JP2010259414A - Substance production method, cell proliferation promoter, expression enhancer, and method for producing erythroascorbic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance production method capable of easily improving a substance production efficiency of a cell, a new use of erythroascorbic acid, and a method for producing erythroascorbic acid in high efficiency. <P>SOLUTION: There are provided: a method for producing a target substance by using a substance producing cell, which includes a production step to produce the target substance by the substance producing cell in the presence of erythroascorbic acid as a cell activation agent, and a recovering step to recover the target substance produced by the production step; a cell proliferation promoting agent containing erythroascorbic acid in an amount effective for promoting the proliferation, and an expression promoting agent containing erythroascorbic acid in an amount effective for promoting the expression; and an erythroascorbic acid production method including formation of erythroascorbic acid by bringing ascorbic acid-decomposing bacteria into contact with ascorbic acid or its dehydroascorbic acid in the presence of ubiquitin, and recovery of erythroascorbic acid from a reaction system after the contacting step. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a substance production method, a cell growth promoter and an expression enhancer, and a method for producing erythroascorbic acid.

Technology that produces bioactive substances such as insulin, growth hormone, and antibodies that are originally biosynthesized in the body in E. coli, yeast, or animal cells using genetic recombination technology has been put into practical use. Physiologically active substances are used as pharmaceuticals.
In this context, the development of technologies to improve the productivity of recombinant proteins in cultured cells includes the selection of cell lines, the design of vectors used for genetic recombination, the search for compounds that promote cell growth, and the study of medium composition It is advanced from such a side.

For example, a method of introducing a molecular chaperone into a mammalian host cell that expresses a recombinant protein is known (see, for example, Patent Document 1). According to this method, it is said that the production amount of a secretory recombinant protein product can be increased.
In addition, a method is known in which an endogenous signal peptide sequence of DNA encoding a target protein is replaced with a signal peptide sequence of human growth hormone (see, for example, Patent Document 2). This method is said to improve the homogeneity and / or secretion of recombinant proteins expressed in mammalian cells.
As an example of a compound that promotes cell growth, cell growth is promoted by adding linear polyphosphoric acid or fructan to the medium (see, for example, Patent Documents 3 and 4).

Erythroascorbic acid is a compound obtained by chemical synthesis using glucose as a raw material or by oxidative decomposition of ascorbic acid (see, for example, Non-Patent Documents 1 and 2). As for erythroascorbic acid, an antioxidant action in yeast and a vitamin C (L-ascorbic acid) -like action in larvae of Tobacco hornworm have been reported (for example, see Non-Patent Documents 3 and 4). Furthermore, recently, it has become possible to produce erythroascorbic acid by a conversion reaction from ascorbic acid using a microorganism (Patent Document 5 ).

JP-T-2007-52481 Special table 2004-516830 gazette JP 2000-069961 A JP 2008-228587 A JP 2007-135445 A

Carbohydr. Res. 220, 117-125 (1991) Arch. Biochem. Biophys. , 355, 9-14 (1998) Mol. Microbiol. , 30, 895-903 (1998) J. et al. Agric. Food Chem. , 41, 1391-1396 (1993)

However, in the method described in Patent Document 1, it is necessary to transform a mammalian cell line by introducing an expression vector containing DNA encoding a chaperone protein, and there remain problems in work efficiency and cost. .
The method described in Patent Document 2 is a method of newly introducing an expression vector in which a DNA encoding a human growth hormone signal peptide and a DNA encoding a target protein are fused, into a mammalian cell, Mammalian cells that have already been transformed to express the target protein are not used as they are, and there are still problems in work efficiency and cost.
Further, since the effect of a compound that promotes cell proliferation may vary depending on the cell type, it is important to develop many usable candidate compounds.

  On the other hand, regarding erythroascorbic acid, no knowledge has been obtained regarding the effects on the proliferation and protein expression of cultured cells.

  Regarding the production of erythroascorbic acid, in the method described in Patent Document 5, ascorbic acid is added to the culture broth of the microorganism to perform a conversion reaction, so that the erythroascorbine produced by the culture solution and the microorganism product used for the conversion is produced. Mixed with acid. For this reason, the purification operation after conversion is complicated, and there remains a problem to be solved in terms of manufacturing cost.

  The present invention has been made in view of the above, and an object of the present invention is to provide a substance production method capable of easily increasing the substance production efficiency of cells and a novel use of erythroascorbic acid.

  The present invention has been made in view of the above, and provides a simple method for producing erythroascorbic acid.

  The substance production method of the present invention is a substance production method for producing a target substance using substance-producing cells, wherein the substance-producing cells produce the target substance in the presence of erythroascorbic acid as a cell activator. It is a substance production method including a production process to be performed and a recovery process for collecting the target substance produced in the production process.

  The concentration of the erythroascorbic acid is preferably 0.1 to 1000 μg / ml. The substance producing cell is preferably an animal cell. Furthermore, it is preferable that the substance producing cell has a recombinant protein expression mechanism for expressing the target substance.

The cell growth promoter of the present invention is a cell growth promoter containing a proliferation promoting effective amount of erythroascorbic acid.
The expression enhancer of the present invention is an expression enhancer containing an effective expression enhancing amount of erythroascorbic acid.

  The method for producing erythroascorbic acid according to the present invention comprises producing erythroascorbic acid by contacting ascorbic acid or dehydroascorbic acid with an ascorbic acid degrading bacterium in the presence of ubiquitin, and erythroascorbic acid from the reaction system after the contact. Recovering the acid.

  According to the present invention, it is possible to provide a substance production method capable of easily increasing the substance production efficiency of cells and a novel use of erythroascorbic acid.

  Moreover, according to this invention, the method of producing erythroascorbic acid simply can be provided.

It is the result of TLC which shows the production | generation of erythro ascorbic acid from L-ascorbic acid using the refinement | purification ubiquitin derived from a bovine erythrocyte. 2 is a HPLC chromatogram confirming the production of erythroascorbic acid according to Example 1. It is the result of TLC which shows the production | generation of erythro ascorbic acid from dehydroascorbic acid using the refinement | purification ubiquitin derived from a bovine erythrocyte. It is a result of TLC showing erythroascorbic acid production from ascorbic acid or dehydroascorbic acid using recombinant human ubiquitin.

Hereinafter, the substance production method, cell growth promoter and expression enhancer of the present invention will be described in detail.
(Material production method)
The substance production method of the present invention is a substance production method for producing a target substance using substance-producing cells, wherein the substance-producing cells produce the target substance in the presence of erythroascorbic acid as a cell activator. A production process to be performed, and a collection process to collect the target substance produced in the production process.
In the substance production method of the present invention, the target substance can be easily and efficiently produced by producing the target substance using the substance-producing cells in the presence of erythroascorbic acid.

  In the production step, the target substance is produced by the substance-producing cells in the presence of erythroascorbic acid as a cell activator.

  The substance-producing cell according to the present invention refers to a cell that produces a target substance. The cell type is not particularly limited, and may be any of bacteria, yeast, fungi, algae, plant cells, animal cells, and the like. An animal cell is preferable in that it can be used for production of a protein having a higher-order folded structure and / or post-translational modification, and examples thereof include insect cells and mammalian cells.

Examples of insect cells include cell lines such as sugar beet, silkworm, and the like, and specifically, Sf9 cells, Sf21 cells, and the like.
Examples of mammalian cells include cell lines derived from various tissues such as humans, monkeys, mice, rats, hamsters, rabbits, and dogs. Specifically, CHO cells (cells derived from Chinese hamster ovary) NSO cells (mouse myeloma-derived cells), BHK cells (hamster kidney-derived cells), HEK293 cells (human embryonic kidney-derived cells), COS cells (African green monkey kidney-derived cells), and the like.

  The substance-producing cell preferably has a recombinant protein expression mechanism for expressing the target substance. The recombinant protein expression mechanism is a mechanism for selectively expressing a target protein. In the present invention, a well-known recombinant protein expression mechanism can be used. By providing a recombinant protein expression mechanism, a target protein can be expressed in large quantities. A substance-producing cell having a recombinant protein expression mechanism can be obtained, for example, by introducing an expression vector having a DNA encoding a target protein into the cell.

  The target substance produced by the substance producing cell is not particularly limited as long as it is a compound produced by the cell. It may be organic or inorganic, and may be retained in the cell or secreted outside the cell, regardless of whether it is synthesized or decomposed. Examples include nucleic acids, amino acids, sugars, glycoproteins, fatty acids, vitamins, peptides, antibodies, hormones, and the like.

The erythroascorbic acid can act as a cell activator for substance-producing cells in the production process.
The cell activator means a drug having a function of activating cells. The activation may be cell proliferation or expression enhancement, and both may not be clearly distinguished. When the substance-producing cells are brought into contact with erythroascorbic acid, cell proliferation and / or expression are enhanced, and the production amount of the target substance can be increased.

The above-mentioned cell proliferation is generally used in the meaning used in the art. For example, it means that the number of cells in a cell population has increased due to repeated cell division. Cells may be dead. Cell proliferation may be evaluated by a method generally used for this purpose, and may be measurement of the number of cells, measurement of the amount of an indicator compound, or the like.
With the above expression enhancement, an increase in the amount of the target substance may be recognized either in the cell producing the target substance or in the secretion from the cell. Expression enhancement can be evaluated based on, for example, the concentration of the target substance contained in the liquid medium after culturing the cells.
In addition, the expression enhancement of the target substance means that the expression level of the cell population increases as the expression level of the cell unit increases, the expression level of the cell population increases, and the cell proliferation is promoted and the number of cells increases. The amount may be increased, and the two need not be clearly distinguished.

  The erythroascorbic acid according to the present invention may be a commercially available product or a product of a fungus having an ascorbic acid decomposing ability. When erythroascorbic acid, which is a fungal product, is used, this erythroascorbic acid is produced from ascorbic acid by, for example, contacting ascorbic acid with an ascorbic acid-degrading fungus to produce erythroascorbic acid (production process). The erythroascorbic acid recovered (recovery step) can be obtained by a production method.

  The fungus used for the production of erythroascorbic acid may be a fungus having an ability to resolve ascorbic acid, and may be an asexual generation or a sexual generation. Among them, the genus Penicillium, the genus Aspergillus, the genus Chaetomim, the genus Paecilomyces, the genus Eupenicillium, the genus Fusium, the genus Fusium, A fungus selected from the group consisting of, in particular, the genus Penicillium is preferred. Examples of the Penicillium genus include Penicillium No. 196F (National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center Accession No. FERM P-20598, received on July 21, 2005) is particularly preferable from the viewpoint of production ability.

  The medium used for the fungal culture is not particularly limited as long as the medium is known to grow fungi. For example, it can be maintained and grown in a stationary culture under aerobic conditions at 25 ° C. in a medium such as potato dextrose agar medium (medium composition: 1000 ml of potato (200 g) extract, 20 g glucose, 20 g agar). Either a liquid culture medium or a solid culture medium may be used, but when recovering bacterial cells, it is a liquid culture medium from the viewpoint of recovery of bacterial cells and from the viewpoint of recovery of products when contacting with ascorbic acid. It is preferable.

  The medium used for fungal liquid culture may be any medium that is known to grow fungi. For example, glucose 2 mass%, starch 2 mass%, dry broth 0.5 Although the culture medium by mass%, yeast extract 0.2 mass%, soybean meal 0.5 mass%, and an antifoamer 0.01 mass% can be mentioned, it can change suitably according to the kind and state of a microbe. .

  The culture conditions vary depending on the type of fungus and the growth state / number of fungi in the preculture, but it is generally preferable to culture until the wet weight reaches 0.01 to 0.1 g / ml. However, it can be increased further.

  The production | generation process in this manufacturing method can be performed by adding ascorbic acid to the culture medium which culture | cultivates fungi. For example, the manufacturing method of erythro ascorbic acid of Unexamined-Japanese-Patent No. 2007-135445 is mentioned.

Ascorbic acid to be added to the liquid containing microbial cells may be either powder or liquid as long as it can be metabolized by contact with fungi. As a solvent for obtaining an ascorbic acid solution, any solvent may be used, and water, methanol, ethanol and the like can be mentioned.
The amount of ascorbic acid to be added varies depending on the type and growth state of the fungus, but is generally 0.1 to 200 mg / ml, more preferably 0.1 to 100 mg / ml, and even more preferably 3 to 30 mg / ml. do it. If it is less than 0.1 mg / ml, erythroascorbic acid may not be obtained. On the other hand, if it exceeds 200 mg / ml, conversion may be incomplete, which is not preferable.

  After addition of ascorbic acid, it may be left at room temperature (25 ° C.) or shaken. When shaking, it may be carried out at a speed normally applied in fungal culture. For example, a TB-25S (amplitude 70 mm) rotary shaker manufactured by Takasaki Kagaku Co., Ltd. makes 100 ml of liquid in a 500 ml flask. In some cases, it can be 30 to 240 rpm, preferably 160 to 200 rpm. The temperature at the time of shaking can be appropriately set according to fungal growth conditions at various temperatures, but is generally 4 to 50 ° C, preferably 15 to 37 ° C, more preferably 20 to 30 ° C, and most preferably. Room temperature (25 ° C.). When the temperature is lower than 4 ° C, conversion to erythroascorbic acid may not occur, which is not preferable. On the other hand, when the temperature is higher than 50 ° C, enzyme activity for conversion may decrease, which is not preferable.

  The standing or shaking described above can be continued until all ascorbic acid has been converted to erythroascorbic acid, depending on the type and growth state of the fungus, but generally 10 to 80 hours, preferably 24 to 48 hours. I just need it. During this time, the fungus comes into contact with ascorbic acid, metabolizes ascorbic acid, converts it into erythroascorbic acid, generates it, and releases it outside the cell. In addition, the said continuation time can be suitably changed with the conversion state from ascorbic acid to erythro ascorbic acid. Confirmation of such a conversion state can be easily performed using known means such as thin layer chromatography.

In the recovery step in this production method, erythroascorbic acid obtained in the above generation step is recovered.
As a method for recovering erythroascorbic acid released to the outside of the cell, any means known to be able to recover or purify a compound in a liquid may be used. Liquid chromatography (ion exchange chromatography) ), Solvent extraction, crystallization, and the like. The product is preferably recovered and purified in two or more stages from the viewpoint of recovery efficiency.
Before using each recovery means, it is preferable to remove the cells from the solution from which erythroascorbic acid has been released. Filtration or the like may be used to remove the cells.
The obtained erythroascorbic acid can be confirmed by known chemical analysis and instrumental analysis.

  In the production method described above, from the viewpoint of efficiently recovering erythroascorbic acid in the recovery step, the generation step is performed by bringing the ascorbic acid-decomposing fungus washed with the cleaning liquid into contact with ascorbic acid in the generation buffer. It is preferable to set it as the process of producing | generating an acid. Since erythroascorbic acid is produced using the cells thus washed in the production buffer, erythroascorbic acid can be recovered from a system with few impurities. As a result, the purification process of erythroascorbic acid can be simplified.

The washing solution for washing the cells may be any solution generally used for washing the cells, and examples thereof include physiological saline, phosphate buffer, Tris buffer, and the like. Among these, physiological saline is preferable from the viewpoint of isotonic pressure and further having few constituent components.
Washing is not particularly limited as long as it can remove the culture solution components adhering to the cells.

  In addition, before being used for this production | generation process, the washing | cleaning microbial cell was used for preculture, in order to make it a predetermined | prescribed number of bacteria before using for manufacture of erythroascorbic acid, or to make it a production | generation active state It is preferable that By using the washed cells after such preculture, the production efficiency of erythroascorbic acid can be increased. The pre-culture conditions may be normal culture conditions, and the culture conditions described above can be applied as they are.

  The production buffer is not limited as long as it can maintain the washed cells to the extent that erythroascorbic acid production by the washed cells can be maintained, and examples thereof include physiological saline, phosphate buffer, Tris buffer, and the like. These may be the same as or different from the cleaning liquid. Further, the pH of the production buffer is usually 3.0 to 10.0, and preferably 4.5 to 8.0 from the viewpoint of suppressing non-specific decomposition of ascorbic acid.

The suspension concentration of the microbial cells in the production buffer solution varies depending on the type of fungus or the growth state, but is usually 0.01 to 1.0 g / ml in wet weight. However, it can be increased further.
The production reaction in the production buffer solution varies depending on the number and type of washing cells used, but it may usually be 18 hours to 72 hours. From the viewpoint of the ratio between the amount of erythroascorbic acid produced and the amount of residual ascorbic acid 36 Time to 48 hours is preferable.

  In the production step of the substance production method of the present invention, it is sufficient that erythroascorbic acid is present in the reaction system during substance production by substance producing cells. Contact in the reaction system between erythroascorbic acid and substance-producing cells may be performed by any method depending on the culture conditions such as the type of target substance, the type of cells, and the culture temperature. For example, erythroascorbic acid may be added to a medium in which substance-producing cells are being cultured, or substance-producing cells may be added to a culture solution in which erythroascorbic acid is dissolved. Further, erythroascorbic acid may be continuously added to the reaction system, or may be periodically added.

The erythroascorbic acid is not particularly limited as long as it is present in the reaction system in an amount effective for substance production, and can be used at 0.1 to 1000 μg / ml, although it varies depending on the cell type. If it is 0.1 μg / ml or more, the substance-producing cells can be sufficiently activated to produce the substance, and if it is 1000 μg / ml or less, an efficient substance corresponding to the amount added without impairing the cell activity. Production can be expected. From the viewpoint of producing a sufficient amount of substance by substance-producing cells, the use concentration is preferably 1 to 500 μg / ml, more preferably 10 to 500 μg / ml, and 30 to 300 μg / ml. Particularly preferred.
In the present invention, the use concentration refers to a final concentration that is an erythroascorbic acid concentration in an environment where cells are placed and can be artificially adjusted. For example, when substance-producing cells are used with erythroascorbic acid added to the culture medium, it means the erythroascorbic acid concentration in the medium immediately after the addition.

In the collection step in the substance production method of the present invention, the target substance produced in the production step is collected. Any method may be used as long as it is known to be able to extract and purify compounds in cells or extracellular (for example, in a medium). Cell lysis or disruption can be carried out by a known method, and examples of a method for purifying a target substance in a solution include liquid chromatography (ion exchange chromatography), solvent extraction, and crystallization. The purification of the target substance is preferably performed in two or more stages from the viewpoint of recovery efficiency. The recovery step in the substance production method of the present invention may be recovery of cells themselves.
The obtained target substance can be confirmed by known chemical analysis or instrumental analysis according to the type of the target substance.

(Cell growth promoter and expression enhancer)
As described above, erythroascorbic acid acts as an activator for cells, and can promote cell proliferation and enhanced expression.
Therefore, the cell growth promoting agent of the present invention contains a growth promoting effective amount of erythroascorbic acid. The expression enhancer of the present invention contains an effective expression enhancing amount of erythroascorbic acid.

The cell to be used for the cell growth promoter of the present invention is not particularly limited as long as it is a cell that exhibits a growth promoting action when contacted with erythroascorbic acid, and is limited to the substance-producing cell used in the substance production method. Not. For example, the present invention may be applied to applications intended for cells themselves to promote cell proliferation and to obtain large amounts of target cells. Examples of such target cells include epidermal cells, epithelial cells, blood cells and the like constituting the cell sheet.
In addition, the cell to be used for the expression enhancer of the present invention is not particularly limited as long as it is a cell that produces the target substance and has an expression enhancing action when contacted with erythroascorbic acid, It is not limited to the substance production cell used for the said substance production method.
There is no particular limitation on the cell type, and the same cells as those in the substance production method can be targeted, and preferred embodiments are also the same.

  The cell growth promoter and expression enhancer of the present invention may be brought into contact with cells at the time of use. There is no restriction | limiting in particular in the use aspect, According to culture conditions, such as a kind of cell and culture | cultivation temperature, it can select arbitrarily.

The cell growth promoter and expression enhancer of the present invention may be a growth promoting effective amount and an expression enhancing effective amount, respectively. The proliferation-promoting effective amount in the present invention means an amount capable of promoting the growth of the target cells of interest during use. On the other hand, the expression-enhancing effective amount means an amount capable of enhancing the function of the gene expression system for producing a substance in the target cell of interest at the time of use. In particular, the target substance based on the transgene in the genetically modified cell This means the amount of production that can be increased.
The proliferation promoting effective amount and the expression enhancing effective amount in the present invention vary depending on the cell type, but from the viewpoint of obtaining a sufficient effect without impairing the cell activity, it can be used at a concentration of 0.1 to 1000 μg / ml. 1 to 500 μg / ml, preferably 10 to 500 μg / ml, particularly preferably 30 to 300 μg / ml. Here, the use concentration is as described above.

The cell growth promoter and expression enhancer of the present invention may be composed of erythroascorbic acid alone or may be composed of known pharmaceutical additives. Known additives for pharmaceutical preparations can include bulking agents, excipients, emulsifiers, wetting agents, and the like, and can be appropriately selected depending on the usage form of the cell growth promoter and expression enhancer.
Moreover, the form of the cell growth promoter and expression enhancer of the present invention is not particularly limited and can be arbitrarily selected by those skilled in the art. For example, it can be in the form of emulsion, solution, aqueous solution, wettable powder, powder, granule, fine granule, and the like.
In the case of such various use forms, if the final concentration can be adjusted to the use concentration described above, even if the use concentration itself contains erythroascorbic acid, it is contained in a concentrated form. There may be.

(Method for producing erythroascorbic acid)
Next, the erythroascorbic acid production method of the present invention will be described in detail.
In addition, the description mentioned above regarding substance production, the manufacturing method of said erythroascorbic acid, etc. is applicable also to the manufacturing method of erythroascorbic acid described below suitably.

  The method for producing erythroascorbic acid of the present invention comprises producing erythroascorbic acid by contacting ascorbic acid or dehydroascorbic acid with an ascorbic acid-degrading fungus in the presence of ubiquitin (hereinafter referred to as a production step), and after the contact Recovering erythroascorbic acid from the reaction system (hereinafter referred to as recovery step).

  In the erythroascorbic acid production method of the present invention, ubiquitin is present as a conversion reaction activator when an ascorbic acid-degrading fungus converts erythroascorbic acid from ascorbic acid or the like, so that erythroascorbine can be more efficiently converted from ascorbic acid or the like. Acid can be produced.

  The ubiquitin according to the present invention is a ubiquitin contained in eukaryotic cells, and is a protein known to be composed of 76 amino acids in organisms such as humans, cows and yeasts. The eukaryotic species is not particularly limited, and may be any of fungi, yeast, algae, plants, animals, and the like. Ubiquitin is a highly conserved protein in the course of evolution, and the difference in amino acid sequence between species is extremely small, but ubiquitin derived from fungi, yeast or animals is preferred.

  Examples of fungi or yeast-derived ubiquitin include the genera Penicillium and Saccharomyces, and examples of animal-derived ubiquitin include bovine. These may be naturally derived ubiquitin or ubiquitin produced using E. coli as a host as a recombinant.

  Such ubiquitin is preferably a natural ubiquitin (SEQ ID NO: 1 and SEQ ID NO: 2) or a ubiquitin variant in which the sixth lysine (hereinafter, “K6”) is conserved. SEQ ID NO: 1 matches, for example, human and bovine sequences, and SEQ ID NO: 2 matches, for example, yeast sequences. The “ubiquitin mutant in which K6 is conserved” in the present invention may be any mutant as long as the sixth lysine of natural ubiquitin is conserved and has a function as a conversion reaction activator. A mutant in which lysine other than the sixth is substituted with another amino acid is applicable.

  Ascorbic acid or dehydroascorbic acid serving as a substrate for the production reaction can be used without any problem as long as it is commercially available ascorbic acid or dehydroascorbic acid. Also, any of L-ascorbic acid and D-ascorbic acid can be used. Furthermore, dehydroascorbic acid can also be prepared by oxidizing ascorbic acid with an oxidizing agent such as bromine.

  Ubiquitin used in the production process may be highly purified, contain impurities such as cell extracts, or may be used as production cells as long as the cells can produce ubiquitin. it can.

In order to bring these ubiquitins into contact with the above-mentioned ascorbic acid-decomposing fungi, the production method may include adding ubiquitin or ubiquitin-producing cells to the culture system of the ascorbic acid-degrading bacteria. Here, the ubiquitin-producing cell is not particularly limited as long as it has ubiquitin-producing ability, and may be an introduced cell into which a recombinant having a ubiquitin-producing system is introduced in addition to cells that are naturally produced. . In addition, if it is single cell organisms, such as algae, adding microorganisms itself is also included by addition of a ubiquitin producing cell.
These ubiquitins may be a solution (composition) or a solid. In the case of a solid, the ubiquitin may be immobilized on a carrier such as a polymer. In the case of those immobilized on a carrier, arranging the carrier itself in the reaction system is also included as one form of “addition”.

  Ascorbic acid or dehydroascorbic acid to be contacted may be in the form of powder or liquid as long as it can be metabolized by contact with the aforementioned ubiquitin, a composition containing ubiquitin, or a cell containing ubiquitin. Good. Any solvent may be used as a solvent for obtaining a solution of ascorbic acid or dehydroascorbic acid, and examples thereof include organic solvents such as water, buffer solution, methanol, and ethanol.

  The amount of ascorbic acid or dehydroascorbic acid to be added varies depending on the addition form of ubiquitin, such as a purified product, a composition, or a produced cell, but is generally 0.01 to 200 mg / ml, more preferably 0.1 to 100 mg / ml, More preferably, it may be added in an amount of 0.3 to 30 mg / ml. If it is less than 0.01 mg / ml, erythroascorbic acid may not be obtained. On the other hand, if it exceeds 200 mg / ml, conversion may be incomplete, which is not preferable.

  The solvent used in the production step may be a normal culture solution, but as described above, from the viewpoint of simplification of the purification step and efficiency, a production buffer solution is preferable. As described above, the production buffer is not particularly limited as long as it can maintain erythroascorbic acid production, and examples thereof include physiological saline, phosphate buffer, Tris buffer, and the like. Moreover, the pH of the buffer used for a production | generation process is 3.0-12.0 normally, and 4.0-10.0 are preferable from a viewpoint of the production | generation efficiency of erythro ascorbic acid, and nonspecific decomposition | disassembly suppression of ascorbic acid.

  The amount of ubiquitin added to the buffer used in the production step varies depending on the form of ubiquitin, but is usually 0.001 μg / ml to 1000 μg / ml as a content corresponding to purified ubiquitin. However, it can be further increased or decreased.

  After addition of ascorbic acid or dehydroascorbic acid, it may be left still or shaken. When shaking, it may be carried out at a speed normally applied in fungal culture. For example, a TB-25S (amplitude 70 mm) rotary shaker manufactured by Takasaki Kagaku Co., Ltd. makes 100 ml of liquid in a 500 ml flask. In some cases, it can be 30 to 240 rpm, preferably 160 to 200 rpm. The temperature at the time of shaking can be appropriately set according to the progress of the reaction at various temperatures, but in the reaction using ubiquitin or a composition containing ubiquitin, generally 4 to 100 ° C. is a cell containing ubiquitin. In the reaction using, 4 to 50 ° C. can be used. If the temperature is lower than 4 ° C, conversion to erythroascorbic acid may not occur, which is not preferable. In a reaction using cells containing ubiquitin, if the temperature is higher than 50 ° C, conversion activity may decrease, which is not preferable.

  The standing or shaking described above can be continued until all ascorbic acid or dehydroascorbic acid is converted to erythroascorbic acid, depending on the state of the conversion reaction, but generally 1 minute to 24 hours, preferably 3 minutes to 12 If it is time. During this time, ubiquitin comes into contact with ascorbic acid or dehydroascorbic acid to convert and produce erythroascorbic acid. In addition, the said continuation time can be suitably changed with the conversion state from ascorbic acid or dehydroascorbic acid to erythro ascorbic acid. Confirmation of such a conversion state can be easily performed using known means such as thin layer chromatography.

In the recovery step in this production method, erythroascorbic acid obtained in the above generation step is recovered. As a method for recovering the produced erythroascorbic acid, any means known to be able to recover or purify a compound in a liquid may be used. Liquid chromatography (ion exchange chromatography), solvent extraction And crystallization. The product is preferably recovered and purified in two or more stages from the viewpoint of recovery efficiency.
The obtained erythroascorbic acid can be confirmed by known chemical analysis and instrumental analysis.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

[Example 1]
<Production of erythroascorbic acid using fungi>
Penicillium No. which is a fungus of the genus Penicillium. 196F (FERM P-20598) was added to a seed medium (glucose 20 g / L, starch 20 g / L, dried bouillon (or peptone) 5 g / L, yeast extract 2 g / L, soybean meal 5 g / L, antifoam CB442. (Nippon Yushi Co., Ltd., 0.1 g / L) A 500-ml Ehrlenmeyer flask with baffle containing 100 ml was inoculated and cultured at 25 ° C. for 4 days with shaking.

Next, a part (5 ml) of this culture solution was added to a growth medium (starch 30 g / L, yeast extract 3 g / L, NaNO ₃ 10 g / L, KCl 0.5 g / L, KH ₂ PO ₄ 30 g / L, MgSO ₄ · 7H ₂ O 0.5 g / L, FeSO ₄ · 7H ₂ O 0.05 g / L, CaCl ₂ · 2H ₂ O 0.05 g / L, Antifoam CB442 0.1 g / L, pH 5.5) 100 ml Was inoculated into a 500-ml Ehrlenmeyer flask with baffle and cultured at 25 ° C. for 1 day with shaking.

  The culture was filtered to collect the cells and washed twice with physiological saline. The total amount of the washed cells was suspended in 100 ml of 0.22 M potassium phosphate buffer (pH 5.5), and 10 mg. Vitamin C (L-ascorbic acid) was added so as to be / ml. Subsequently, the mixture was shaken in a baffled 500-ml Ehrlenmeyer flask at 25 ° C. for 36 to 48 hours.

The culture solution for 10 flasks obtained by the above treatment was filtered to obtain a total of 1.1 L of filtrate. To this was added 5.5 L of methanol and left at 4 ° C. overnight. The filtrate obtained by filtering this was concentrated and freeze-dried to obtain 15.75 g of a dried solid product.
This dried product was suspended in 525 ml of chloroform: methanol: acetic acid (3: 1: 1), and the supernatant was collected by centrifugation and concentrated to obtain 13.8 g of a residue.

The residue was dissolved in water and purified by preparative HPLC using Asahipak NH2 P-130 28F (28.0 × 300 mm). Using 0.05% (v / v) aqueous formic acid containing 40% (v / v) acetonitrile as a developing solvent, elution was carried out at 20 ml / min, and an eluate having a retention time of 75 to 85 minutes was collected. A total of 8 fractions yielded 490 mg of purified product. It was confirmed by ¹ H-NMR and ¹³ C-NMR, mass spectrometry, ultraviolet absorption, infrared absorption, and specific rotation that the purified product was L-erythroascorbic acid.

<Evaluation of cell growth promoting action of erythroascorbic acid>
The cell growth promoting action of erythroascorbic acid obtained above was examined by the following method.

A rhBRAS production strain producing recombinant human type BL angiostatin (rhBRAS) was prepared by the following method. BL angiostatin is a known protein produced by the specific cleavage of plasminogen.
A reverse transcription reaction was performed using HepG2 Total RNA 2 as a template, and a DNA fragment of a plasminogen partial sequence corresponding to the target BL angiostatin was obtained by PCR.
After blunting the ends of this DNA fragment, pCAGGS 1. dhfr. Cloning into the Sal I site of the neo vector gave a plasmid construct. A large amount of plasmid DNA for transfection was prepared from this construct by a conventional method.
The plasmid DNA was introduced into CHO / dhfr-cells using the transfection reagent TransIT-CHO, and a rhBRAS production strain was established by a conventional method.

The rhBRAS-producing cells obtained from the above rhBRAS-producing strain were suspended in Dulbecco's MEM (DMEM) medium supplemented with 10% fetal bovine serum to prepare a suspension of 1 × 10 ⁴ cells / ml. This suspension was seeded on a 24-well plate at 500 μl / well.

The seeded cells were statically cultured at 37 ° C. for 72 hours under 5% carbon dioxide gas. Thereafter, the DMEM medium was removed from each well with an aspirator, and DMEM medium (serum-free) was added at 500 μl / well.
Next, erythroascorbic acid or vitamin C diluted with DMEM medium is added to each well, the total amount of the culture medium in each well is 550 μl, and the final concentration of erythroascorbic acid or vitamin C is 0 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml, 10 μg / ml, and 100 μg / ml.
Subsequently, the cells were statically cultured at 37 ° C. for 72 hours under 5% carbon dioxide gas.

  After culturing, the supernatant of each well was removed with an aspirator. Each well was washed three times with 500 μl of PBS, and then 0.1 N NaOH was added at 250 μl / well to lyse rhBRAS-producing cells.

  150 μl of bradford reagent was added to 30 μl of the solution obtained by lysing the cells, and the absorbance was measured at 595 nm by the bradford method. The protein concentration was calculated based on the calibration curve. The results are shown in Table 1 below. Table 1 shows the relative values, with the protein concentration in the supernatant of each well having an erythroascorbic acid or vitamin C concentration of 0 μg / ml as 100.

  As shown in Table 1, the cell increase rate of rhBRAS-producing cells when erythroascorbic acid (EAA) was added was higher than that when vitamin C (AsA) was added, and the cells increased in proportion to the amount of EAA added. Growth was promoted.

[Example 2]
<Evaluation of recombinant protein expression enhancing effect of erythroascorbic acid>
In order to investigate the effect of erythroascorbic acid on enhancing the expression of recombinant protein, rhBRAS produced by rhBRAS-producing cells was quantified by the following competitive ELISA.

  Human natural BL angiostatin prepared according to a known method (BioTechniques 40: 590-594, 2006) was diluted to 2 μg / ml in PBS, and this was placed in a 96-well Eliza plate at 50 μl / well at 4 ° C. Immobilized overnight. After immobilization, each well is washed 3 times with 100 μl of PBS, PBS containing 50 mg / ml BSA (hereinafter referred to as “BSA-containing PBS”) is added at 50 μl / well, and left at room temperature for 1 hour. Blocked by.

Culture was carried out in the same manner as in Example 1 except that the suspension concentration of rhBRAS-producing cells was seeded in each well at 2 × 10 ⁴ cells / ml, and the cell supernatant was collected from each well as a supernatant for production amount measurement. . The obtained supernatant is diluted with BSA-containing PBS so that the final concentration is 10% and the anti-plasminogen antibody (manufactured by Wako Pure Chemical Industries, Ltd.) is 1.5 μg / ml and placed at room temperature for 1 hour. Incubated by

Each well of the plate after blocking was washed 3 times with 100 μl of PBS, sample solution or standard solution was added at 50 μl / well, and incubated at room temperature for 1 hour.
After incubation, wash once with 100 μl PBS, and add 50 μl / well of secondary antibody (HRP-anti-rabbit IgG, manufactured by Wako Pure Chemical Industries, Ltd.) diluted with BSA-containing PBS to 3 μg / ml. Incubated by allowing to stand at room temperature for 1 hour.

After incubation, each well was washed three times with 200 μl of PBS, and o-Phenylenediamine (Sigma Aldrich Co., Ltd., 1/2500 hydrogen peroxide solution diluted in PBS to 0.4 mg / ml was added immediately before. (Addition) was added at 100 μl / well and allowed to react at room temperature for 30 minutes. Subsequently, 3N sulfuric acid was added to stop the reaction.
The absorbance value was measured at a wavelength of 490 nm, and the rhBRAS concentration (μg / ml) was calculated from the standard curve. The results are shown in Table 2 below. The numerical values in Table 2 indicate the production amount of rhBRAS (μg / ml).

  As shown in Table 2, the amount of rhBRAS produced by the rhBRAS-producing cells increased about 2.4 times that when no erythroascorbic acid (EAA) was added at 100 μg / ml.

  From these facts, it is clear that erythroascorbic acid exhibits cell growth promoting action and expression enhancing action. Therefore, erythroascorbic acid can be expected to have an effect as a cell growth promoter and expression enhancer, and can be used for substance production using cells.

  From the above, according to the present invention, the substance production efficiency of cells can be easily increased. Moreover, according to this invention, the novel use of erythroascorbic acid called a cell growth promoter and an expression enhancer can be provided.

[Example 3]
<Conversion reaction of ascorbic acid to erythroascorbic acid by ubiquitin>
Highly purified ubiquitin derived from bovine erythrocytes (SEQ ID NO: 1) (SIGMA, St. Louis, MO, USA (catalog number U6253)) (5 μg / ml) or buffer and L-ascorbic acid (Wako Pure Chemical Industries) ( 17 mM) was incubated in buffer (10 mM potassium phosphate, pH 7.4, 100 mM KCl, 20 mM NaCl, and 10 mM EDTA) at 37 ° C. for the times shown in FIG. Thereafter, 15 μl of the reaction solution was mixed with 1 μl of butylhydroxytoluene (576 mM in DMSO), and a part (3 μl) was spotted on a silica gel plate (Merck Silica gel 60 F254, Merck KGaA, Darmstadt, Germany). The silica gel plate on which the sample was spotted was developed with chloroform-methanol-acetic acid (6: 3: 1), and the spot was detected with silver nitrate or ultraviolet rays.

  As a result, as shown in FIG. 1, ubiquitin led to erythroascorbic acid production from ascorbic acid. This conversion could be detected after 5 minutes of reaction. After 12 hours of reaction, ascorbic acid disappeared, but erythroascorbic acid also disappeared, and a substance having no UV absorption (marked with *) was generated instead. On the other hand, ascorbic acid hardly changed in the absence of ubiquitin. Ori, SF, AsA, eAsA, and DHAsA in the figure indicate the origin, solvent tip, standard ascorbic acid, standard erythroascorbic acid, and standard dehydroascorbic acid, respectively (the same applies to FIGS. 3 and 4).

[Example 4]
<Confirmation of erythroascorbic acid generated from ascorbic acid by the action of ubiquitin>
A part of the reaction solution obtained by reacting ubiquitin and ascorbic acid for 6 hours under the same conditions as in FIG. 1 was analyzed by HPLC using Asahipak NH2 P-50 4D. Mobile phase: 0.05% aqueous formic acid containing 40% acetonitrile, flow rate: 1 ml / min, detection: UV (255 nm), temperature: room temperature.

  As a result of HPLC analysis, as shown in FIG. 2, the produced substance was completely in agreement with standard L-erythroascorbic acid.

[Example 5]
<Conversion reaction of dehydroascorbic acid to erythroascorbic acid by ubiquitin>
In the experiment whose results are shown on the left side of FIG. 3, highly purified ubiquitin derived from bovine erythrocytes (SEQ ID NO: 1) (SIGMA, St. Louis, MO, USA (Catalog No. U6253)) (5 μg / ml) or buffer Liquid and L-ascorbic acid (Wako Pure Chemicals) (17 mM), dehydroascorbic acid (17 mM) (CH Jun, WW Wells, Spontaneous conversion of L-dehydroascorbic acid to L-ascorbic acid-L-ascorbic acid acid acid.Arch.Biochem.Biophys.355 (1998) 9-14.), or 2,3-diketo-L-gulonic acid (16 mM) (Y. Kagawa. Enzymati) c studies on ascorbic acid catabolism in animals.I. Catabolism of 2,3-diketo-L-gulonic acid, J. Biochem. 51 (1962) 134-144.) was prepared using a buffer (10 mM phosphasate). (pH 7.4, 100 mM KCl, 20 mM NaCl, and 10 mM EDTA) at 37 ° C. for 0 or 10 minutes. Thereafter, 15 μl of the reaction solution was mixed with 1 μl of butylhydroxytoluene (576 mM in DMSO), and a part (3 μl) thereof was spotted on a silica gel plate (Merck Silica gel 60 F254, Merck KGaA, Darmstadt, Germany). The silica gel plate on which the sample was spotted was developed with chloroform-methanol-acetic acid (6: 3: 1), and the spot was detected with silver nitrate. In addition to the annotation in FIG. 1, DKG indicates the development position of standard 2,3-diketo-L-gulonic acid.

  In the experiment whose results are shown on the left side of FIG. 3, highly purified ubiquitin from bovine erythrocytes (SEQ ID NO: 1) (SIGMA, St. Louis, MO, USA (Catalog No. U6253)) (5 μg / ml) and dehydro Ascorbic acid (17 mM) (C. H. Jung, W. W. Wells, Spontaneous conversion of L-dehydroascorbic acid to L-ascorbic acid and L-erythroascorbic acid. Biol. 98. Biochem. Buffer solution (10 mM potassium phosphate, pH 7.4, 100 mM KCl, 20 mM NaCl, and 10 mM EDTA) Incubated for 10 minutes at 37 ° C. At that time, a reaction solution containing butylhydroxytoluene (BHT) (36 mM) or a reaction solution substituted with nitrogen gas was also used. Thereafter, 15 μl of the reaction solution was mixed with 1 μl of butylhydroxytoluene (576 mM in DMSO), and a part (3 μl) was spotted on a silica gel plate (Merck Silica gel 60 F254, Merck KGaA, Darmstadt, Germany). The silica gel plate on which the sample was spotted was developed with chloroform-methanol-acetic acid (6: 3: 1), and the spot was detected with silver nitrate. However, TLC spotted with the nitrogen-substituted reaction solution was developed with chloroform-methanol-acetic acid (5: 4: 1).

  As shown on the left of FIG. 3, erythroascorbic acid was produced by ubiquitin from ascorbic acid or dehydroascorbic acid, but not from 2,3-diketo-L-gulonic acid.

As shown on the right side of FIG. 3, erythroascorbic acid production from dehydroascorbic acid by ubiquitin is carried out by replacing the antioxidant butylhydroxytoluene (BHT in the figure) or nitrogen gas in the reaction liquid (N ₂ in the figure). Completely inhibited.

[Example 6]
<Conversion reaction of ascorbic acid and dehydroascorbic acid to erythroascorbic acid by recombinant ubiquitin mutant>
Highly purified ubiquitin derived from bovine erythrocytes (SEQ ID NO: 1) (SIGMA, St. Louis, MO, USA (Cat. No. U6253)) (5 μg / ml) or buffer, or recombinant human ubiquitin (K6R, Or K6 only [K11R / K27R / K29R / K33R / K48R / K63R], both purchased from Boston Biochem, Cambridge, MA, USA, catalog number UM-K6R or UM-K6O, respectively, and L-ascorbic acid (Wako Pure) Drug) (17 mM), or dehydroascorbic acid (17 mM) (CH Jun, WW Wells, Spontaneous conversion of L-dehydroascorbic acid to L-ascorbic cid and L-erythroascorbic acid.Arch.Biochem.Biophys.355 (1998) 9-14.) in buffer (10 mM potassium phosphate, pH 7.4, 100 mM KCl, 20 mM NaCl, and 10 mM EDTA). Incubated at 37 ° C. for 0 or 10 minutes. Thereafter, 15 μl of the reaction solution was mixed with 1 μl of butylhydroxytoluene (576 mM in DMSO), and a part (3 μl) was spotted on a silica gel plate (Merck Silicagel 60 F254, Merck KGaA, Darmstadt, Germany). The silica gel plate on which the sample was spotted was developed with chloroform-methanol-acetic acid (6: 3: 1), and the spot was detected with silver nitrate.

  As shown in FIG. 4, the K6R ubiquitin mutant did not have the ability to generate erythroascorbic acid from ascorbic acid or dehydroascorbic acid, but the K6 only mutant had the same activity as wild-type ubiquitin. . From these results, it was shown that lysine, which is the sixth amino acid from the N-terminal of ubiquitin, is important for erythroascorbic acid conversion activity from ascorbic acid or dehydroascorbic acid.

  From these facts, it is clear that ubiquitin exhibits the activity of converting ascorbic acid or dehydroascorbic acid to erythroascorbic acid based on structural features including the 6th lysine residue from the N-terminal in the molecule. It is. Therefore, utilization of ubiquitin is extremely effective in an efficient method for producing erythroascorbic acid.

From the above, erythroascorbic acid can be produced effectively by using ubiquitin.
Thus, according to the present invention, a novel method for producing erythroascorbic acid can be provided.

Claims (11)

A substance production method for producing a target substance using substance producing cells,
A production process for producing a target substance by the substance-producing cells in the presence of erythroascorbic acid as a cell activator;
A recovery step of recovering the target substance produced in the production step;
A material production method comprising:   The method for producing a substance according to claim 1, wherein the concentration of the erythroascorbic acid is 0.1 µg / ml to 1000 µg / ml.   The method for producing a substance according to claim 1 or 2, wherein the substance-producing cell is an animal cell.   The method for producing a substance according to any one of claims 1 to 3, wherein the substance-producing cell has a recombinant protein expression mechanism for expressing the target substance.   A cell growth promoter comprising a growth promoting effective amount of erythroascorbic acid.   An expression enhancer comprising an effective expression enhancing amount of erythroascorbic acid. Contacting ascorbic acid or dehydroascorbic acid with ascorbic acid degrading bacteria in the presence of ubiquitin to produce erythroascorbic acid;
Recovering erythroascorbic acid from the reaction system after the contact;
A process for producing erythroascorbic acid comprising   The method for producing erythroascorbic acid according to claim 7, wherein the ubiquitin is an ubiquitin derived from an animal.   The method for producing erythroascorbic acid according to claim 7, wherein the ubiquitin is bovine-derived ubiquitin.   The method for producing erythroascorbic acid according to any one of claims 7 to 9, comprising adding ubiquitin or ubiquitin-producing cells to the culture system of the ascorbic acid-degrading bacterium.   The method for producing erythroascorbic acid according to any one of claims 7 to 10, wherein the ubiquitin is a natural ubiquitin or a ubiquitin mutant containing K6.

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