JP4240558B2 - Methanol-utilizing yeast culture method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for culturing a methanol-utilizing yeast transformed by introducing a gene encoding a heterologous protein, and to a method for efficiently producing the heterologous protein.
[0002]
[Prior art]
Advances in recombinant DNA technology have made it possible for microorganisms to produce a wide variety of useful peptides and proteins. Such a protein is produced by culturing a transformed host microorganism by introducing a gene encoding the target protein into the host microorganism.
[0003]
In general, E. coli is used as the host microorganism. Escherichia coli has a large amount of expression of heterologous proteins and has excellent characteristics. However, when E. coli is used as a host, it is not without problems.
[0004]
One of the problems is that the expressed protein forms a cell inclusion body (inclusion body), becomes insoluble, and is folded into a structure having no activity. As a result, in order to obtain a human protein having physiological activity, it is necessary to first perform a solubilization treatment using a denaturing agent or the like, and then perform a reactivation treatment. However, structural analogs of the target protein may be generated due to these operations. Difficulties in recovering proteins produced using E. coli as a host and the problems that the produced proteins sometimes have low physiological activity are associated with post-translational processing of proteins produced by genetic recombination (eg, glycosylation). ) May not be possible.
[0005]
Unlike E. coli, yeast, which is a eukaryotic microorganism, has an endoplasmic reticulum, Golgi apparatus, etc., as well as animal cells, and has a higher-order structure capable of expressing physiological activity of heterologous proteins derived from eukaryotic cells. It is known that it can be produced, and has an advantage that it can be grown in a single cell and has a high growth rate compared to animal cells and the like, so that it can be easily cultured in large quantities.
[0006]
[Problems to be solved by the invention]
In yeast, Saccharomyces cerevisiae (Saccharomyces cerevisiae) is widely used as a host for expressing a gene encoding a heterologous protein.
[0007]
However, Saccharomyces cerevisiae is difficult to grow to a high cell density, and the production amount per culture solution is not large.
[0008]
On the other hand, methanol-assimilating yeasts such as Pichia pastoris, Hansenula polymorpha, and Candida bodiniii are industrially used in terms of production amount per culture solution. Suitable for use in production of heterologous proteins on a scale. These yeasts can be cultured to high cell densities using either glycerol or methanol as the sole carbon source.
[0009]
For expression of heterologous proteins in methanol-utilizing yeast such as Pichia pastoris, a promoter inducible by methanol is usually used. That is, after culturing until reaching a high cell density by adding glycerol by fed-batch culture method, methanol is continuously or periodically added to induce protein expression. At this time, the addition rate is determined so that the methanol concentration falls within a certain range.
[0010]
Methanol assimilating yeasts such as Pichia pastoris can grow using methanol as a single carbon source and ammonia as a single nitrogen source. Methanol is oxidized into formic acid via formaldehyde in the cell, and finally decomposed into carbon dioxide and water. Methanol-assimilating yeast acquires energy by using the above reaction as a catabolic metabolism of a carbon source, but in order to connect it to the synthesis reaction of biological components such as amino acids, that is, anabolic metabolism, it passes through a metabolic system called a serine cycle. is required. In order for yeast to produce a heterologous protein, it is considered necessary to acquire energy exceeding the energy required for growth and to enhance the ability to synthesize amino acids as constituent components. To this end, from the viewpoint of metabolic engineering, it is necessary to activate the catabolic metabolic system for energy acquisition and to activate the anabolic metabolic system such as amino acid synthesis.
[0011]
Therefore, an object of the present invention is to express a heterologous protein by culturing a methanol-utilizing yeast transformed by introducing a gene placed under the control of a promoter in which the gene encoding the heterologous protein can be induced by addition of methanol. It is an object of the present invention to provide a method capable of improving the productivity of heterologous proteins.
[0012]
[Means for Solving the Problems]
The present invention, which has been accomplished to achieve the above object, is a method for culturing a methanol-utilizing yeast transformed with a gene encoding a heterologous protein to express the heterologous protein, wherein methanol is added. On the other hand, a method for cultivating methanol-assimilating yeast, characterized in that an amount of aspartic acid having a certain correlation with the amount is added. Hereinafter, the present invention will be described in detail.
[0013]
In the present invention, methanol-assimilating yeast transformed with an expression vector linked with a gene encoding a heterologous protein under the control of a promoter that can be induced by methanol is used.
[0014]
In the present invention, methanol-assimilating yeast is used as a host. Examples of the methanol-assimilating yeast include those belonging to the genus Pichia, Hannucella or Candida. Among them, Pichia pastoris, Hansenula polymorpha, Candida boidini and the like can be exemplified as particularly preferable yeasts.
[0015]
By using the method of the present invention, various heterologous proteins can be efficiently produced. The heterologous protein to be expressed is not particularly limited, and examples thereof include human and other mammalian proteins. Among them, a human protein having physiological activity is an important protein in the field of pharmaceuticals and the like, and can be exemplified as an important heterologous protein in the present invention. Examples of such a human protein having physiological activity include those selected from the group consisting of interleukin 6, interleukin 6 receptor and derivatives thereof.
[0016]
The gene encoding the heterologous protein as described above is used by being linked under the control (downstream side) of a promoter that can be induced by methanol. The promoter is a promoter for expression of a gene encoding an enzyme of methanol metabolism, and examples thereof include an alcohol oxidase gene promoter isolated from Pichia pastoris. As a method for transforming yeast using an expression vector containing a gene in which a gene encoding a heterologous protein is linked under the control of a promoter, a usual method can be used.
[0017]
The present invention monitors and controls one or more factors involved in yeast growth such as pH, dissolved oxygen concentration, stirring speed, temperature or aeration rate in order to monitor the state of yeast during culture. However, it is preferable to carry out. A culture apparatus for carrying out such culture is conventionally known, and a commercially available apparatus can be used. The culture method is not particularly limited except that methanol is added continuously or periodically, and conventionally used methods such as a batch culture method, a fed-batch culture method, and a continuous culture method can be used. .
[0018]
The addition of methanol may be performed using a pump or the like while monitoring the factors involved in yeast growth as described above, but it is particularly preferable to automatically control the addition amount according to the result of the monitoring. . In addition, it is preferable to start the addition after expressing the heterologous protein, that is, usually after the yeast culture is started and the number of cells is sufficient to produce the heterologous protein. The invention is not limited to this. That is, even when methanol or aspartic acid is added from the beginning of the culture, it is within the scope of the present invention. As for the addition of methanol, it can be exemplified that it is added continuously or periodically. However, even if it is added irregularly according to the results of the monitoring described above, the amount of methanol added at that time is constant. It is within the scope of the present invention to add an amount of aspartic acid having the following correlation.
[0019]
The present invention relates to a culture method in which methanol is added while a yeast is cultured to express a gene encoding a heterologous protein. When the heterologous protein is expressed, that is, when the heterologous protein is produced by expressing at least the introduced gene. An amount of aspartic acid having a certain correlation with the amount of methanol added is added, thereby enhancing the production of heterologous proteins by methanol-utilizing yeast.
[0020]
The addition of aspartic acid may be performed using a pump or the like, for example, as in the case of methanol, but it is preferable that the amount added is automatically controlled in relation to the amount of methanol added.
[0021]
The amount of aspartic acid added can be exemplified by, for example, obtaining the amount of methanol added as the amount added per unit time and determining the amount of aspartic acid to be added per unit time so as to correlate with the value. Of course, if methanol is added periodically or irregularly, it is possible to determine and add the amount of aspartic acid to be added so as to correlate with the amount of methanol added for each addition. Moreover, the addition of aspartic acid does not need to be performed completely simultaneously with the addition of methanol, and the addition may be slightly shifted in time.
[0022]
The correlation between the addition amount of methanol and the addition amount of aspartic acid is such that the addition amount of aspartic acid / the addition amount of methanol is within a range of 1/10 to 1/500 by weight, preferably 1/50 to 1/200. Within range. This correlation does not need to be constant during one culture operation, and can be changed within the above range.
[0023]
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to an Example.
[0024]
Example 1
Human interleukin 6 receptor (hereinafter abbreviated as IL-6R) was expressed as a heterologous protein using Pichia pastoris as a methanol-assimilating yeast and alcohol oxidase 1 promoter (AOX1) as a methanol-inducible promoter.
[0025]
100 mL medium (3 g of H3PO4, 2.38 g of K2SO4, 0.65 g of KOH, 0.15 g of CaSO4 · 2H2O, 1.9 g of MgSO4 · 7H2O, 1.4 mg of CuSO4 · 5H2O, 0.1 mg of KI, 0.7 mg of MnSO · H2O per liter) , NaMoO4 · 2H2O 0.05 mg, H3BO30.005 mg, CoCl2 · 6H2O 0.12 mg, ZnSO4 · 7H2O 4.7 mg, FeSO4 · 7H2O 15.2 mg, biotin 0.046 mg, glycerol 10 g) A shake flask was inoculated with 20% glycerol frozen strain and shake-cultured at 30 ° C. for 20 hours to prepare a preculture.
[0026]
10 L medium (18 g H3PO4, 14.28 g K2SO4, 3.9 g KOH, 0.9 g CaSO4 · 2H2O, 11.7 g MgSO4 · 7H2O, 8.4 mg CuSO4 · 5H2O, 1.1 mg KI, 4.2 mg MnSO4H2O, NaMoO4) 16L capacity jar fermenter (NBS) containing 0.3 mg of 2H2O, 0.03 mg of H3BO3, 0.7 mg of CoCl2 · 6H2O, 28 mg of ZnSO4 · 7H2O, 91 mg of FeSO4 · 7H2O, 0.28 mg of biotin and 25 g of glycerol SF116) manufactured by the company was inoculated with 100 mL of the preculture solution, and aeration and stirring culture was started at 30 ° C. Ammonia water was added for the purpose of pH adjustment and nitrogen source addition.
[0027]
From 24 hours after the start of the culture, methanol was added for the purpose of inducing expression and supplementing the carbon source. The methanol concentration in the culture solution is continuously measured with an on-line methanol sensor for fermentation (alcohol sensor manufactured by Able Co., Ltd.), and the methanol is automatically adjusted with a tubing pump so that the methanol is in the range of 0.8 to 1.2%. Was added. A 10% aqueous solution of sodium aspartate was prepared and charged into the reservoir. Asparagine in conjunction with the addition of methanol so that 1/10 weight of sodium aspartate 10% (weight / weight) aqueous solution is added per unit time with respect to the weight of methanol added per unit time. Sodium acid aqueous solution was added. Thereby, in this example, the addition weight of the sodium aspartate aqueous solution per unit time becomes 1/100 of the addition amount of methanol (the addition amount per unit time of aspartic acid itself is 1/100 of methanol by weight ratio). 130).
[0028]
The culture was completed 210 hours after the start of the culture. As a result of measuring the OD of the culture solution, the value of OD 600 nm was 300.
[0029]
The culture solution was centrifuged at 10,000 × g, and the concentration of IL-6R in the supernatant was quantified by the EISA method, which was 50 mg / L. For quantification by ELISA, a rabbit anti-human IL-6R monoclonal antibody was used as a secondary antibody for the mouse anti-human IL-6R monoclonal antibody, and an alkaline phosphatase-labeled goat anti-rabbit IgG antibody was used for detection.
[0030]
For comparison, culturing with a 16 L capacity jar fermenter was performed under the same conditions as described above using the same strain as described above. However, the feed of the carbon source and the nitrogen source was only methanol and ammonia, and no aspartic acid was added.
[0031]
In the same manner as described above, the culture was terminated 210 hours after the start of the culture, and the OD of the culture solution was measured. As a result, the value of OD 600 nm was 330. Further, the culture broth was centrifuged, and the concentration of IL-6R in the supernatant was quantified by ELISA, which was 20 mg / L.
[0032]
As described above, when transformed yeast is cultured without adding aspartic acid but only methanol, the amount of IL-6R produced is less than half that of when aspartic acid is fed in conjunction with methanol. There wasn't.
[0033]
Example 2
Using the same strain as in Example 1, culturing with a 2 L jar fermenter (manufactured by Oriental Yeast) was performed under the same conditions. The composition of the medium was the same as in Example 1, and the amount charged was 1 L. In this example, the addition amount per unit time of the sodium aspartate aqueous solution was 1/10 of the addition amount per unit time of methanol.
[0034]
As in Example 1, the culture was completed 210 hours after the start of culture, and the OD of the culture solution was measured. As a result, the value of OD 600 nm was 320. Further, the culture solution was centrifuged, and the concentration of IL-6R in the supernatant was quantified by ELISA, which was 45 mg / L. For comparison, the same strain as described above was used, and culture was performed using a 2 L jar fermenter under the same conditions as described above. However, the feed of the carbon source and the nitrogen source was only methanol and ammonia, and no aspartic acid was added.
[0035]
In the same manner as described above, the culture was terminated 210 hours after the start of the culture, and the OD of the culture solution was measured. As a result, the value of OD 600 nm was 310. Further, the culture broth was centrifuged, and the concentration of IL-6R in the supernatant was quantified by ELISA, which was 17 mg / L.
[0036]
As described above, when transformed yeast is cultured without adding aspartic acid but only methanol, the amount of IL-6R produced is less than half that of when aspartic acid is fed in conjunction with methanol. There wasn't.
[0037]
【The invention's effect】
According to the present invention, when cultivating a methanol-assimilating yeast transformed with a gene encoding a heterologous protein and expressing the heterologous protein, methanol is added and an amount correlated with the amount of methanol added. The expression level of the heterologous protein can be enhanced by a simple operation of adding asparagine.
[0038]
Therefore, according to the present invention, when methanol-assimilating yeast is used as a host, a heterologous protein can be produced more efficiently than before.

Claims (5)

A method of culturing a methanol-assimilating yeast transformed with a gene encoding a heterologous protein to express the heterologous protein, wherein methanol is added so that the methanol concentration in the culture solution falls within a certain range. It was added to obtain the amount of methanol addition amount per unit time, of methylotrophic yeast which comprises adding a 1 / 10-1 / 500 aspartic acid in a weight ratio for the said amount Culture method. The method according to claim 1, wherein the yeast is a yeast belonging to the genus Pichia, Hansenula or Candida. 3. A method according to claim 2, characterized in that the yeast is Pichia pastoris, Hansenula polymorpha or Candida bodiniii species. The method according to any one of claims 1 to 3, wherein the heterologous protein encoded by the gene is a human protein having physiological activity. The method according to claim 4, wherein the physiologically active protein is a physiologically active protein selected from the group consisting of interleukin 6, interleukin 6 receptor and derivatives thereof.