JP2005312463A - Method for producing human serum albumin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing human serum albumin, by which the production of the human serum albumin is increased. <P>SOLUTION: This method for producing the human serum albumin is characterized by culturing a human serum albumin (HSA)-producing host prepared in a genetic operation under a temperature condition of 21 to 29°C. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an improvement in a method for producing human serum albumin (hereinafter referred to as HSA) by culturing a host transformed by genetic manipulation.

  HSA is a major protein component of plasma and is used as a medicine, for example, as a therapeutic agent for hemorrhage, shock or burn patients, hypoproteinemia, fetal erythroblastosis and the like.

  Currently, HSA is mainly produced as a product from a collected blood fraction. However, this production method is uneconomical and it is difficult to supply raw blood. Moreover, there is a problem that blood contains an undesirable substance such as hepatitis virus.

  In recent years, with the advent of recombinant DNA technology, it has become possible to produce a wide variety of useful polypeptides by microorganisms and cells, and research and development of mass production of genetically modified technology for HSA has also been actively conducted. However, the production yield is low, and the problem is how to establish a high-purity and low-cost industrial production technology.

In culturing an HSA-producing host prepared by genetic manipulation in a medium, conventionally, in particular, when the host is yeast, it has been usually cultured at 30 ° C. (Patent Documents 1 and 2).
JP-A-3-83595 JP-A-4-299984

  An object of the present invention is to further increase the production amount of HSA by improving the culture conditions under such technical background.

  As a result of repeated studies to solve the above-mentioned problems, the present inventors have conducted HSA production by culturing an HSA-producing host prepared by genetic manipulation in a medium under a temperature condition of 21 to 29 ° C. The inventors have found that the degree of growth of the sex host and the production amount of HSA can be increased, and that the coloring degree of HSA can be suppressed, and the present invention has been completed.

  The present invention is characterized in that an HSA-producing host prepared by genetic manipulation is cultured at 21 to 29 ° C. According to the method, the amount of HSA produced and the HSA-producing host There is an effect that the degree of proliferation increases, and at the same time, the degree of coloring of HSA can be reduced.

The HSA-producing host prepared by genetic manipulation used in the present invention is not particularly limited as long as it is prepared through genetic manipulation and can produce HSA. Even if it is a thing, it can utilize suitably. Specifically, bacteria (for example, Escherichia coli, yeast, Bacillus subtilis, etc.), animal cells, etc. that have been made HSA-productive through genetic manipulation can be mentioned. In particular, in the present invention, it is preferable to use yeast, especially Saccharomyces spp., Pichia spp. Also, auxotrophic strains and antibiotic sensitive strains can be used. Furthermore, Saccharomyces cerevisiae AH22 strain (a, his 4, leu 2, can 1), Pichia pastoris GTS115 (his 4), Kluyveromyces lactis which are G418 sensitive strains MW98-8C (α, uraA, arg, lysK ⁺ , pKD1 °) and the like are preferably used.

The methods for preparing these HSA-producing hosts, the methods for producing HSA by culturing them, and the methods for separating and collecting HSA from the culture are all carried out by adopting known methods and similar methods. For example, as a method for preparing an HSA-producing host (or HSA-producing strain), for example, a method using a normal human serum albumin gene (Japanese Patent Laid-Open Nos. 58-56684, 58-90515, 58-150517) ), A method using a novel human serum albumin gene (Japanese Unexamined Patent Publication Nos. 62-29985 and 1-98486), a method using a synthetic signal sequence (Japanese Unexamined Patent Publication No. 1-2240191), a serum albumin signal A method using a sequence (Japanese Patent Laid-Open No. 2-167095), a method of incorporating a recombinant plasmid onto a chromosome (Japanese Patent Laid-Open No. 3-72889), a method of fusing hosts together (Japanese Patent Laid-Open No. 3-53877), methanol method where mutation is containing medium, a method using a mutant AOX ₂ promoter (Japanese Patent Laid-Open No. 6-90768 JP-A-4-299984), expression of HSA by Bacillus subtilis (JP-A 62-25133), expression of HSA by yeast (JP-A 60-41487, 63-39576, 63-74493) And the expression of HSA by Pichia yeast (JP-A-2-104290).

Among these, the method of causing mutation in a methanol-containing medium is specifically performed as follows. That is, first, a plasmid having a suitable host, preferably Pichia yeast, specifically, a transcription unit that expresses HSA under the control of the AOX ₁ promoter in the AOX ₁ gene region of GTS115 strain (NRRL deposit number Y-15851) by a conventional method. To obtain a transformant (see JP-A-2-104290). This transformant has a weak growth ability in methanol medium. Therefore, according to the method described in JP-A-4-299984, this transformant is cultured in a methanol-containing medium to cause mutation, and only viable strains are recovered. In this case, the methanol concentration is exemplified by about 0.0001 to 5%. The medium may be a synthetic medium or a natural medium. Examples of culture conditions include 15 to 40 ° C. and about 1 to 1000 hours.

  Moreover, as a method for culturing an HSA-producing host (ie, a method for producing HSA), in addition to the methods described in the above-mentioned publications, high-concentration glucose is supplied in small and moderate amounts by fed batch culture. A method of obtaining a high concentration of bacterial cells and products while avoiding high-concentration substrate inhibition on the bacterial cells (Japanese Patent Laid-Open No. 3-83595), and a method of enhancing the production of HSA by adding fatty acids to the medium (Japanese Patent Laid-Open No. 4) -293495) and the like.

  Further, as a method for separating and collecting HSA, in addition to the methods described in the above-mentioned publications, protease inactivation by heat treatment (JP-A-3-103188), anion exchanger, hydrophobic carrier and activated carbon are used. Examples thereof include a coloring suppression method (JP-A-4-54198) by separating HSA and a coloring component using at least one selected from the group.

  As the medium used for culturing the transformed host, a medium known in this field is usually used, and the culture conditions are carried out according to a general ordinary method. The medium may be either a synthetic medium or a natural medium, and is preferably a liquid medium. For example, as a synthetic medium, various sugars as a carbon source, urea, ammonium salt, nitrate, etc. as a nitrogen source, various vitamins, nucleotides, etc. as micronutrients, and Mg, Ca, Fe, Na, K, Mn as inorganic salts , Co, Cu and the like. YNB liquid medium [0.7% yeast nitrate base (Difco), 2% glucose] and the like. Examples of the natural medium include YPD liquid medium [1% yeast extract (Difco), 2% bactopeptone (Difco), 2% glucose]. The pH of the medium may be neutral, weakly basic, or weakly acidic. Preferably it is pH 5.7-6.5. In the case of a methanol-assimilating host, a methanol-containing medium can be used. In this case, the methanol concentration is about 0.01 to 5%.

  With respect to the culture temperature, a preferable range particularly when the production amount of HSA is used as an index is 21 to 29 ° C, preferably 21 to 28 ° C, more preferably 21 to 25 ° C, and further preferably 21 to 23 ° C. When culturing at a temperature higher than 29 ° C., cell growth and HSA production are suppressed, and problems such as an increase in HSA coloration occur. Also, when culturing at a temperature lower than 21 ° C., It is not preferable because cell growth and HSA production are suppressed. On the other hand, up to about 21 ° C., the cell growth degree and the amount of HSA production tend to increase as the culture temperature decreases. In practice, it is not preferable because temperature management is costly and cumbersome because a separate device is required. In particular, when commercializing an invention, it is necessary to consider economics such as cost and labor as important factors in addition to the effects of increasing the production amount of HSA and suppressing the degree of HSA coloring. Therefore, a suitable culture temperature when comprehensively determined from such a viewpoint is 23 to 28 ° C, more preferably 25 to 27 ° C.

  When an HSA-producing host is cultured at the above culture temperature, for example, in the case of yeast, the amount of HSA produced can be increased by 1.2 to 1.5 times compared to the conventional culture at 30 ° C., and HSA The coloring degree of can also be reduced by 10 to 30%.

  The culture time is about 1 to 1000 hours, and the culture is carried out by a batch culture method, a semi-batch culture method or a continuous culture method with standing, shaking, stirring or aeration. In addition, it is preferable to perform preculture prior to the culture. As the medium at this time, for example, a YNB liquid medium or a YPD liquid medium is used. The culture conditions for pre-culture are as follows. That is, the culture time is preferably 10 to 100 hours, and the temperature is preferably about 30 ° C. for yeast and about 37 ° C. for bacteria.

  Thus, after completion of the culture, HSA is collected from the culture filtrate or the cells and cells by known separation and purification means, respectively.

  EXAMPLES Hereinafter, examples and reference examples will be shown to specifically describe the present invention, but the present invention is not limited to these examples and the like.

Example 1
(1) Preparation of strain to be used HSA expression plasmid pPGP1 obtained by removing the 5 ′ non-coding region of the HSA gene from plasmid pHSA113 having a transcription unit expressing HSA under the control of the AOX ₁ promoter described in JP-A-2-104290 It was created. pPGP1 has cDNA encoding the amino acid sequence of normal HSA [The amino acid sequence of normal HSA and the chromosomal DNA sequence encoding normal HSA are described in J. Biol. Chem., 261, 6747-6757 (1986). Yes]. Then, according to the method described in JP-A-2-104290, a fragment obtained by cleaving the AOX ₁ gene region of Pichia pastoris GTS115 (his4) with the HSA expression plasmid pPGP1 with Not1 restriction enzyme To obtain transformant PC4130. Since this strain does not have the AOX ₁ gene, the growth ability in a medium containing methanol as a carbon source is low (Mut-strain).

PC4130 was inoculated into 3 ml of YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose), and inoculated into 50 ml of YPD medium so that the initial OD ₅₄₀ = 0.1 after 24 hours. The cells were inoculated into 50 ml of YPD medium so that the initial OD ₅₄₀ = 0.1 after culturing at 30 ° C. for 3 days. Further, the same passage was repeated every 3 days. At each passage, the cells were diluted with sterilized water to 10 ⁷ cells / plate, and 2% MeOH-YNBw / oa. a. Plates (0.7% yeast nitride base without amino acid, 2% methanol, 1.5% (agar powder)) were cultured at 30 ° C. for 5 days to determine the presence or absence of colonies. As a result, 12 days 20 colonies were generated from the 2% MeOH-YNBw / oa.a plate applied after passage, on which the mut-strain could hardly grow and the mut + strain could grow, i.e., the plate had no colonies. This indicates that the assimilation of methanol was increased, and a strain converted to Mut + was obtained, and one of the resulting colonies was appropriately diluted with sterile water to obtain 2% MeOH-YNBw. Spread on a plate and isolated to a single colony, one of which was named GCP101.

  AOX2 promoter isolated from GCP101 strain [mutant. Using the natural AOX2 promoter (YEAST, 5, 167-177 (1988) or Mol. Cell, Biol., 9, 1316-1323 (1989) with the 255th base upstream of the start codon mutated from T to C) The plasmid pMM042 for HSA expression was constructed and introduced into the Pichia pastoris GTS115 strain to obtain a transformant UHG42-3 strain (JP-A-4-29984).

(2) Medium composition YPD medium (2% bactopeptone, 1% yeast extract, 2% glucose) was used for preculture. The composition of the batch medium is shown in Table 1, and the composition of the feed medium is shown in Table 2.

(3) Cultivation method using 3 liter fermenter <1> Pre-culture 1 ml was inoculated from a frozen stock vial into a 300 ml Erlenmeyer flask with baffle containing 50 ml of YPD medium, and cultured with shaking at 30 ° C. for 24 hours. . 14 ml of the culture solution after 24 hours of culture was inoculated into 700 ml of a batch medium.
<2> Main Culture 14 ml of the preculture was inoculated into 700 ml of the batch medium, and cultured with aeration and stirring using a 3 liter mini jar fermenter. The culture temperature was set to 15, 21, 23, 25, 27, 28, and 30 ° C. The lower limit of the stirring speed was set to 200 rpm, and the upper limit was set to 1000 rpm. The batch culture was performed while controlling the dissolved oxygen concentration to maintain about 50% of the saturation concentration. Addition of the feed medium was started when glycerol in the medium was consumed in batch culture. The pH was controlled at a constant value of 6.2. The defoaming was carried out by appropriately adding an antifoaming agent to the medium as necessary. The culture was performed for 360 hours.

(4) Culture method using 1200 liter fermenter <1> Pre-culture 1 ml of glycerol frozen stock strain was inoculated into a 1,000 ml Erlenmeyer flask with baffle containing 200 ml of YPD medium and shaken at 30 ° C. for 24 hours. Cultured.
<2> Pre-culture The culture solution was inoculated into a 10-liter jar fermenter containing 5 liters of YPD medium, and cultured with aeration and stirring at 30 ° C. for 24 hours. Aeration was controlled so that the dissolved oxygen concentration maintained about 50% of the saturated dissolved oxygen concentration. Moreover, pH control was not performed in the preculture.

<3> Main culture The preculture was inoculated into 250 liters of a batch culture medium, and aerated and stirred with a 1,200 liter fermenter. The culture temperature was set to 23, 25, 27, 29, and 30 ° C. Batch culture was started while controlling the stirring speed so that the dissolved oxygen concentration maintained about 50% to 30% of the saturated dissolved oxygen concentration. Addition of the feed medium was started when glycerol in the medium was consumed in batch culture. The pH was controlled at a constant value of 5.85. Antifoaming was performed by adding an antifoaming agent as appropriate to the medium. The culture was performed for 360 hours.

Test Example 1 Measurement of Bacterial Cell Concentration In the culture at each culture temperature in Example 1, the culture solution is sampled at an arbitrary culture time and appropriately diluted with distilled water so that the OD ₅₄₀ value at the time of measurement is 0.3 or less. After that, the absorbance at 540 nm was measured using a spectrophotometer (UV200 type, manufactured by Shimadzu Corp.). In this experiment, the conversion from the absorbance to the dry cell mass was OD ₅₄₀ value / 5.2. Tables 3 and 4 show the maximum cell concentration at each culture temperature.

Test Example 2 Evaluation of HSA Production Amount and Color Degree In the culture at each culture temperature in Example 1, the culture solution was sampled at an arbitrary culture time and centrifuged at 15,000 rpm for 5 minutes. The obtained supernatant was clarified through ultrafree C3HV and then subjected to gel filtration analysis by HPLC.
Column: Tosoh TSKgel G3000SW _X1
Mobile phase: 0.3M NaCl, 50mM Na-Phosphate, 0.1% NaN _3, pH6.5
Flow rate: 0.7 ml / min.
Injection volume: 50 μl
Detection: A _28O , A ₃₅₀ (2 wavelengths)

Tables 3 and 4 also show the evaluation of HSA production amount and coloring degree. The production amount is expressed as% of the total production amount (g) at each temperature when the total HSA production amount (g) of culture at 30 ° C. is defined as 100. In addition, the degree of coloring was evaluated by the absorbance ratio (A ₃₅₀ / A _{28 O} ) at ₃₅₀ nm and 280 nm for the culture solution sampled immediately before the end of the culture (after 359 hours).

Claims (1)

  A method for producing human serum albumin, comprising culturing a human serum albumin-producing host prepared by genetic manipulation under a temperature condition of 21 to 29 ° C.