JPH0436190A - Medium and production of protein or peptide thereby - Google Patents

Abstract

PURPOSE:To obtain a medium capable of suppressing decomposition of secreted and produced protein and peptide and producing a large amount of protein and peptide stably and efficiently, containing an amino acid, a salt thereof and an inorganic salt and having ionic strength of >= a specific value. CONSTITUTION:The objective medium of culturing a bacterium to secrete and to produce protein or peptide in an extracellular way, containing at least one of an amino acid, a salt thereof and an inorganic salt and having >=1 ionic strength. Bacillus subtilis, specific a strain having extracellular protease productivity reduced by mutation is preferable as the bacterium.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides a medium useful for efficiently producing proteins or peptides secreted and produced by microorganisms outside the cells without degrading them, and a medium for producing proteins produced by the medium without decomposing them. Or it relates to a method for producing a peptide.

``Conventional technology and invention to solve a!fi] Gene recombination Gene technology uses enzymes etc. to create a heterogeneous DNA in a test tube.
By integrating NA into a vector to create a recombinant, and transferring it into a living cell host, the integrated DNA
This is a technology to produce heterogeneous proteins and peptides based on Advances in these genetic engineering techniques have made it possible to prepare large quantities of proteins and peptides that conventionally exist in trace amounts in nature.

However, when Escherichia coli, the most frequently used host, produces large amounts of proteins, peptides, etc. within its cells, it produces insoluble aggregates (inclusion bodies), and the proteins and peptides become inactive. Become. Additionally, E. coli produces endotochin, which is a pyrogen. Therefore, in order to use the product as a medicine,
It is necessary to completely remove and purify these by a complicated process, which has the drawback of requiring cost and time.

On the other hand, there are various advantages when using microorganisms that produce useful substances extracellularly as hosts. In other words, in microorganisms that produce endodoxins, such as Escherichia coli, Pseudomonas bacteria, and Staphylococcus bacteria, which produce useful substances outside the bacterial body, these pyrogen products By using highly safe microorganisms such as Bacillus subtilis, actinomycetes, yeast, and molds that have a high ability to secrete proteins outside the bacterial body, it is possible to produce products in large quantities and It has various advantages, such as being able to obtain it in an active form without being insolubilized, and (2) easy separation and purification of the product.

However, since many microorganisms secrete protease, which is a protein-degrading enzyme, out of their cells, there is a problem that useful proteins and peptides produced by the microorganisms are degraded and cannot be accumulated.

In order to suppress the degradation of products by host-derived butyase, it has been proposed to grow protease-deficient strains (
J, Bacteriol, ] 60.44
2-444 (1984); Appl, Envir
on, Microbiol, 53.379-384
．． (1987), Japanese Unexamined Patent Publication No. 84-37285) However, even in the hosts with low protease productivity that have been reported to date, they are effective in degrading proteins and peptides, which are gene products produced outside the bacterial body. It is difficult to suppress this situation.

On the other hand, in order to suppress the decomposition of products by host-derived proteases (e.g., β-lactamase derived from Escherichia coli), the applicants have discovered that collic acid, maleic acid, fumaric acid, acetic acid, or their respective We discovered that culturing the host in a medium containing organic acids such as salts was useful, and filed an application earlier (Japanese Patent Application Laid-Open No. 61-52297).

An object of the present invention is to provide a culture medium that can effectively suppress the degradation of proteins and peptides by host-derived proteases.

Another object of the present invention is to provide a method for producing proteins or peptides that can be obtained stably and in high yields.

[Structure of the Invention] The present inventors have further searched for additives that are inexpensive, easy to handle, and can effectively suppress the decomposition of proteins and peptides by host-derived proteases. As a result, amino acids and their salts, We also found that the degradation of the produced gene product by host-derived proteases is effectively suppressed in a medium containing inorganic salts. It has also become clear that increasing the ionic strength of the medium is effective in effectively suppressing the degradation of gene products by host-derived proteases, leading to the present invention.

That is, the present invention provides a medium for culturing microorganisms that secrete and produce proteins or peptides extracellularly, comprising at least one component of an amino acid, a salt thereof, and an inorganic salt;
In addition, a culture medium having an ionic strength of 1 or more is provided.

The present invention also provides a method for producing a protein or peptide, which involves culturing a microorganism that secretes and produces a protein or peptide extracellularly in the above-mentioned medium.

The present invention is applicable to a wide range of media containing conventional carbon sources, nitrogen sources, inorganic salts, growth factor components, etc. The type of medium is not particularly limited, and can be selected from natural media, semi-synthetic media, and synthetic media depending on the microorganism to be cultured.

The medium may be a solid medium containing agar, seratin, etc., or preferably a liquid medium.

Examples of amino acids include L-glutamic acid, L-aspartic acid, arginine, and lysine.

The amino acid may also be a salt with sodium, potassium, lithium, ammonium, or the like.

Specific examples of inorganic salts include sodium sulfate, potassium sulfate, lithium sulfate, ammonium sulfate, sodium chloride, potassium chloride, lithium chloride, ammonium chloride, sodium nitrate, potassium nitrate, lithium nitrate,
Ammonium nitrate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate,
Dipotassium hydrogen phosphate, potassium dihydrogen phosphate, lithium phosphate, lithium dihydrogen phosphate, ammonium phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, sodium ammonium hydrogen phosphate, sodium carbonate, potassium carbonate , ammonium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, ammonium hydrogen carbonate, and the like. Preferred inorganic salts include sodium sulfate, ammonium sulfate, sodium chloride, potassium chloride, ammonium chloride.

The medium only needs to contain at least one of the above-mentioned amino acids, amino acid salts, and inorganic salts, and may contain an organic acid and its salt in addition to the above-mentioned components.

As the organic acid, carboxylic acid is preferred. Examples of the carboxylic acid include acetic acid, succinic acid, maleic acid, fumaric acid, glutaric acid, 3,3-dimethylgeltanic acid, malic acid, adipic acid, suberic acid, and trans-aconitic acid.

Examples of organic acid salts include sodium, potassium, lithium, and ammonium salts.

Among these components, high electrolyte compounds are preferred. These components can be used singly or in combination of two or more of the same or different types, except for the case where organic acids and their salts are used alone. In addition, when adding a combination of two or more amino acids and their salts, inorganic salts, or organic acids and their salts to the medium, especially a combination of two or more different components, the gene product may be degraded by host-derived proteases. is effectively suppressed.

The ionic strength of the medium is set at a value that allows the host microorganism to grow in the medium and effectively inhibits the degradation of gene products produced in the medium by host-derived proteases. Ru.

Such ionic strength is greater than or equal to 1, preferably 1°25
-3, more preferably about 1.5-2.5.

The ionic strength of the medium can be controlled by adjusting its concentration depending on the number of charges of the ionic species of the electrolyte component.

Further, the pH of the medium can be adjusted to a value suitable for culturing microorganisms, for example, about pH 6 to 8, using a basic compound such as sodium hydroxide or an acidic compound such as an organic acid.

In order to further stabilize secreted proteins and peptides, a protease inhibitor such as phenylmethylsulfonylfluoride F may be added to the culture medium of the present invention. The amount of protease inhibitor added is, for example,
It is about 0.01-1.0mM.

The culture medium of the present invention can be suitably applied to the cultivation of microorganisms that secrete and produce proteins or peptides outside of their cells, and significantly suppresses the degradation of useful proteins or peptides secreted by the microorganisms.

Therefore, proteins or peptides can be obtained stably and efficiently.

In the method for producing proteins or peptides of the present invention, the microorganism to be cultured is not particularly limited as long as it secretes and produces proteins or peptides outside of its cells. In order to efficiently obtain useful proteins and peptides, recombinant DNA is produced by integrating a heterologous DNA fragment encoding the desired gene product into a vector using conventional methods using genetic recombination technology. It is preferable to use a microorganism that has been transformed by transferring the microorganism into a host by a conventional method.

As long as the gene product is a protein or peptide,
Regardless of whether it is derived from animals, plants, microorganisms, etc.
It can be produced by the method of the present invention. Examples of gene products include α-interferon, β-interferon, γ-interferon, interleukin-1,
Examples include peptide hormones such as interleukin-2, interleukin-3, prostaglandin, growth hormone, urokinase, insulin, and calcitonin, and protein enzymes such as β-lactamase, amylase, and isomerase. Gene products also include fusion proteins in which a useful peptide with a relatively small molecular weight and a carrier protein are cleavably linked. Such fusion proteins include vasoactive intestinal polypeptide (hereinafter referred to as VI), which has a vasodilatory effect.
P), for example, Staphylococcus aureus
protein A derived from eus) (JP-A-83-24587
After Asp, the 404th amino acid (excluding the signal peptide) in the structural gene (see Publication No. 7), VIP-glycine, That is, the following amino acid sequence H-His-Ser-Asp-Ala-Val-Phe
-Th r-As p-As n-Tyr -Thr
-Arg-Leu-Arg-Lys-Gln-Met-
Ala-Val-Lys-Lys-Tyr-Leu-
Asn-5er-11e-Leu-Asn-Gly-
OH-bound fusion protein (hereinafter referred to as protein A-VI)
(referred to as P-Gly).

Examples of hosts that can be used include bacteria such as Escherichia coli and Bacillus subtilis, actinobacteria, yeast, and mold. Among these hosts, microorganisms belonging to the genus Bacillus that are particularly safe and secrete large amounts of proteins outside the bacterial body, especially Bacillus subtilis, are highly safe.
tilis) is preferred. Furthermore, among Bacillus subtilis, a strain whose extracellular protease productivity is reduced by a technique such as mutation is preferred. When a host with low protease productivity is cultured in combination with the above medium, degradation of gene products can be significantly suppressed, and proteins and peptides can be efficiently obtained in large amounts.

Examples of Bacillus subtilis with low protease productivity include (1) Bacillus subtilis 104HL, as proposed by the applicant in JP-A-64-37284;
Bacillus subtilis D with the pap gene introduced into the strain
Strain Y-16 (Feikoken Kyoiku No. 9488); (2) lacks the ability to produce alkaline protease and neutral protease;
Examples include a strain in which the spo OAΔ677 mutant gene is introduced into Bacillus subtilis, which has a protease activity of 3% or less of that of the wild strain. Particularly preferred strains are those belonging to (2) above, and such strains include, for example,
-281440, spo OA to Bacillus subtilis 104HL strain as proposed by the present inventors.
Bacterial strains into which the △677 mutant gene has been introduced, especially Bacillus subtilis 5P011 strain (Feikoken Bacterial Serial No. 10987),
・Spo O^Δ to Cutilus subtilis DY-16 strain
This includes strains into which the 877 mutant gene has been introduced, particularly Bacillus subtilis SPL 14 strain (Feikoken Bibori No. 1098111). Among strains with low protease productivity, Bacillus subtilis 5PL14 strain is particularly preferred.

In addition, as a strain having the pap gene, for example, Bacillus subtilis 72N26 strain [Agricultural and Biological Chemistry (Agric,
Biol, Chew, ), 43; 2348-2
Examples include 349.1979.

Examples of strains having the spo OAΔ677 gene include Bacillus subtilis ATCC35138 strain, A
TCC39090 strain, ATCC39091 strain, ATCC
39092 strain, ATCC39094 strain, ATCC390
Examples include 96 stocks. These stocks are American
Type Culture Collection (American
Type Culture Correction)
Available from.

The host microorganism transformed by the recombinant DNA is cultured under the usual conditions used for culturing microorganisms, such as a temperature of 15 to 45°C, preferably 25 to 40°C, and a culture time of 1.
This can be carried out for about 0 to 60 hours. When the medium is a liquid medium, shaking culture or aerated agitation culture can usually be adopted. The ionic strength of the medium should be 1 or more at the start of culture, and during culture, amino acids and their salts,
Inorganic salts, organic acids and their salts, etc. may be added.

Proteins and peptides secreted and produced during culture are separated and collected from the culture medium by conventional methods.

Note that preferred embodiments of the present invention are as follows.

(a) At least one of an amino acid, its salt, and an inorganic salt
A culture medium having an ionic strength of 1.25 to 3, preferably 1.5 to 2.5.

(b) at least one of an amino acid, a salt thereof, and an inorganic salt
It contains two components, an organic acid and its salt, and has an ionic strength of 1.
．． 25-3, preferably 1.5-25.

(e) A medium in which the amino acids and their salts are L-glutamic acid, L-aspartic acid, and their sodium, potassium, and ammonium salts.

(tl) A medium in which the inorganic salts are sulfuric acid, hydrochloric acid, nitric acid, and their sodium, potassium, and ammonium salts.

(e) Organic acids and their salts include succinic acid, maleic acid, fumaric acid, glutaric acid, 3,3-dimethylgluconic acid, malic acid, adipic acid, suberic acid, trans-aconitic acid, and their sodium and potassium , a medium that is an ammonium salt.

(f) A microorganism that secretes and produces proteins or peptides extracellularly is a Bacillus genus, preferably a Bacillus subtilis genus, more preferably a bacterial cell, which has been transformed with a DNA recombinant of a heterologous DNA and a vector DNA. A method for producing a protein or peptide produced by Bacillus subtilis with low external protease productivity.

(g) Bacillus subtilis, which has low protease productivity, lacks the ability to produce alkaline protease and neutral protease, and the spo OAΔ677 mutant gene was introduced into Bacillus subtilis, which has a protease activity of 3% or less of the wild strain. Strain, preferably Bacillus subtilis 5P011
Bacillus subtilis strain 5PL14 strain (Feikoken Bokuyori No. 10987), more preferably Bacillus subtilis 5PL14 strain (Feikoken Bibori No. 1098).
No. 8) A method for producing a protein or peptide.

〔Effect of the invention〕

According to the culture medium of the present invention and the method for producing proteins or peptides produced therein, it is possible to significantly suppress the decomposition of proteins and peptides secreted and produced outside the bacterial cell by host-derived proteases, so that proteins and peptides can be produced stably and in large quantities. can be obtained efficiently.

[Examples] The present invention will be described in more detail below based on Examples.

Bacillus subtilis ATCC 39096 strain, which is a Bacillus subtilis having the spo OA8877 gene and the lincomycin resistance gene (hereinafter referred to as Linr), was grown at a temperature of 37°C until the logarithmic growth phase using the LB medium shown in Table 1. Cultured with shaking.

(Hereinafter, blank space) Table 1 The bacterial cells contained in 50 ml of the culture solution were collected, and the cells were collected using the Saito-Miura method (TL, 5Aito, K, Miura, Bi).
ochjm, Biophy's, Acta, 72
．． 619 (1963)), chromosomal DNA was extracted and purified.

Using the obtained chromosomal DNA, Bacillus subtilis strain DY-16 was transformed by the competent cell transformation method. Next, the obtained transformant was incubated with LB containing 5 μmz/mI lincomycin.
The cells were cultured using an agar plate medium, and strains into which Lin' had been introduced were selected.

As a result, about 800 lincomycin-resistant transformants were obtained. Next, 51 strains lacking sporulation ability were isolated from these lincomycin-resistant transformed strains as follows.

The transformed Bacillus subtilis was spread on a minimal agar medium containing 50+ag/J of histidine and cultured at a temperature of 37°C for 48 hours.

Strains that met the following indicators 1 to 3 were selected as spore-forming ability-deficient strains.

1. The transformed strain was inoculated into the sporulation medium, and the colony lost the ability to produce melanin pigment. 2. At a temperature of 80°C, 1.
3. No spore formation is observed by microscopic examination.

Among the obtained 173 strains deficient in sporulation ability, the strain with the lowest protease activity was selected as described below, and the protease activity was measured.

The spore-forming ability-defective transformant strain and the parent strain Bacillus subtilis DY-16 strain were separately inoculated onto an LB agar plate medium containing 1% by weight of casein, and cultured at a temperature of 37°C for 24 hours. Since casein is degraded by protease secreted by the bacterial body, the size of the halo around the bacterial body formed by the decomposition of casein is used as an indicator to determine whether Bacillus or
We isolated 400 strains whose protease production ability was lower than that of the I. subtilis DY-16 strain.

Bacillus subtilis strain DY-16 and 40 transformed strains with reduced protease activity were each cultured overnight in 10 ml of LB medium, and 1 ml of the culture was centrifuged. Protease activity was measured. That is, 0.2% FITC-casein (manufactured by Sigma), 100mM Tris-HCl buffer (pH 7.5)
), 100 μl of the supernatant test solution containing protease was added to 100 μl of the substrate solution containing 2 mM calcium chloride, and the mixture was reacted at 37° C. for 3 hours. After the reaction was completed, the reaction was stopped by adding 200 µm of 7.5% trichloroacetic acid to the reaction solution, and the supernatant was obtained by centrifugation (500 mM).
After neutralization with lithium-hydrochloric acid buffer (pH 8.5), fluorescence at an excitation wavelength of 490 nm and an emission wavelength of 525 nm was measured.

In addition, the enzyme activity that releases trichloroacetic acid soluble peptide equivalent to 1 μM Tyr from casein per minute is expressed as IU.
It was converted using Pronase (manufactured by Kaken Pharmaceutical Co., Ltd.) as a standard.

As a result, we obtained four strains with extremely reduced protease activity compared to the parent strain Bacillus subtilis DY-16, and these strains were respectively inoculated with No\ subtilis 5P.
They were LQ strain, 5PLII strain, 5pL14 strain, and 5PL39 strain.

Then, after culturing Bacillus subtilis DY-16 strain and the transformants with reduced protease activity in 10 ml of LB medium for 18 hours, they were cultured in 50 ml of Medi.
uIllA medium (Journa 1 inch Bacteriol
After the cells were transferred to a culture medium (Japanese), and cultured for 24 hours and 48 hours, the protease activity in the culture supernatant was measured in the same manner as above.

The results are shown in Table 2.

(Hereinafter, blank space) Table 2 (In the table, OD 600 indicates the absorbance of the culture supernatant at a wavelength of 600 ns.
Relative values are shown when the protease activity of S. subtilis DY-16 strain is set as 100. ) From Table 2, it was confirmed that the four transformed strains had a protease activity that was 1/20 or less lower than that of the parent strain. In particular, it was confirmed that the protease activity of the 5PL14 strain was suppressed to about 3% of that of the parent strain DY-16 strain. This 5PL14 strain has the above-mentioned genetic marker. Furthermore, all of these strains have auxotrophies for histidine and leucine, and multiple auxotrophies required by the host were confirmed in gene recombination experiments.

In addition, Bacillus subtilis DY-1 subjected to transformation
When the protease activities of the six strains were compared with Bacillus subtilis strain 20725, which is a wild protease strain, the results shown in Table 3 were obtained. The protease activity was measured by culturing the strain for 24 hours in the same manner as described above.

Table 3 From Table 3, the Bacillus subtilis DY-16 strain produces only about 1% of the protease of the wild type 207-25 strain.

Bacillus subtilis 5PL obtained as above
14 strains were used as hosts for the following plasmid pMD235.

(1) Obtaining protein A gene Escherichia colt MV transformed with a plasmid containing protein A gene
13 C14/pDcP2411 (Unexamined Japanese Patent Publication No. 63-24
5677)), the alkaline method [Nuel, ^
cids Res, 7.1513-1523. (1
979)] was used to prepare plasmid pDCP2411. The obtained plasmid DNA (10 μg) was treated with restriction enzymes EcoRI and BamHI (20 units each).
2.2kb DNA containing the protein A gene was digested with
Fragment A was obtained.

(2) Preparation of plasmid pMD200 A 2.2 kb Ec containing the protein A gene.

RI/BamHI fragment (lμg) and EcoRI/BamHI long fragment of plasmid pUB110 (
0.5μg) and T4 DNA ligase (100un
Bacillus subtilis
5ubtilis P S L 1 strain (available from the Bacillus Genetic Stock Center, Ohio State University) was transformed by the protoplast method [Mo1. G
en.

Genet, 16111. lit ~
115. (1979) Ko.

The obtained kanamycin-resistant (Km) strain was transferred to LB medium (
1.0% tryptone, 0.5% yeast extract, 0.5% N
a(1!, pH 7,2) 10mM (Kanamycin K
(containing lOug/ml) and cultured at 37°C for 16 hours. Regarding the culture supernatant, ELISA [Pr
oc, Natl, ^cad, Set, USA,
80.697-701. (1983)] investigated the productivity of protein A, and as a result, the plasmid p
B containing MD200, 5ubtilis P S L
I got 1 share.

(1) Chemical synthesis of VIP-Gly gene A DNA sequence encoding VIP-Guy consisting of 29 amino acid residues,
At its N-terminus, there is a restriction enzyme, a DNA sequence of a spacer region containing the PstI cleavage site, and Phe-Arg, which is a recognition sequence for kallikrein, and a fudon TAA from beginning to end at its C-terminus.
A 122 bp vIp-cty gene with added TAG and restriction enzyme B a m HI cleavage sites was generated using a DNA synthesizer (Applied Biosystems Model 381).
Chemically synthesized in A).

(4) Preparation of protein A-V I P-G ly fusion protein expression plasmid pMD235 Chemically synthesized V I P-G 1 3. Gene (5μg
), and the restriction enzymes Pstl, Ba
The long chain fragment (1 μg) of plasmid pMD200 digested with mH1 was digested with TaDNA ligase (200 units).
) and ligated using Bacillus subtilis B, 5ubtilisSP
The L14 strain was transformed by the protoplast method.

Plasmid pMD235 was obtained from the obtained Km strain. When DNA sequencing was performed on the part inserted into pMD235 using the M13 dideoxy method,
The VIP-Guy gene was inserted into position 9 as intended.

Examples 1 to 12 and comparative examples and Bacillus subtilis DY-16 strain, 5PL1
4 strains (containing pMD235) were cultured in medium A (tryptone 33
g1 Yeast extract 20g1 Sodium chloride 7.4g, Casamino acid 20g1 Glucose 10g, Disodium hydrogen phosphate 8g, Potassium dihydrogen phosphate 4g1 Manganese chloride 0
．． 06mM to 11! 100 m
11 and cultured in a pleated Erlenmeyer flask under conditions of 37°C and 120" pH. A portion of the culture solution was sampled at 12 and 24 hours of incubation, and vrp- The amount of cty was measured.The results are shown in Table 4 in mg per gram of the medium.The ionic strength of the medium was measured as follows.

Quantification of VIP-G by enzyme immunoassay of VIP-Gly
The quantification of ly by enzyme immunoassay was carried out by the method described in "Enzyme Immunoassay" (edited by Eiji Ishikawa et al., Igaku Shoin).

First, a rabbit was immunized with VIP to obtain anti-VIP serum. From this anti-VIP serum, the 8th test of "enzyme immunoassay"
Maleimide-hinge method + described on pages 3-92:, Nori anti-V
IP-1gG, anti-VIP-F(ab')2, anti-VI
P-peroxidase labeled-Fab' was prepared.

VIP-Gly was measured by enzyme immunoassay according to the following procedure.

Anti-VIP-F (a
b') z was adsorbed and blocked with 1% bovine serum albumin. Add the test solution to this plate and VIP-
After binding Gly to anti-VIP-F(ab')2 adsorbed on the solid phase, it was washed, and further anti-VIP-peroxidase-labeled Fab2 was added to bind VIP-G bound to the solid phase.
I made a sandwich of ly. After removing free labeled antibody, 10mM ortho-phenylenediamine (OPD),
A reaction solution containing 0.025% hydrogen peroxide and 50 mM sodium acetate buffer (pH 5.0) was added, and the dye produced by the peroxidase reaction was measured by absorption at a wavelength of 490 nm.

Applied Biosystems (AB
VIP-Gly, whose amino acid composition was confirmed by solid-phase synthesis using a peptide synthesizer manufactured by I) and purified by reverse-phase high-performance liquid chromatography, was used.

Ionic strength of the medium The ionic strength of the medium was determined by measuring the electrical conductivity of the medium using an electrical conductivity meter, and is shown in Table 4 as the ionic strength in terms of sodium chloride.

(below, margin) From Table 4, If the ionic strength of the medium is 1 or more, In case, The gene product V ■ large amount of y is accumulated in

Claims (1)

[Scope of Claims] 1. A medium for culturing microorganisms that secrete and produce proteins or peptides extracellularly, which contains at least one component of amino acids, salts thereof, and inorganic salts, and has an ionic strength of 1 or more. A culture medium characterized by: 2. A method for producing a protein or peptide, which comprises culturing a microorganism that secretes and produces a protein or peptide extracellularly in the medium according to claim 1.