JPH02234673A - Production of protein with yeast as host - Google Patents

Abstract

PURPOSE:To obtain the title protein of higher organism origin markedly improved in its secretory productivity by feeding a carbon source and/or nitrogen source at amount(s)s more than the minimum amount required for the proliferation of yeast in producing a foreign protein using the transformed yeast. CONSTITUTION:When a foreign protein is to be produced using yeast transformed by secretory manifestation vector containing promoter and alien gene, a carbon source and/or nitrogen source is (are) fed to a culture medium, at amount(s) more than the minimum amount required for the proliferation of the transformed yeast. For example, as the carbon source, 80-150g/l of glucose or succharose, and as the nitrogen source, 10-30g/l of ammonium sulfate are fed, respectively. And, as inorganic nutritive salts, for example, 1-3g/l of dipotassium phosphate, 1-3g/l of magnesium sulfate heptahydrate, 0.5-2g/l of potassium chloride, 10-50mg/l of ferrous sulfate heptahydrate, etc., are fed, respectively.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing useful proteins by using recombinant DNA technology, and more specifically, the present invention relates to a method for producing useful proteins using recombinant DNA technology. The present invention relates to a method for secreting and producing useful proteins derived from higher organisms, such as , in a high yield.

[Prior art]

In recent years, many attempts have been made to use recombinant DNA technology to produce large amounts of useful proteins of human and biological origin using microorganisms. Among these, yeast can avoid the contamination of toxic substances such as pyrogens compared to the conventionally commonly used Escherichia coli, making the products safer and ensuring that genetic products derived from higher organisms have the correct structure. It has become one of the hosts that deserves attention due to its ease of use.

Furthermore, methods of secreting and producing target proteins outside the bacterial cells are being actively attempted for reasons such as preventing degradation by proteases and simplifying purification.

However, research on the secretory production of proteins derived from higher organisms using yeast as a host mainly focuses on the construction of secretory expression vectors,
The research is limited to basic studies at the genetic level, such as improving promoters and secretion signals, and there is no study aimed at industrial production, that is, studies on stabilizing transformants, or culture engineering studies such as medium composition and culture conditions. The current situation is that not much is being done. Therefore, the secretory production level remains low.

In this regard, the present inventors have attempted to mass-produce human lysozyme using recombinant DNA technology, and have already succeeded in expressing the synthetic human lysozyme gene in yeast and secreting the produced human lysozyme outside of the cells (particularly Kaisho 62-2
48488), but at this stage the secretory production of human lysozyme is at its maximum level of 0.48488). The level was as low as 6 tng/j2, which was still insufficient to meet the demands of industrial production. On the other hand, regarding the secretory productivity of human lysozyme, there have been some attempts to improve the DNA sequence of the signal peptide and succeeded in increasing the amount of secretory production of human lysozyme (Japanese Patent Application Laid-Open No. 63-233789). At present, there are no examples of studies that have been carried out from the perspective of culture engineering in order to improve the production, and it must be said that the secretory production volume is still at a low level, failing to meet the demands of industrial production. do not have.

[Problem to be solved by the invention]

The problem of the present invention is to solve the problem from a viewpoint that has not been considered in the past, in order to improve the secretion productivity of proteins derived from higher organisms when yeast is used as a host. By focusing on the relationship between efficiency and external environmental factors, especially the culture medium composition such as carbon sources, nitrogen sources, and inorganic nutrients, and preparing the optimal culture medium composition, it is possible to improve the ability of the above-mentioned proteins derived from higher organisms when using yeast as a host. The aim is to dramatically improve the secretory biology of .

[Means to solve the problem]

Conventionally, the culture medium composition for secretory production of proteins derived from higher organisms by yeast consists of the minimum nutritional components necessary for the growth of the host yeast, and there have been no studies that have been conducted from the viewpoint of productivity of the target heterologous protein.

In contrast, the present invention relates to improving and reconfiguring the culture medium composition, including carbon sources, nitrogen sources, and inorganic nutrient salts, from the viewpoint of protein productivity.

Specifically, the carbon source and nitrogen source are supplied in an amount higher than the minimum amount necessary for the growth of the transformed yeast.
For example, ammonium sulfate is supplied as a nitrogen source at 10 to 30 g/ffi. Also, as inorganic nutritional salts, for example, dipotassium phosphate 1 to 3 g/! !
．． , magnesium sulfate heptahydrate 1-3 g/l, potassium chloride 0.5-2 g/l, ferrous sulfate heptahydrate 1.0
~50+ng/p! etc.

With such a medium composition, it is possible to significantly improve the secretion productivity of proteins derived from higher organisms by yeast. Here, proteins applicable to the present invention include proteins derived from higher organisms such as human lysozyme, human epidermal growth factor, and hirudin, but it is of course effective for secretory production of any protein other than these. .

In particular, lysozyme has the action of lysing various bacteria, so it can be used as a preservative for foods, medicines, etc., and as an antibacterial agent, either alone or in combination with antibiotics. It also has anti-inflammatory effects, anti-bleeding effects, and sputum-expurgating effects for people with respiratory disorders, so it can be used as a pharmaceutical for those patients.

Lysozyme is an enzyme protein that is widely distributed in the secretions and organ tissues of the human body and animals, but chicken egg white contains a particularly large amount of lysozyme, and it is relatively easy to isolate highly pure lysozyme. Therefore, lysozyme currently used as a preservative in foods, etc., or as a pharmaceutical agent, such as in anti-inflammatory enzyme preparations and cold medicines, is derived from chicken eggs.

However, when these chicken egg white-derived lysozymes are used for medicinal purposes, hypersensitivity symptoms such as rash and redness often appear, which are considered as side effects of the immune response caused by foreign proteins.

In view of this situation, the present inventors attempted mass production of human lysozyme using recombinant DNA technology.
We have already succeeded in expressing the synthetic human lysozyme gene in yeast and secreting the produced human lysozyme extracellularly. (JP 62-248488) Next, as a host yeast that can be used in the present invention, Saccharomyces cerevi
siaeX2180-IA strain, Saccharomyc
S. cerevisiae X2180-IB strain, Sa
ccharomyces cerevisiae S28
8 C strain (both Yeast GeneticSt
ock Center (Department of Biophysics and Medical Physics, University of California, Berkeley, USA) and mutant derivatives derived from these strains. Promoters include enolase 1 promoter, 3-phosphoglycerate kinase promoter, etc. Yeast glycolytic enzyme genes such as , glyceraldehyde-3-phosphate dehydrogenase promoter can be used.

Enolase is one of the glycolytic enzymes that catalyzes the interconversion of 2-phosphoglyceric acid and phosphoenolpyruvate, and is a highly expressed enzyme that accounts for several percent of the total proteins in yeast.

By the way, it is known that there are often several types of isozymes in glycolytic enzymes, and their expression is controlled differently depending on the type of carbon source and culture conditions, and enolase also has two types, ENO 1 and ENO 2. There are several genes. Among these, ENO 1 has a strong promoter that induces expression under aerobic continuous culture conditions for producing baker's yeast, and is suitable for practical production of useful proteins using yeast. be.

Furthermore, it goes without saying that as the secretion signal, a signal suitable for the production of each protein can be used, and as the expression vector, any type of vector that can be replicated in yeast can be used.

The following example is an example in which human lysozyme was secreted and expressed in the medium of the present invention using egg white lysozyme signals under the control of the ENO 1 promoter, but the present invention is not limited to these in any way. do not have.

〔Example〕

1. Conversion from the galactose-inducible GAL 10 promoter of promoter 1 to the enolase 1 promoter (hereinafter abbreviated as ENO 1 promoter), which is one of the glycolytic enzyme genes that is generally considered to have high expression efficiency. was performed as follows. (See Figure 1) (]) Introduction of BamllI site into GALIO promoter Plasmid YEp for secretory expression of human lysozyme, which has been used conventionally
-11In order to extract the entire GALIO promoter from LYsIG, the 5′ of the GALIO promoter must be
A unique restriction enzyme site is also required at the 3' end. Therefore, the Sau3AI site present at the 5' end of the GALIO promoter was converted to a BamHI site that does not exist on the vector.

(2) Sall sit to ENOI promoter
Introducing only a portion of the ENO 1 gene promoter into G
pB to completely replace the ALIO promoter
Sall not present on R-MENO plasmid
site was introduced into the 3' end of the ENO 1 promoter.

(3) Bam of YCp-11LYsIG plasmid
}II, Salr digestyEp-nt. ysr
r; Dig the plasmid with Bamll and Sall
A fragment in which a portion of the estL GALIO promoter was removed was recovered.

(4) BamHI of pBR-M-ENO plasmid
, SalI digestpBR-M-HNO plasmid was digested with Bam and Sall.
A fragment containing the O1 promoter was recovered.

(5) Ligation using T4-DNA ligase (3) and [
The fragments obtained in step 4) were ligated with T4-DNA ligase to construct a new secretory expression plasmid pESII containing the ENO 1 promoter. This plasmid was transferred to E, col
i introduced into C600 strain, and the resulting transform
The plasmid in ant was digested with various restriction enzymes and confirmed to be the desired plasmid.

2. A plasmid pEslI-2 in which two human lysozyme secretion expression units of Uni1 were linked was constructed as shown in FIG. Specifically, after completely digesting pEsll with Smal,
Partial digestion was performed with al, and a 2188 bp human lysozyme secretion expression unit was separated and recovered by 0.7% agarose gel electrophoresis. Next, this 2188bp fiamllI
and Xbar end with Klenow enzyme and dNTP (N
:^, G, C, T) in the presence of fillir+Ltebl
After setting unt end, pEsIl Small s
ite to construct a new plasmid pESH-2.

This plasmid was transferred to E. introduced into coli C600 strain,
The plasmid in the obtained transformant was digested with various restriction enzymes and confirmed to be the desired plasmid.

3. Plasmids pEsll and pE obtained in Nos. 1 and 2 of
sH-2 was converted to S. sH-2 by the lithium method. cerevi
siae A2-1-IA strain (I eu2) (X
A leucine auxotrophic strain was generated by inducing a leucine auxotrophic mutant strain from Y.2180-IA, and then backcrossing it with X2180-B. Jigami et
al. J. Biochem. Vol 99Pllll
1125 (1986)) and selective medium (see
-1 eU) was obtained.

4. (1) Evaluation of transformants containing pEsH in conventional medium. Transformants were cultured with shaking at 30°C for 3 days in L-shaped test tubes containing 5 ml of selective medium containing 2% glucose. , 1 mR of the resulting culture solution. was inoculated into 19 ml of fresh selective medium with the same composition and cultured with shaking at 30°C for an additional 3 days (2
An Erlenmeyer flask with a capacity of 00 mR was used) to prepare a preculture solution.

For the main culture, a 500-volume Sakaguchi flask was used.

After adding 147 ml of the evaluation medium with the conventional composition shown in Table 1, 3 ml of the preculture solution was inoculated and cultured with shaking at 30° C. for 5 days.

The activity of human lysozyme secreted and produced outside the bacterial cell was measured using Micrococcus lys, a lysozyme-sensitive bacterium.
The experiment was carried out as follows using P. odeikticus as a substrate.

13 Micrococcus Iysodeikt
icus cell suspension (lyophilized cell suspension 15mg/100m
°C 50mM sodium phosphate buffer (pH 6.5
))800μ! Add 200μ℃ of culture supernatant to
After mixing quickly, reduce the turbidity of the bacterial cell suspension using a quartz cell with an optical path length of 1 cm and absorbance at 450 nm at room temperature (25°C).
Each measurement was followed up.

The enzyme activity was determined by measuring the decrease in absorbance at 450 nm for 3 minutes using this measurement system, and the amount of enzyme required to decrease the absorbance by 0.001 per minute was defined as 1 unit.

As a result, the secreted amount of HiL-lysozyme after 120 hours of culture was 0.26 mg/ff.

(2) Similarly, in the evaluation medium with the conventional composition shown in Table 1, p
F. When the transformant containing stl-2 was cultured and analyzed in the same manner as in evaluation (1), it was found that the culture
The amount of human Lyduzyme secreted after h was 0.46 rng/l. Met.

(3) Evaluation of the transformant containing pESl+-2 using the medium of the present invention. Culture and analytical quantification were performed in the same manner as in (1) except that the medium of the tree invention shown in Table 2 was used.
After 0 hours, the secreted amount of Hi1.Lidzyme was 6.2 mg/l. In addition, almost no difference was observed in the amount of bacterial cells in the cultures (1) to (3).

As a result of the above, the secretion production amount of Hi-1 lysozyme per unit cell has been significantly improved, and the medium composition of the present invention is extremely excellent for mass-producing lysozyme using yeast.

Plant = 1 glucose ammonium sulfate monopotassium phosphate Magnesium sulfate First chloride Sodium chloride, lithium Calcium chloride trihydrate manganese sulfate Sodium molybdate zinc sulfate Boric acid copper sulfate 20g 5g 1g 0.5g 200μg 100mg 1. O O mg 400μg Thorium 200μg 400μg 500μg 40μg potassium iodide biotin Pan I/calcium thenate folic acid Boar 1-le Niacin P-aminobenzoic acid Pyridoxine hydrochloride Ribofurahin Thiamine hydrochloride methionine isoleucine Hallin 1・Reply 1 fan histicine glutamic acid aspartic acid Tyrosine lysine Phenylalanine 100μg 2μg 400μg 2μg 2mg II00μg 200μg 400μg 200μg 400μg 20mg 30mg 150mg 20mg 20mg 100n+g 100mg 30mg 30mg 50mg Threonine Shirin arginine adenine Uracil 200mg 3 7 5 mg 20mg 20mg 20mg Add ion-exchanged pure water to make 1r.

Table - Glucose Ammonium Sulfate Dipotassium Phosphate Magnesium Sulfate Heptahydrate Potassium Chloride Ferrous Sulfate Heptahydrate Monopotassium Phosphate Magnesium Sulfate Ferrous Chloride Sodium Lium Chloride Dihydrate Calcium Chloride Sulfate 100g of manganese]. Og 2g 1.5g 0.5g 20mg 1g 0.58 200μg 100mg 100mg 400μg Sodium molybdate Zinc sulfate Borate Copper sulfate Potassium iodide Biotin Calcium pantothenate Folic acid Inositol Niacin P-Aminobenzoic acid Acid Pyridoxine Riboflavin Hydrochloride Thiamine Methionine Isoleucine Palin Tryptophan Hisytidine glutamic acid 200μg 400μg 500μg 40μg 100μg 2μg 400μg 2μ8 2mg 400μg 200μg 4001tB 200μg 400μg 20■ 30n+g 150mg 20■ 20mg 100mg Aspartic acid 100mg tyrosine
30mg lysine
30mg phenylalanine 50mg threonine 200mg serine
375 ■ Arginine 20mg Adenine
20mg uracil
20■ Add ion-exchanged heavy water and add 1. 0! [Effects of the Invention] According to the present invention, the amount of secretion and production of proteins, such as human lysozyme, per unit cell by transformed yeast is greatly improved, and the present invention utilizes yeast to produce useful proteins derived from higher organisms. It is extremely effective in manufacturing.

[Brief explanation of drawings]

FIG. 1 is a flow chart of the process of converting the GALIO promoter to the ENO 1 promoter. FIG. 2 is a flowchart of the steps for enhancing the human lysozyme secretion expression unit.

Claims (1)

[Claims] 1. When producing a heterologous protein using a yeast transformed with a secretion expression vector containing a promoter, a foreign gene, etc., the minimum amount required in a medium for the growth of the transformed yeast A method for producing a heterologous protein, which comprises supplying the above carbon source and/or nitrogen source. 2. The method for producing a heterologous protein according to claim 1, wherein the heterologous protein is human lysozyme. 3. The yeast is Saccharomyces cerevisiae (Saccha)
2. The method for producing a heterologous protein according to claim 1, wherein the protein is S. cerevisiae. 4. Claim 1, wherein the promoter is a glycolytic enzyme gene.
A method for producing the described heterologous protein. 5. Glycolytic enzyme genes include enolase 1 gene, 3-phosphoglycerokinase gene, and glyceraldehyde-3
- The method for producing a heterologous protein according to claim 4, wherein the protein is selected from phosphate dehydrogenase genes. 6. Glucose or sucrose as carbon source 80~
The method for producing a heterologous protein according to claim 1, wherein 150 g/l is supplied. 7. 10-30g/l of ammonium sulfate as a nitrogen source
2. A method for producing a heterologous protein according to claim 1, wherein the heterologous protein is supplied. 8. Claim 1 in which one or more of dipotassium phosphate, magnesium sulfate heptahydrate, potassium chloride, and ferrous sulfate heptahydrate is supplied to the medium as inorganic nutrient salts.
A method for producing the described heterologous protein. 9. A culture medium for secretory production of a heterologous protein by the transformed yeast according to claim 1, which contains a carbon source and/or nitrogen source in an amount more than the minimum amount required for the growth of the transformed yeast. 10. Glucose or sucrose as a carbon source 80
The medium for secretory production of a heterologous protein according to claim 9, comprising ~150 g/l. 11. Ammonium sulfate as a nitrogen source at 10-30g/
10. The medium for secretory production of a heterologous protein according to claim 9. 12. The medium contains dipotassium phosphate, magnesium sulfate heptahydrate, potassium chloride, and dibasic sulfate as inorganic nutrient salts.
10. The medium for secretory production of a heterologous protein according to claim 9, which contains at least one of iron and heptahydrate. 13. When producing a heterologous protein by secretion using yeast as a host and using a gene encoding a glycolytic enzyme as a promoter, glucose or sucrose is used as a carbon source.
0-150g/l, dipotassium phosphate as inorganic nutrient salts 1-3g/l, magnesium sulfate heptahydrate 1-3
1. A method for producing a heterologous protein, characterized in that a desired protein is obtained in high yield by supplying the desired protein at a high yield. 14. The production method according to claim 13, wherein the heterologous protein is human lysozyme. 15. Yeast is Saccharomyces cerevisiae (Saccharomyces
14. The production method according to claim 13, characterized in that the plant is aromyces cerevisiae. 16. The gene encoding the glycolytic enzyme is elanose 1
14. The production method according to claim 13, characterized in that the gene is selected from the group consisting of a 3-phosphoglycerokidase gene and a glyceraldehyde-3-phosphate dehydrogenase gene.