JPH0480680B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to β-interferon (hereinafter referred to as β-IFN).
This invention relates to a method for producing physiologically active peptides derived from eukaryotic cells, such as (abbreviated as ), by fermentation. In recent years, with the use of genetic engineering technology that has rapidly developed, genes encoding useful physiologically active peptides derived from eukaryotic cells such as β-IFN have been cloned, and they have been linked to promoters derived from microorganisms to express the desired purpose within microorganisms. It has become possible to efficiently produce physiologically active peptides (T. Taniguchi et al.: Proc. Jap. J. Acad.
~469 (1979), JP-A-57-77654, JP-A-58-
110600). Techniques for efficiently culturing microorganisms containing recombinant plasmids thus obtained according to their properties and fermentatively producing the desired physiologically active peptides at high yields are extremely important. In general, the optimal production conditions for proteins and enzymes produced by bacterial cells are not necessarily the same as the optimal growth conditions for the bacteria themselves, and the culture conditions such as culture temperature, pH, aeration and agitation, and culture medium conditions. It fluctuates greatly depending on In addition, the production method varies greatly depending on whether the target protein or enzyme is an inducible type or a constitutive type. For example, in the case of an inducible type, the production amount varies greatly depending on the conditions of addition of the inducer, etc. . Furthermore, if the target protein or enzyme is unstable, it is necessary to consider a stable production method. The present inventors used a microorganism having a recombinant plasmid that produces β-IFN etc. to fermentatively produce the desired production active peptide in high yield.
After investigating various culture conditions as mentioned above, we succeeded in finding conditions that allow production of active peptides such as β-IFN to be produced extremely efficiently.
The present invention has now been completed. The present invention will be explained in detail below. The present invention belongs to the genus Etsuchierichia and contains a recombinant DNA consisting of a gene encoding a peptide derived from eukaryotic cells, a vector, and a promoter,
In addition, when culturing a microorganism capable of producing the peptide in a medium, accumulating the peptide in the culture, and collecting the peptide from the culture, the culture is
Provided is a method for producing a peptide, characterized in that it is carried out at a temperature of ~27°C. To date, many bioactive peptides have been expressed in microorganisms, and in most cases, Escherichia coli has been used as the microorganism. In the case of E. coli, the culture temperature commonly used is 37°C.
Furthermore, the actual production of these physiologically active peptides is often carried out at 37°C. According to the method of the present invention, 10 to 25
Peptides can be efficiently produced by culturing at a low temperature, preferably 15 to 20°C. For example, in the case of E. coli, good results can be obtained by culturing at 15-30°C, preferably 20-25°C. Examples of the eukaryotic cell-derived peptides of the present invention include insulin, interferon, and growth hormone, preferably human β-interferon peptide. As vectors, vectors derived from Escherichia coli, Bacillus subtilis, and yeast are used, and preferably Escherichia coli-derived pBR322, pBR313, pMB9, etc. are used.
Examples of promoters include lac promoter, phoA promoter, trp promoter, and preferably trp promoter.
A promoter is used. As the medium for the method of the present invention, a medium commonly used for culturing host microorganisms is used. Generally, a rich medium is better for improving the growth of bacteria, but a peptone-based medium is not preferable, for example, when using the trp promoter as a promoter. It is known that when peptide expression is controlled by the trp promoter, IAA can be added as an inducer. The present invention also provides a method for dramatically improving the effect of adding IAA. In other words, when using E. coli from the latter half of the logarithmic growth phase during microbial culture to the time of maximum growth, the dry cell concentration is 3.
The production of the peptide can be improved by adding IAA to the culture medium when the microorganism grows in the range of ~30 mg/ml. The IAA to be added is
It is used in the range of 10 to 200 mg/. In order to further improve the production of peptides, IAA is added continuously or intermittently after the above-mentioned addition of IAA. When added continuously, the total amount is 200mg/
When adding intermittently, it may be added 3 to 10 times at a concentration of 5 to 50 mg/each. Improvements in culture methods such as those described above, i.e., improvements in peptide production by low-temperature culture, selection of IAA addition timing, and additional supplementation of IAA, are not known in peptide production using recombinant DNA technology as well as production using general microorganisms. This was first discovered by the present inventors. Examples of the present invention will be shown below. Example 1 The human β-IFN gene was transduced using the trp promoter (hereinafter referred to as
Escherichia coli ILV-1 ATCC39025 (abbreviated as Ptrp) containing the constructed recombinant plasmid pLT-1.
58-110600) using the method described below,
Examine the productivity of human β-IFN. The above bacterial cells are cultured in LG medium (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 2 g of glucose dissolved in 1 part water and adjusted to pH 7.0 with NaOH) at 30°C for 17 hours. 50 ml of this culture solution was mixed with 1 MGC medium (Na ₂ HPO ₄ 0.6%, KH ₂ PO ₄ 0.3%, NaCl 0.5%,
_NH4Cl0.1 %, glucose 0.5%, casamino acids 0.5
%, MgSO ₄ 1mM, and thiamine 4mg/) for main culture. Note that ampicillin is added to the culture solution at a concentration of 50 mg/ml. Under aeration and stirring conditions of 750 rpm and 1 vvm, pH is controlled to 6.5, and culture is performed at various temperatures from 15 to 37°C. Glucose concentration is adjusted by feeding so that it is in the range of 0 to 1.0% while checking it with a glucose analyzer. Casamino acids are also fed in the same amount as glucose. When the bacteria have grown to a dry cell concentration of 3mg/ml
Add 20 mg/ml of IAA (manufactured by Wako Pure Chemical Industries, Ltd.) and continue culturing for an additional 12 to 72 hours. β-IFN is extracted from bacterial cells as follows. 1 ml of the culture solution was centrifuged at 8000 rpm for 10 minutes to collect bacteria, and diluted with 30mM NaCl, 30mM, Tris-HCl (PH
7.0) Wash with buffer. The washed bacterial cells were suspended in the above buffer solution, and 100 μg of lysozyme, 0.25M,
Add 25μ of EDTA to make a 1ml suspension, leave it at 0°C for 30 minutes, and repeat freezing and thawing three times to destroy the bacterial cells. This is centrifuged at 15,000 rpm for 30 minutes to obtain a supernatant, and the amount of β-IFN in the supernatant is quantified according to Armstrong's method [Armstrong et al.
Appl. Microbiol., vol. 21, 723-725 (1971)] [However, as a virus, Vesicular Stomatitis
Virus, animal cells derived from human amniotic cells
Use Wish cell]. The results are shown in Table 1.

[Table] As is clear from Table 1, β-IFN productivity varies significantly depending on culture temperature, and extremely high β-IFN productivity can be obtained by culturing at a temperature around 20°C. Example 2 Using strain ILV-1, the culture temperature was fixed at 20°C, and the culture conditions were the same as in Example 1, except that IAA was added at the times shown in Table 2, and the timing of IAA addition was investigated. I do. The concentration of IAA added was 20 mg/. The results are shown in Table 2.

[Table] As is clear from Table 2, β-IFN productivity is
It varies markedly depending on the bacterial cell concentration at the time of adding IAA, and when added from the latter half of the logarithmic growth period to the time when growth peaks, extremely high β-IFN
Gain productivity. Example 3 Using bacterial cells ILV-1, at a culture temperature of 20°C, 20 mg/IAA was added at a dry bacterial cell concentration of 3 mg/ml,
Some of it is left as is, and some of it is taken every 12 hours for a total of 5 times each time.
The cells were cultured in the same manner as in Example 1, except that IAA was additionally added at a concentration of 20 mg/ml, and the productivity of β-IFN was examined. The results are shown in Table 3.

【table】

Claims (1)

[Scope of Claims] 1. Cultivating in a medium a microorganism belonging to the genus Escherichia that contains a recombinant DNA consisting of a gene encoding a peptide derived from eukaryotic cells, a vector, and a promoter, and has the ability to produce the peptide,
When accumulating the peptide in the culture and collecting the peptide from the culture, the culture was maintained at 15-27°C.
A method for producing a peptide, characterized in that it is carried out at a temperature of . 2. The method according to claim 1, wherein the promoter is a tryptophan promoter. 3. Claims 1 or 2, characterized in that 3-indoleacrylic acid (hereinafter abbreviated as IAA) is added to the culture medium from the latter half of the logarithmic growth phase to the time when the microorganism exhibits maximum growth during cultivation of the microorganism. Method described. 4. The method according to claim 1, 2 or 3, characterized in that after adding IAA, IAA is further added continuously or intermittently. 5. The method according to claim 1, 2, 3 or 4, wherein the peptide is a peptide of human β-type interferon.