation reaction tank 1, a membrane separation tank 12 having a separation membrane element 2 therein, and a water head difference control device 3. A porous membrane is incorporated in the separation membrane element 2 in the membrane separation tank 12. As the porous membrane, for example, it is preferable to use a separation membrane and a separation membrane element disclosed in International Publication No. 2002/064240 pamphlet. The membrane separation tank 12 is connected to the fermentation reaction tank 1 via a fermentation culture medium circulation pump 11.

  In FIG. 2, the medium is fed into the fermentation reaction tank 1 by the medium supply pump 7, the fermentation culture solution in the fermentation reaction tank 1 is stirred by the stirrer 5 as necessary, and the gas supply device 4 as needed. , And the pH of the fermentation broth is adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8 if necessary, and by the temperature controller 10 if necessary. By adjusting the temperature of the fermentation broth, fermentative production with high productivity can be performed. Further, the fermentation broth in the apparatus is circulated between the fermentation reaction tank 1 and the membrane separation tank 12 by the fermentation broth circulation pump 11. The fermentation broth is filtered and separated by the separation membrane element 2 into a fermentation broth containing microorganisms and a filtrate not containing microorganisms, and the filtrate is removed from the apparatus system.

  Moreover, the microorganisms filtered and separated remain in the apparatus system, so that the microorganism concentration in the apparatus system can be maintained high, and fermentation production with high productivity is possible. Here, the filtration / separation by the separation membrane element 2 is performed by the water head differential pressure with the water surface of the membrane separation tank 12, and no special power is required. If necessary, the filtration / separation speed of the separation membrane element 2 and the amount of fermentation broth in the apparatus system can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3. Moreover, the gas required by the gas supply apparatus 4 can be supplied in the membrane separation tank 12 as needed. As described above, the filtration / separation by the separation membrane element 2 is exemplified by the water head differential pressure. However, if necessary, the filtration / separation may be performed by suction filtration using a pump or the like, or pressurizing the inside of the apparatus system. You can also.

  For protein recovery, the fermentation broth is appropriately extracted and recovered at a time when the protein is highly expressed or active. A fermentation broth with high protein expression or activity is extracted and only the cells are recovered. For cell collection, it is possible to collect the cells by centrifugation using, for example, a centrifuge. Next, the collected cells are crushed. The crushing method can be performed using a mechanical method such as a homogenizer or an ultrasonic wave. The disrupted cells can be suspended in an appropriate buffer such as a phosphate buffer, and the target protein can be purified and recovered using column chromatography, liquid chromatography, ion exchange chromatography, or the like.

  Next, the separation membrane element used in the present invention will be described.

  The outline of the separation membrane and the separation membrane element disclosed in International Publication No. 2002/064240, which is an example of a preferred embodiment of the separation membrane element used in the present invention, will be described below with reference to the drawings. FIG. 3 is a schematic perspective view for explaining one embodiment of the separation membrane element used in the present invention.

  As shown in FIG. 3, the separation membrane element is configured by arranging a flow path material 14 and the separation membrane 15 (porous membrane) in this order on both surfaces of a rigid support plate 13. The support plate 13 has recesses 16 on both sides. The separation membrane 15 filters the fermentation broth. The flow path member 14 is for efficiently flowing the filtrate filtered through the separation membrane 15 to the support plate 13. The filtrate that has flowed to the support plate 13 is taken out through the recess 16 of the support plate 13. The outside is taken out of the continuous fermentation apparatus through the water collecting pipe 17.

  The separation membrane element shown in FIG. 4 will be described. FIG. 4 is a schematic perspective view for illustrating another separation membrane element used in the present invention. As shown in FIG. 4, the separation membrane element is mainly composed of a separation membrane bundle 18 composed of a hollow fiber membrane (porous membrane), an upper resin sealing layer 19, and a lower resin sealing layer 20. The separation membrane bundle 18 is bonded and fixed in a bundle by an upper resin sealing layer 19 and a lower resin sealing layer 20. Adhesion / fixation by the lower resin sealing layer 20 has a structure in which the hollow portion of the hollow fiber membrane (porous membrane) of the separation membrane bundle 18 is sealed to prevent leakage of the fermentation broth. On the other hand, the upper resin sealing layer 19 does not seal the inner hole of the hollow fiber membrane (porous membrane) of the separation membrane bundle 18 and has a structure in which permeate flows through the water collecting pipe 22. This separation membrane element can be installed in the continuous fermentation apparatus via the support frame 21. The filtrate filtered by the separation membrane bundle 18 passes through the hollow portion of the hollow fiber membrane and is taken out to the outside of the continuous fermentation apparatus via the water collecting pipe 22. As the power for taking out the filtrate, a method such as a differential pressure at the water head, a pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used.

  The member constituting the separation membrane element of the continuous fermentation apparatus used in the method for producing a protein by continuous fermentation of the present invention is preferably a member resistant to high-pressure steam sterilization operation. If the inside of the continuous fermentation apparatus can be sterilized, it is possible to avoid the risk of contamination by undesirable microorganisms during continuous fermentation, and more stable continuous fermentation is possible. The members constituting the separation membrane element are preferably resistant to 15 minutes at 121 ° C., which is the condition of the high-pressure steam sterilization operation.

  Examples of the separation membrane element member include metals such as stainless steel and aluminum, polyamide resins, fluorine resins, polycarbonate resins, polyacetal resins, polybutylene terephthalate resins, PVDF, modified polyphenylene ether resins, and polysulfone resins. These resins can be preferably selected.

  In the continuous fermentation apparatus used in the protein production method by continuous fermentation of the present invention, the separation membrane element may be installed in the fermentation reaction tank as shown in FIG. 1 or outside the fermentation reaction tank as shown in FIG. May be installed. When the separation membrane element is installed outside the fermentation reaction tank, a separate membrane separation tank can be provided and the separation membrane element can be installed inside it, and the culture fluid is circulated between the fermentation reaction tank and the membrane separation tank. The culture solution can be continuously filtered by the separation membrane element.

  In the continuous fermentation apparatus used in the method for producing a protein by continuous fermentation according to the present invention, the membrane separation tank is preferably capable of high-pressure steam sterilization. If the membrane separation tank can be autoclaved, it is easy to avoid contamination with germs.

When continuous fermentation is performed according to the method for producing a protein by continuous fermentation of the present invention, a higher bacterial cell concentration is obtained compared to conventional batch culture, and extremely efficient protein production becomes possible. Here, the production rate in continuous culture is calculated by the following (Equation 3).
Fermentation production rate (g / L / hr) = Product concentration in extraction liquid (g / L) × Fermentation culture liquid extraction speed (L / hr) ÷ Operation liquid volume of apparatus (L) (Equation 3 )
In addition, the fermentation production rate in batch culture is calculated by dividing the amount of product (g) at the time when all of the raw carbon source is consumed by the time (h) required for consumption of the carbon source and the amount of culture solution (L) at that time. Is required.

  Hereinafter, in order to describe the present invention in more detail, canine interferon γ is selected as a protein, Escherichia coli is selected as a microorganism capable of producing protein, and protein using the continuous fermentation apparatus shown in the schematic diagrams of FIGS. 1 and 2 is used. Manufactured. The present invention is not limited to the description of these examples. Here, Escherichia coli MC1061 strain (manufactured by ATCC) was used as Escherichia coli. An E. coli strain capable of producing recombinant canine interferon γ was constructed and introduced by introducing a gene encoding canine interferon γ into E. coli strain MC1061. Specifically, a transformed Escherichia coli having the ability to produce recombinant canine interferon γ by introducing a plasmid in which a gene encoding canine interferon γ is linked downstream of a phosphate binding protein gene derived from Escherichia coli K-12. A stock was created and used.

(Reference Example 1) Construction of vector containing upstream region gene of phosphate binding protein gene (phoS) (1) Preparation of genomic DNA of E. coli K-12 KLF / KL159 (Yale University E. E. coli. GeneticResourceCenter CGSC Strain # 4302) is cultured in T medium (bactotryptone 10 g / L, NaCl 5 g / L) for one day, and then precipitated by centrifugation. Using nucleic acid extractant “Sepagene” (manufactured by Sanko Junyaku Co., Ltd.) Genomic DNA was recovered. That is, Reagent 1 composed of Tris buffer was added to the precipitate, and after stirring, allowed to stand at room temperature for 10 minutes. Next, after adding reagent 2 composed of guanidine thiocyanate and mixing gently, reagent 3 composed of adsorbent and chloroform was added and mixed vigorously. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was recovered, reagent 4 composed of acetate buffer was added, and isopropanol was further added and mixed by inversion. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was removed and the precipitate was dissolved in sterile distilled water.

(2) Acquisition of Phosphate Binding Protein PhoS Upstream Sequence Gene Based on E. coli K12 strain genomic sequence information (MEDLINE), two types of primers of SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized with a DNA synthesizer. 2 μL of the genomic DNA obtained in (1) above was added to a 0.5 mL microcentrifuge tube, and each primer was added at 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl ₂ , 25 mM KCL, 100 μg / Reagents were added to make mL gelatin, 50 μM each dNTP, 4 units LA-Taq DNA polymerase to make a total volume of 100 μL. 35 cycles using a DNA thermal cycler from Perkin-Elmer Cetus under conditions of denaturation of DNA at 94 ° C for 1 minute, primer annealing at 55 ° C for 1 minute, and primer extension at 72 ° C for 1 minute. Reacted. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 300 bp was prepared according to a conventional method. Further, two kinds of primers of SEQ ID NO: 3 and SEQ ID NO: 4 were synthesized with a DNA synthesizer. 2 μL of the DNA fragment prepared from the agarose gel was added to a 0.5 mL microcentrifuge tube, and each primer was 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl2, 25 mM KCL, 100 μg / mL gelatin, Each reagent was added so as to be 50 μM each dNTP, 4 units LA-Taq DNA polymerase to make a total volume of 100 μL. 35 cycles using a DNA thermal cycler from Perkin-Elmer Cetus under conditions of denaturation of DNA at 94 ° C for 1 minute, primer annealing at 55 ° C for 1 minute, and primer extension at 72 ° C for 1 minute. Reacted. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 300 bp was prepared according to a conventional method.

  The DNA fragment thus obtained was transferred to T-Vector of Invitrogen by Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 1 was used for ligation. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, after confirming that the PCR fragment has been inserted into this plasmid by PCR under the same conditions as described above, a fluorescent DNA sequencer was used and a Perkin Elmer dye terminator cycle sequencing kit was used according to the attached protocol. The DNA fragment was confirmed to be a sequence encoding a gene derived from Escherichia coli K12 (SEQ ID NO: 5).

(3) Construction of vector added with upstream sequence of phosphate binding protein gene (phoS) and SD sequence Phosphate binding protein phoS was prepared using the vector containing upstream sequence gene of phosphate binding protein phoS prepared in (2) above. An SD sequence was added to the 3 'end of the upstream sequence gene. That is, SEQ ID NO: 3 as a primer on the sense side and a primer SEQ ID NO: 6 having an antisense side, that is, an SD sequence were prepared. Next, PCR using the combination of primers of SEQ ID NO: 3 and SEQ ID NO: 6 was performed. That is, 2 μL of the vector DNA was added to a 0.5 mL microcentrifuge tube, and each primer was 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl ₂ , 25 mM KCL, 100 μg / mL gelatin, 50 μM each. Each reagent was added so as to be dNTP, 4 units LA-Taq DNA polymerase to make a total volume of 100 μL. 35 cycles using a DNA thermal cycler from Perkin-Elmer Cetus under conditions of denaturation of DNA at 94 ° C for 1 minute, primer annealing at 55 ° C for 1 minute, and primer extension at 72 ° C for 1 minute. Reacted. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 300 bp was prepared according to a conventional method.

This DNA fragment was transferred to T-Vector of Invitrogen by Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 1 was used for ligation. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, after confirming that the PCR fragment has been inserted into this plasmid by PCR under the same conditions as described above, a fluorescent DNA sequencer was used and a Perkin Elmer dye terminator cycle sequencing kit was used according to the attached protocol. The DNA fragment was confirmed to be a sequence encoding a gene derived from E. coli K12. (SEQ ID NO: 7)
The vector containing the upstream sequence gene of the phosphate binding protein phoS prepared in (2) above and pBR322 from Takara Bio Inc. are each subjected to restriction enzyme treatment with EcoRI, each is subjected to agarose gel electrophoresis, and the gene is obtained according to a conventional method. Cut out. For pBR322, after dephosphorylation treatment with Alkaline Phospatase Shrimp (manufactured by Nippon Roche), upstream sequence gene of phosphate binding protein phoS and DNA Ligation Kit Ver. of Takara Shuzo Co., Ltd. 1 was used for ligation. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, after confirming that the PCR fragment has been inserted into this plasmid by PCR under the same conditions as described above, a fluorescent DNA sequencer (DNA sequencer 373S manufactured by PerkinElmer) was used, and according to the attached protocol, Using a terminator cycle sequencing kit, it was confirmed that the obtained DNA fragment was a sequence encoding the upstream sequence gene of the phosphate binding protein phoS and the newly added SD sequence. Regarding the name of the vector, SEQ ID NO: 7 is designated as pPho2.

Reference Example 2 Acquisition of Canine IFN-γ Gene (1) Preparation of Canine cDNA Canine lymphocytes were isolated from canine peripheral blood and stimulated with phytohemaglutinin (PHA) at a final concentration of 50 ug / mL for 48 hours. Thereafter, total RNA was prepared using “ISOGEN” (registered trademark) (manufactured by Nippon Gene). The obtained RNA was dissolved in 10 mM Tris-HCl buffer (pH 7.5) (hereinafter abbreviated as TE) containing 1 mM EDTA, treated at 70 ° C. for 5 minutes, and then equilibrated with TE containing 1 M LiCl. The RNA solution was applied to an oligo dT cellulose column and washed with the same buffer. Further, after washing with TE containing 0.3 M LiCl, the poly (A) RNA adsorbed with 1 mM EDTA (pH 7.0) containing 0.01% SDS was eluted. Single-stranded cDNA was synthesized using the poly (A) RNA thus obtained. Specifically, 5 μg of poly (A) RNA and 0.5 μg of oligo dT primer (12-18mer) were placed in a sterilized 0.5 mL microcentrifuge tube, and sterilized water treated with diethyl pyrocarbonate was added to 12 μL. After incubating at a temperature of 10 minutes, it was placed in ice for 1 minute. To this was added 2 μL of 200 mM MgCl ₂ and 1 μL of 10 mM dNTP and 2 μL of 0.1 M DTT, respectively, and incubated at 42 ° C. for 5 minutes. Then, 1 μL of 200 units of Super Script II RT manufactured by GibcoBRL was added, and the temperature was 42 ° C. The cDNA synthesis reaction was carried out by further incubation for 50 minutes. Furthermore, the reaction was stopped by incubating at a temperature of 70 ° C. for 15 minutes, and placed on ice for 5 minutes. In this reaction solution, 1 μL of E. coli was added. E. coli RNase H (2 units / mL) was added and incubated at a temperature of 37 ° C. for 20 minutes.

(2) Acquisition of canine IFN-γ gene Based on the nucleotide sequences of N-terminal and C-terminal of canine IFN-γ, two types of primers of SEQ ID NO: 8 and SEQ ID NO: 9 were synthesized with a DNA synthesizer. 2 μL of cDNA obtained from the canine lymphocytes of (1) above was added to a 0.5 mL microcentrifuge tube, and each primer was added at 20 pmol, 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl2, 25 mM KCL, 100 μg. Each reagent was added so as to be / mL gelatin, 50 μM each dNTP, 4 units Taq DNA polymerase to make a total volume of 100 μL. 35 cycles using a DNA thermal cycler from Perkin-Elmer Cetus under conditions of DNA denaturation at 94 ° C for 1 minute, primer annealing at 55 ° C for 2 minutes, primer extension at 72 ° C for 3 minutes Reacted. This was electrophoresed on a 1% agarose gel, and a DNA fragment of about 500 bp was prepared according to a conventional method.

  The DNA fragment thus obtained was transferred to T-Vector of Invitrogen by Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 1 was used for ligation. Using this, Escherichia coli was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, after confirming that the PCR fragment has been inserted into this plasmid by PCR under the same conditions as described above, use a fluorescent DNA sequencer and a PerkinElmer dye terminator cycle sequencing kit according to the attached protocol. It was confirmed that the obtained DNA fragment was a sequence encoding canine IFN-γ.

(3) Acquisition of canine IFN-γ mutant gene The canine IFN-γ mutant gene is obtained by deleting the C-terminal of the canine IFN-γ protein by 16 residues. Based on the terminal base sequence, two kinds of primers of SEQ ID NO: 10 and SEQ ID NO: 11 were synthesized with a DNA synthesizer. Using the cDNA obtained from canine lymphocytes of (1) above as a template, a DNA fragment of about 500 bp is obtained in the same manner as in (2) above, inserted into T-Vector, and a sequence encoding canine IFN-γ. Was confirmed (SEQ ID NO: 12).

(Reference Example 3) Construction of a vector containing the upstream region gene of a phosphate binding protein gene (phoS) and a canine IFN-γ gene The pPho2 vector and the canine IFN-γ mutant gene prepared in Reference Example 1 and Reference Example 2, respectively, Restriction enzyme treatment with NdeI and XhoI was performed, each was subjected to agarose gel electrophoresis, and the gene was excised according to a conventional method. The gene was transferred to Takara Shuzo Co., Ltd. DNA Ligation Kit Ver. 1 was used for ligation. Using this, Escherichia coli JM109 was transformed according to a conventional method, and plasmid DNA was prepared from the obtained transformant by a conventional method. Next, E. coli MC1061 (manufactured by ATCC) was transformed with this plasmid DNA according to a conventional method.

Reference Example 4 Production of porous membrane (Part 1)
Polyvinylidene fluoride (PVDF) resin was used as the resin and N, N-dimethylacetamide (DMAc) was used as the solvent, and these were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
[Undiluted solution]
・ PVDF: 13.0% by weight
DMAc: 87.0% by weight
Next, after cooling the above stock solution to a temperature of 25 ° C., a polyester fiber non-woven fabric (porous substrate) having a density of 0.48 g / cm ³ and a thickness of 220 μm, which was previously pasted on a glass plate. And immediately immersed in a coagulation bath having a temperature of 25 ° C. having the following composition for 5 minutes to obtain a porous film having a porous resin layer formed on a porous substrate.
[Coagulation bath]
-Water: 30.0% by weight
DMAc: 70.0% by weight
After peeling this porous membrane from the glass plate, it was immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc to obtain a separation membrane. When the surface of the porous resin layer was observed with a scanning electron microscope at a magnification of 10,000 within the range of 9.2 μm × 10.4 μm, the average diameter of all observable pores was 0.1 μm. . Next, when the pure water permeation coefficient of the separation membrane was evaluated, it was 50 × 10 ⁻⁹ m ³ / m ² / s / Pa. The pure water permeation amount was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.035 μm, and the membrane surface roughness was 0.06 μm.

Reference Example 5 Production of porous membrane (part 2)
A vinylidene fluoride homopolymer having a weight average molecular weight of 41,000 and γ-butyrolactone were dissolved at a temperature of 170 ° C. at a rate of 38% by weight and 62% by weight, respectively, to prepare a stock solution. This stock solution was discharged from the base while stirring γ-butyrolactone as a hollow portion forming liquid, and solidified in a cooling bath composed of an 80% by weight aqueous solution of γ-butyrolactone at a temperature of 20 ° C. to produce a hollow fiber membrane.

Next, 14% by weight of vinylidene fluoride homopolymer having a weight average molecular weight of 284,000, 1% by weight of cellulose acetate propionate (Eastman Chemical Co., CAP482-0.5), and N-methyl-2-pyrrolidone 77% by weight, polyoxyethylene coconut oil fatty acid sorbitan (manufactured by Sanyo Chemical Co., Ltd., trade name “IONET T-20C” (registered trademark)) 5% by weight, and water 3% by weight at a temperature of 95 ° C. The stock solution was prepared by mixing and dissolving. This undiluted solution was uniformly applied to the surface of the hollow fiber membrane obtained above, and a hollow fiber membrane (porous membrane) used in the present invention was immediately solidified in a water bath. The average pore diameter of the treated water side surface of the obtained hollow fiber membrane (separation membrane) was 0.05 μm. Next, the pure water permeation rate of the hollow fiber membrane as the separation membrane was evaluated and found to be 5.5 × 10 ⁻⁹ m ³ / m ² · s · Pa. The amount of water permeation was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.006 μm.

(Comparative Example 1)
The most typical batch culture as a culture form using microorganisms was performed using a 1.5 L jar fermenter, and the protein productivity was evaluated. The medium was autoclaved at 121 ° C. for 20 minutes and used. In Comparative Example 1, Escherichia coli MC1061-canine interferon γ strain was used as a microorganism, the evaluation of the product recombinant canine interferon γ was evaluated using a Western blotting method, and the glucose concentration was measured using “Glucose Test Wako C”. “(Registered trademark)” (manufactured by Wako Pure Chemical Industries, Ltd.) and “Phospha C-Test Wako” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) were used for the phosphoric acid concentration measurement. The operating conditions of Comparative Example 1 are as follows.
-Fermentation reaction tank capacity (initial medium amount): 1.0 (L)
・ Temperature adjustment: 37 (℃)
-Fermentation reactor aeration rate: 1.5 (L / min) air-Fermentation reactor agitation speed: 800 (rpm)
PH adjustment: pH 7.0 adjusted with 2N H ₂ SO ₄ and 14N aqueous ammonia.

  First, E. coli MC1061-canine interferon γ strain was cultured in a test tube with shaking overnight in a medium having the composition shown in Table 1 and tetracycline (10 μg / ml) added (pre-culture). The obtained culture broth was inoculated into 50 ml of an initial medium supplemented with fresh tetracycline (10 μg / ml), and cultured in a 500 ml Sakaguchi flask for 24 hours at a temperature of 37 ° C. with an amplitude of 30 cm and 180 rpm. (Pre-culture). The preculture was inoculated into a 1.0 L starting medium of a jar fermenter. Batch culture was performed using the initial medium. As a result of the culture, the growth stopped at the cell concentration OD600 = 7, which was 1/4 or less compared to the result of Example 1 described later. In addition, the bacterial cell concentration OD600 = 10 in each fraction of the appropriately sampled culture solution was unified, and the bacterial cell solution was used as a sample, using Konica Immunostain HRP (manufactured by Konica), and in accordance with the attached protocol, dog interferon γ Was detected. The signal intensity was measured using a molecular imager (manufactured by BioRad). In addition, non-transformed E. coli was used as a negative control sample, and Interdock (Toray Industries, Inc .: dog interferon γ) was used as a positive control sample. As a result, the expression of canine interferon γ was not confirmed.

Example 1 Production of Protein (Part 1)
In order to investigate whether or not a continuous fermentation production system can be obtained by operating the continuous fermentation apparatus of FIG. 1, a continuous fermentation test of this apparatus was conducted using the composition medium shown in Table 1 and the additional medium having the composition shown in Table 2. went. The medium was autoclaved at 121 ° C. for 20 minutes, and the additional medium was autoclaved at 121 ° C. for 60 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) produced in Reference Example 4 was used. The operating conditions in Example 1 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: Polyvinylidene fluoride filtration membrane-Separation membrane element Effective filtration area: 120 square cm
・ Temperature adjustment: 37 (℃)
-Fermentation reactor aeration rate: 1.5 (L / min) air-Fermentation reactor agitation speed: 800 (rpm)
PH adjustment: pH 7.0 adjusted with 2N H ₂ SO ₄ and 14N aqueous ammonia.
Sterilization: Fermentation reaction tank containing separation membrane element and medium are autoclaved at 121 ° C. for 20 minutes under high pressure steam sterilization. The additional medium is autoclaved at 121 ° C for 60 min in an autoclave.
-Membrane permeate flow rate control: The flow rate was controlled by the fermentation reaction tank water head difference (the water head difference was controlled within 2 m).

  An Escherichia coli MC1061-canine interferon γ strain was used as the microorganism, a medium having the composition shown in Table 1 was used, and a medium having the composition shown in Table 2 was used as the additional medium. In addition, “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is used for measuring glucose concentration, and “phospha C-test Wako” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is used for measuring phosphoric acid concentration. Was used.

First, the Escherichia coli MC1061-canine interferon γ strain was cultured overnight in a test tube with shaking in a medium having the composition shown in Table 1 and tetracycline (10 μg / ml) added overnight (pre-culture). The obtained culture broth was inoculated into 50 ml of a medium having the composition shown in Table 1 to which fresh tetracycline (10 μg / ml) was added, and was cultured in a 500 ml Sakaguchi flask for 24 hours at a temperature of 37 ° C. with an amplitude of 30 cm. Culturing was performed under the condition of 180 rpm (pre-culture).
The culture broth was inoculated in the continuous fermentation apparatus shown in FIG. 1 in 1.5 L of the medium having the composition shown in Table 1, and the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5. The culture was carried out by adjusting the aeration amount, temperature adjustment and pH adjustment of No. 1. Immediately after completion of the pre-culture, the additional medium having the composition shown in Table 2 is continuously supplied, and the continuous culture is performed while controlling the amount of permeated water so that the amount of the fermentation broth in the continuous fermentation apparatus is 1.5 L. The protein was produced by continuous fermentation while decreasing the acid concentration. Control of the amount of permeated water through the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m at maximum, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 3. went. As appropriate, the phosphoric acid concentration and residual glucose concentration in the membrane permeate filtrate were measured. As a result of the culture, the cells grew to a microbial cell concentration of OD600 = 30 or more, and high concentration culture became possible.

Moreover, the fermentation broth was sampled as appropriate, and it was confirmed by Western blotting whether the protein was expressed when the phosphate concentration decreased. As a method, the cell concentration of each fraction was unified at OD600 = 10, and the cell solution was used as a sample, and canine interferon γ was detected using Konica Immunostain HRP (manufactured by Konica) according to the attached protocol. That is, SDS-PAGE electrophoresis was performed, and the blotted membrane was blocked in a blocking solution (5% skim milk / 0.1% Tween 20 / PBS) at 4 ° C. overnight. The membrane was washed twice with TPBS (0.1% Tween20 · PBS) and treated with an anti-canine interferon γ antibody diluted 1000 times with TPBS for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed 3 times with TPBS for 5 minutes. Thereafter, the resultant was diluted 10,000 times with TPBS and then treated with an HRP-labeled anti-rabbit IgG antibody (manufactured by Cosmo Bio) for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed 3 times with TPBS for 5 minutes, and then a detection reagent (solution A + solution B + solution C + solution D) for Konica Immunostain HRP (manufactured by Konica) was added to cause color development. As a result, the signal intensity was measured using a molecular imager (BioRad). In addition, non-transformed E. coli was used as a negative control sample, and Interdock (Toray Industries, Inc .: dog interferon γ) was used as a positive control sample.
As a result, it was confirmed that canine interferon γ was expressed in a large amount at a phosphate concentration of 1 mM or less. Moreover, it confirmed that it produced 5-10 times compared with the comparative example 1 shown above. By using the continuous fermentation apparatus of FIG. 1, it was confirmed that protein production by continuous fermentation was possible by reducing the concentration of the components in the medium while maintaining the cell growth.

(Example 2) Production of protein (part 2)
In order to investigate whether or not a continuous fermentation production system can be obtained by operating the continuous fermentation apparatus of FIG. 2, the continuous culture test of this apparatus was conducted using the composition medium shown in Table 1 and the additional medium having the composition shown in Table 2. went. The medium was autoclaved at 121 ° C. for 20 minutes, and the additional medium was autoclaved at 121 ° C. for 60 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) produced in Reference Example 4 was used. The operating conditions in Example 2 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
・ Membrane separation tank capacity: 0.5 (L)
・ Separation membrane: Polyvinylidene fluoride filtration membrane ・ Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 37 (℃)
-Fermentation reactor aeration rate: 1.5 (L / min) air-Fermentation reactor agitation speed: 800 (rpm)
-Aeration rate of membrane separation tank: 0.3 (L / min)
- pH Adjustment: _2N H 2 SO ₄ and the circulating liquid amount by the adjustment and the culture solution circulation apparatus pH7.0 by 14N aqueous ammonia: 0.1 (L / min)
-Membrane permeate flow rate control: The flow rate was controlled by the head difference of the fermentation reaction tank (the head difference was controlled within 2 m).

  Escherichia coli MC1061-canine interferon γ strain was used as the microorganism, a medium having the composition shown in Table 1 was used, and a medium having the composition shown in Table 2 was used as the additional medium. In addition, “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is used for measuring glucose concentration, and “phospha C-test Wako” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is used for measuring phosphoric acid concentration. Was used.

First, the Escherichia coli MC1061-canine interferon γ strain was cultured overnight in a test tube with shaking in a medium having the composition shown in Table 1 and tetracycline (10 μg / ml) added overnight (pre-culture). The obtained culture broth was inoculated into 50 ml of a medium having the composition shown in Table 1 to which fresh tetracycline (10 μg / ml) was added, and was cultured in a 500 ml Sakaguchi flask for 24 hours at a temperature of 37 ° C. with an amplitude of 30 cm. Culturing was performed under the condition of 180 rpm (pre-culture).
The culture medium is inoculated in advance in the continuous fermentation apparatus shown in FIG. 2 in 2 L of the medium having the composition shown in Table 1, and the fermentation reaction tank 1 is stirred at 800 rpm by the attached stirrer 5. The culture was performed by adjusting the aeration amount, adjusting the temperature, and adjusting the pH. Immediately after the completion of the pre-culture, the additional medium having the composition shown in Table 2 is continuously supplied, and the continuous culture is performed while controlling the amount of permeated water so that the amount of the fermentation broth in the continuous fermentation apparatus becomes 2 L. The protein was produced by continuous fermentation while decreasing the concentration. The control of the amount of permeated water when performing the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 3. went. As appropriate, the phosphoric acid concentration and residual glucose concentration in the membrane permeate filtrate were measured. As a result of the culture, the cells grew to a microbial cell concentration of OD600 = 30 or more, and high concentration culture became possible.

Moreover, the fermentation broth was sampled as appropriate, and it was confirmed by Western blotting whether the protein was expressed when the phosphate concentration decreased. As a method, the cell concentration of each fraction was unified at OD600 = 10, and the cell solution was used as a sample, and canine interferon γ was detected using Konica Immunostain HRP (manufactured by Konica) according to the attached protocol. That is, SDS-PAGE electrophoresis was performed, and the blotted membrane was blocked in a blocking solution (5% skim milk / 0.1% Tween 20 / PBS) at 4 ° C. overnight. The membrane was washed twice with TPBS (0.1% Tween20 · PBS) and treated with an anti-canine interferon γ antibody diluted 1000 times with TPBS for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed 3 times with TPBS for 5 minutes. Thereafter, the resultant was diluted 10,000 times with TPBS and then treated with an HRP-labeled anti-rabbit IgG antibody (manufactured by Cosmo Bio) for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed 3 times with TPBS for 5 minutes, and then a detection reagent (solution A + solution B + solution C + solution D) for Konica Immunostain HRP (manufactured by Konica) was added to cause color development. As a result, the signal intensity was measured using a molecular imager (BioRad). In addition, non-transformed E. coli was used as a negative control sample, and Interdock (Toray Industries, Inc .: dog interferon γ) was used as a positive control sample.
As a result, it was confirmed that canine interferon γ was expressed in a large amount at a phosphate concentration of 1 mM or less. Moreover, it confirmed that it produced 5-10 times compared with the comparative example 1 shown above. By using the continuous fermentation apparatus shown in FIG. 2, it was confirmed that protein production by continuous fermentation was possible by reducing the concentration of the components in the medium while maintaining cell growth.

(Example 3) Production of protein (part 3)
In order to investigate whether or not a continuous fermentation production system can be obtained by operating the continuous fermentation apparatus of FIG. 1, a continuous fermentation test of this apparatus was conducted using the composition medium shown in Table 1 and the additional medium having the composition shown in Table 2. went. The medium was autoclaved at 121 ° C. for 20 minutes, and the additional medium was autoclaved at 121 ° C. for 60 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) produced in Reference Example 5 was used. The operating conditions in Example 3 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: Polyvinylidene fluoride filtration membrane-Separation membrane element Effective filtration area: 120 square cm
・ Temperature adjustment: 37 (℃)
-Fermentation reactor aeration rate: 1.5 (L / min) air-Fermentation reactor agitation speed: 800 (rpm)
- pH Adjustment: adjusted and sterilized pH7.0 by _2N H 2 SO ₄ and 14N aqueous ammonia fermentation reaction tank containing the separation membrane elements, and media 121 ° C., high-pressure steam sterilization by autoclaving at 20min. The additional medium is autoclaved at 121 ° C for 60 min in an autoclave.
-Membrane permeate flow rate control: The flow rate was controlled by the fermentation reaction tank water head difference (the water head difference was controlled within 2 m).

  Escherichia coli MC1061-canine interferon γ strain was used as the microorganism, a medium having the composition shown in Table 1 was used, and a medium having the composition shown in Table 2 was used as the additional medium. In addition, “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is used for measuring glucose concentration, and “phospha C-test Wako” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is used for measuring phosphoric acid concentration. Was used.

First, the Escherichia coli MC1061-canine interferon γ strain was cultured overnight in a test tube with shaking in a medium having the composition shown in Table 1 and tetracycline (10 μg / ml) added overnight (pre-culture). The obtained culture broth was inoculated into 50 ml of a medium having the composition shown in Table 1 to which fresh tetracycline (10 μg / ml) was added, and was cultured in a 500 ml Sakaguchi flask for 24 hours at a temperature of 37 ° C. with an amplitude of 30 cm. Culturing was performed under the condition of 180 rpm (pre-culture).
The culture broth was inoculated in the continuous fermentation apparatus shown in FIG. 1 in 1.5 L of the medium having the composition shown in Table 1, and the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5. The culture was carried out by adjusting the aeration amount, temperature adjustment and pH adjustment of No. 1. Immediately after completion of the pre-culture, the additional medium having the composition shown in Table 2 is continuously supplied, and the continuous culture is performed while controlling the amount of permeated water so that the amount of the fermentation broth in the continuous fermentation apparatus is 1.5 L. The protein was produced by continuous fermentation while decreasing the acid concentration. The control of the amount of permeated water when performing the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 3. went. As appropriate, the phosphoric acid concentration and residual glucose concentration in the membrane permeate filtrate were measured. As a result of the culture, the cells grew to a microbial cell concentration of OD600 = 30 or more, and high concentration culture became possible.

Moreover, the fermentation broth was sampled as appropriate, and it was confirmed by Western blotting whether the protein was expressed when the phosphate concentration decreased. As a method, the bacterial cell concentration of each fraction was unified at OD600 = 10, and the bacterial cell fluid was used as a sample, and konica immunostain HRP (manufactured by Konica) was used to detect canine interferon γ according to the attached protocol. That is, SDS-PAGE electrophoresis was performed, and the blotted membrane was blocked in a blocking solution (5% skim milk / 0.1% Tween 20 / PBS) at 4 ° C. overnight. The membrane was washed twice with TPBS (0.1% Tween20 · PBS) and treated with an anti-canine interferon γ antibody diluted 1000 times with TPBS for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed 3 times with TPBS for 5 minutes. Thereafter, the resultant was diluted 10,000 times with TPBS and then treated with an HRP-labeled anti-rabbit IgG antibody (manufactured by Cosmo Bio) for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed three times with TPBS for 5 minutes, and then a detection reagent (solution A + solution B + solution C + solution D) of Konica Immunostain HRP (manufactured by Konica) was added to cause color development. As a result, the signal intensity was measured using a molecular imager (BioRad). In addition, non-transformed E. coli was used as a negative control sample, and Interdock (Toray Industries, Inc .: dog interferon γ) was used as a positive control sample.
As a result, it was confirmed that canine interferon γ was expressed in a large amount at a phosphate concentration of 1 mM or less. Moreover, it confirmed that it produced 5-10 times compared with the above-mentioned comparative example 1. By using the continuous fermentation apparatus shown in FIG. 1, it was confirmed that protein production by continuous fermentation was possible by reducing the concentration of components in the culture medium while maintaining cell growth.

(Example 4) Production of protein (part 4)
In order to investigate whether or not a continuous fermentation production system can be obtained by operating the continuous fermentation apparatus of FIG. 2, the continuous culture test of this apparatus was conducted using the composition medium shown in Table 1 and the additional medium having the composition shown in Table 2. went. The medium was autoclaved at 121 ° C. for 20 minutes, and the additional medium was autoclaved at 121 ° C. for 60 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) produced in Reference Example 5 was used. The operating conditions in Example 4 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
・ Membrane separation tank capacity: 0.5 (L)
・ Separation membrane: Polyvinylidene fluoride filtration membrane ・ Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 37 (℃)
-Fermentation reactor aeration rate: 1.5 (L / min) air-Fermentation reactor agitation speed: 800 (rpm)
-Aeration rate of membrane separation tank: 0.3 (L / min)
- pH Adjustment: _2N H 2 SO ₄ and the circulating liquid amount by the adjustment and the culture solution circulation apparatus pH7.0 by 14N aqueous ammonia: 0.1 (L / min)
-Membrane permeate flow rate control: The flow rate was controlled by the head difference of the fermentation reaction tank (the head difference was controlled within 2 m).

  Escherichia coli MC1061-canine interferon γ strain was used as the microorganism, a medium having the composition shown in Table 1 was used, and a medium having the composition shown in Table 2 was used as the additional medium. In addition, “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is used for measuring glucose concentration, and “phospha C-test Wako” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is used for measuring phosphoric acid concentration. Was used.

First, the Escherichia coli MC1061-canine interferon γ strain was cultured in a test tube with shaking overnight in a medium having the composition shown in Table 1 with tetracycline (10 μg / ml) added (cultured in advance). The obtained culture broth was inoculated into 50 ml of a medium having the composition shown in Table 1 to which fresh tetracycline (10 μg / ml) was added, and was cultured in a 500 ml Sakaguchi flask for 24 hours at a temperature of 37 ° C. with an amplitude of 30 cm. Culturing was performed under the condition of 180 rpm (pre-culture).
The culture medium is inoculated in advance in the continuous fermentation apparatus shown in FIG. 2 in 2 L of the medium having the composition shown in Table 1, and the fermentation reaction tank 1 is stirred at 800 rpm by the attached stirrer 5. The culture was performed by adjusting the aeration amount, adjusting the temperature, and adjusting the pH. Immediately after the completion of the pre-culture, the additional medium having the composition shown in Table 2 is continuously supplied, and the continuous culture is performed while controlling the amount of permeated water so that the amount of the fermentation broth in the continuous fermentation apparatus becomes 2 L. The protein was produced by continuous fermentation while decreasing the concentration. The control of the amount of permeated water when performing the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 3. went. As appropriate, the phosphoric acid concentration and residual glucose concentration in the membrane permeate filtrate were measured. As a result of the culture, the cells grew to a microbial cell concentration of OD600 = 30 or more, and high concentration culture became possible.

  Moreover, the culture solution was sampled as appropriate, and the extent to which the phosphoric acid concentration was reduced was confirmed by Western blotting to determine whether the protein was expressed. As a method, the cell concentration of each fraction was unified at OD600 = 10, and the cell solution was used as a sample, and canine interferon γ was detected according to the attached protocol using Konica Immunostain HRP (manufactured by Konica). That is, SDS-PAGE electrophoresis was performed, and the blotted membrane was blocked in a blocking solution (5% skim milk / 0.1% Tween 20 / PBS) at 4 ° C. overnight. The membrane was washed twice with TPBS (0.1% Tween20 · PBS) and treated with an anti-canine interferon γ antibody diluted 1000 times with TPBS for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed 3 times with TPBS for 5 minutes. Thereafter, the resultant was diluted 10,000 times with TPBS, and then treated with an HRP-labeled anti-rabbit IgG antibody (manufactured by Cosmo Bio) for 1 hour at room temperature. The membrane was washed twice with TPBS, and further washed three times with TPBS for 5 minutes, and then a detection reagent (solution A + solution B + solution C + solution D) of Konica Immunostain HRP (manufactured by Konica) was added to cause color development. As a result, the signal intensity was measured using a molecular imager (BioRad). In addition, non-transformed E. coli was used as a negative control sample, and Interdock (Toray Industries, Inc .: dog interferon γ) was used as a positive control sample.

  As a result, it was confirmed that canine interferon γ was expressed in a large amount at a phosphate concentration of 1 mM or less. Moreover, it confirmed that it produced 5-10 times compared with the comparative example 1 shown above. By using this continuous fermentation apparatus, it was confirmed that protein production by continuous fermentation was possible by reducing the concentration of the components in the medium while maintaining the cell growth. In addition, the amounts of bacterial cells and the results of expressed proteins of Comparative Example 1, Example 1 and Example 2 are shown in Table 3, and the amounts of bacterial cells and expressed proteins of Comparative Example 1, Example 3 and Example 4 are also shown. The results are shown in Table 4.

  As can be seen from these comparison results, it has been clarified that protein production is greatly improved by using a continuous fermentation apparatus. By using the continuous fermentation apparatus incorporating the porous membrane disclosed by the present invention and controlling the transmembrane pressure difference, the fermentation broth is separated into the filtrate and the unfiltered solution by the porous membrane, and the concentration of the medium components is adjusted. It was revealed that a continuous fermentation method in which the unfiltered liquid is returned to the fermentation broth while allowing the protein to be expressed by reducing the protein, and the protein can be produced by continuous fermentation while maintaining a high amount of microorganisms.

FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus used in the present invention. FIG. 2 is a schematic side view for explaining one embodiment of another membrane separation type continuous fermentation apparatus used in the present invention. FIG. 3 is a schematic perspective view for explaining one embodiment of the separation membrane element used in the present invention. FIG. 4 is a cross-sectional explanatory diagram for explaining an example of another separation membrane element used in the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fermentation reaction tank 2 Separation membrane element 3 Water head difference control device 4 Gas supply device 5 Stirrer 6 Level sensor 7 Medium supply pump 8 pH adjustment solution supply pump 9 pH sensor / control device 10 Temperature controller 11 Fermentation culture medium circulation pump 12 Membrane Separation tank 13 Support plate 14 Channel material 15 Separation membrane 16 Recess 17 Water collecting pipe 18 Separation membrane bundle 19 Upper resin sealing layer 20 Lower resin sealing layer 21 Support frame 22 Water collection pipe

Claims (10)

  Filtration of a fermentation broth of a microorganism having protein-producing ability through a separation membrane, holding or refluxing the unfiltered liquid in the fermentation broth, adding the culture medium of the microorganism to the fermentation broth, and A method for producing a protein by continuous fermentation in which a protein is recovered from a microbial cell, wherein a porous membrane having pores having an average pore diameter of 0.01 μm or more and less than 1 μm is used as the separation membrane, and the transmembrane pressure difference is determined. A method for producing a protein by continuous fermentation, wherein the filtration treatment is performed in the range of 0.1 to 20 kPa. The continuous water fermentation according to claim 1, wherein the porous membrane has a pure water permeability coefficient of 2 × 10 ⁻⁹ m ³ / m ² / s / pa to 6 × 10 ⁻⁷ m ³ / m ² / s / pa. A method for producing a protein.   The method for producing a protein by continuous fermentation according to claim 1 or 2, wherein the average pore diameter of the porous membrane is 0.01 µm or more and less than 0.2 µm, and the standard deviation of the average pore diameter is 0.1 µm or less.   The method for producing a protein by continuous fermentation according to any one of claims 1 to 3, wherein the membrane surface roughness of the porous membrane is 0.1 µm or less.   The method for producing a protein by continuous fermentation according to any one of claims 1 to 4, wherein the porous membrane is a porous membrane comprising a porous resin layer.   The method for producing a protein by continuous fermentation according to any one of claims 1 to 5, wherein the material of the porous membrane is a polyvinylidene fluoride resin.   The method for producing a protein by continuous fermentation according to any one of claims 1 to 6, wherein the fermentation raw material of the microorganism contains a saccharide.   The method for producing a protein by continuous fermentation according to any one of claims 1 to 7, wherein the gene encoding the protein is linked downstream of the phosphate promoter and the concentration of inorganic phosphate contained in the medium is 1 mM or less.   The method for producing a protein by continuous fermentation according to any one of claims 1 to 8, wherein the protein is canine interferon γ.   The method for producing a protein by continuous fermentation according to any one of claims 1 to 9, wherein the microorganism is Escherichia coli.

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