JPH1189590A - Recovery and purification of protein having epithelial cell growth factor activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply and efficiently recover and purify the subject protein, by culturing Bacillus brevis as a host bacterium, adjusting a culture substance containing a product to proper pH to precipitate an admixture protein and subjecting a clear fraction to salting out. SOLUTION: Bacillus brevis is cultured as a host bacterium. A culture material containing a protein having epithelial cell growth factor activity is adjusted to pH 3.0-4.5, preferably pH 3.5-3.9, an admixture protein contained in the culture substance is specific precipitated and the precipitated protein and the cell are removed to give a clear fraction. Then the obtained clear fraction is mixed with a salt (e.g. sodium chloride) to precipitate a protein having epithelial cell growth factor activity. The precipitated fraction is recovered and the coexisting admixture component is separated and removed to recover and purify the objective protein useful as a therapeutic agent for wound, an antiulcer agent, an auxiliary for anticancer agent, etc., simply and efficiently.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an epidermal growth factor produced by culturing a recombinant using Bacillus brevis as a host bacterium.
r: hereinafter referred to as a method for recovering and purifying a protein having EGF) activity. More particularly, the present invention relates to EGF
Bacillus brevis transformed with a gene encoding an active protein is cultured, and EG is purified from the culture.
The present invention relates to a method for recovering and purifying a protein having F activity, and more particularly to a simple and efficient method for recovering and purifying a protein suitable for industrial mass processing.

[0002]

2. Description of the Related Art EGF has been isolated from urine of humans and horses and submandibular glands of rabbits, rats and mice, and is known to be present in various tissues of mammals ( Adv. Metab. Dis., 8 , 265 (1975)). Human epidermal growth factor (h-EG)
F) has a molecular weight of about 6,000 consisting of 53 amino acids.
Is known to have three disulfide bonds in the molecule and to have the amino acid sequence shown in SEQ ID NO: 1 (H. Gregory, Nature, 257 , 325 (1975)).

The physiological effects of EGF include cell proliferation, gastric acid secretion, anti-ulcer, gastrointestinal mucosa protection, and DN.
It has been reported that A-synthesis promoting action, corneal repair action, calcium release promoting action, wound healing promoting action, anti-inflammatory action, analgesic action, hepatocellular damage inhibitory action, hair follicle retraction action, etc. Growth Factor, page 20, Asakura Shoten 1984). Since EGF has such an effect, its application has been attempted in various fields such as a wound healing agent, an antiulcer agent, and an anticancer agent adjuvant (BIO INDUSTRY, 8 , 275 (199)
1)).

[0004] As described above, various applications of EGF have been studied, and various studies have been made on production methods capable of stably supplying EGF.

For example, Bacillus brevis HPD31
( Bacillus brevis HPD31: This strain is the same strain as Bacillus brevis H102 (FERM BP-1087)) is a strain that secretes and produces proteins outside the cells and does not produce proteolytic enzymes in the culture solution. (JP-B 4-74997), which is used as a host cell for genetic recombination for the production of various proteins. Ebisu et al. Have established a mass production technique of h-EGF using Bacillus brevis HPD31 as a host bacterium, and have succeeded in secreting and producing about 3 g / L of h-EGF in a culture solution (Japanese Unexamined Patent Publication No. 133782). Takagi et al. Have created a novel protein having EGF activity and succeeded in high production thereof using Bacillus brevis HPD31 (Japanese Patent Application No. 8-123970).

However, in order to develop EGF or a protein having EGF activity secreted and produced in a culture solution into a drug or the like by applying the gene recombination technique, it is necessary to use a mixture of cells and media derived from the medium mixed in the culture. It is necessary to remove contaminants such as components, contaminant proteins secreted and produced at the same time, and other metabolites, and to recover and purify only the protein having the desired EGF activity. It is better that this purification step is simple and the number of steps is small. Particularly, when the purification is performed on an industrial scale, if the purification step is complicated, a large capital investment is required, and the production cost is also increased. The problem arises.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above technical and industrial needs, and is directed to the production of a heterologous gene product using Bacillus brevis as a host bacterium. An object of the present invention is to provide an efficient recovery and purification technique from a culture containing a body. That is, the present invention
EGF secreted into Bacillus brevis culture
The present invention provides a method for separating a protein having an activity from coexisting components and recovering and purifying the protein in a simple and high yield. From an industrial point of view, the present invention relates to a method for recovering and purifying a protein having an EGF activity. It has been made for the purpose of newly developing and providing a particularly suitable simple and efficient mass processing system.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies on a method for efficiently recovering and purifying a protein having EGF activity from a Bacillus brevis culture, and as a result, the pH of the culture was determined to be a specific value. When adjusted to the range, most of the contaminating proteins contained in the culture of Bacillus brevis are specifically aggregated and precipitated, and it is possible to efficiently remove the aggregated precipitate fraction and bacterial cells. A useful new finding has been obtained that a protein having EGF activity can be collected and purified very simply and efficiently by membrane filtration or centrifugation by adding a salt to the protein to specifically aggregate and precipitate the protein having EGF activity. . The present invention has been finally completed as a result of further research based on this useful new finding.

That is, the present invention provides a method for preparing a culture of Bacillus brevis containing a protein having EGF activity.
After adjustment, most of the contaminating proteins contained in the culture were specifically precipitated, and after removal of the proteins, salts were added to the clarified fraction to precipitate proteins having EGF activity. The present invention relates to a method for recovering and purifying a protein having EGF activity, which comprises separating and recovering from coexisting contaminants by centrifugation.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, a culture of Bacillus brevis containing a protein having EGF activity is prepared by removing EGF activity from contaminating proteins and other contaminants by the following steps (a) and (b). The present invention relates to a method for recovering and purifying a protein having the same.

That is, in order to carry out the present invention, the culture may be treated according to the following steps (a) and (b), and the protein can be recovered and purified efficiently by these treatments. . (A) -1: adjusting the pH of the culture to 3.0 to 4.5 (preferably 3.5 to 3.9) to precipitate contaminating proteins contained in the culture; ) -2: The precipitate formed in (a) -1 and the cells were separated and removed to obtain a clarified fraction containing a protein having EGF activity; (b) -1: The clarified fraction obtained in (a) -2 Add salt to the fraction,
(B) -2: recovering the precipitate generated in (b) -1.

The method for recovery and purification of the present invention can be applied to a Bacillus brevis culture containing a protein having EGF activity. Specifically, Bacillus brevis HPD31 highly produces h-EGF and a protein having EGF activity outside the cells, and thus the recovery and purification method of the present invention can be effectively applied to the culture of the strain.

Specific examples of proteins having EGF activity recovered and purified by the method of the present invention are as follows: human EGF of SEQ ID NO: 1, mouse EGF of SEQ ID NO: 2, rat EGF of SEQ ID NO: 3, and rat EGF of SEQ ID NO: 4-7. Protein (Japanese Patent Application No. 8
-123970). In addition, these E
The gene encoding the amino acid sequence of the protein having GF activity may be a gene isolated from a cell capable of producing EGF, or a chemically synthesized gene. For example, when a synthetic gene is used, a gene consisting of the most accepted codon in Bacillus brevis is preferred.

A protein gene having EGF activity is
It may be incorporated into Bacillus brevis by a known method. For example, Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Molecular Cloning 2nd ed, A Laboratory Ma
nual, Cold Spring Harbor Laboratory, 1989).

By culturing Bacillus brevis transformed with a gene encoding a protein having EGF activity in this manner, a culture of Bacillus brevis containing a protein having EGF activity can be obtained.

The culture of Bacillus brevis thus obtained is basically treated in the steps (a) to (b) while containing the cells, but the culture is pretreated by membrane treatment or centrifugation. After removing the cells in step (a), the cells may be treated in steps (a) and (b). That is, the present invention relates to (a) pH of the culture.
Is adjusted to 3.0 to 4.5 to specifically precipitate contaminating proteins contained in the culture, and removing the precipitated proteins and cells to obtain a clear fraction;
A salt is added to the clarified fraction obtained in (a) to precipitate a protein having EGF activity, and a method of recovering and purifying a protein having EGF activity in which the precipitated protein fraction is separated and recovered from contaminating components that coexisted therewith It is. Hereinafter, the steps (a) and (b) of the present invention will be described in more detail.

First, a culture of Bacillus brevis containing a protein having EGF activity, or a culture from which cells have been removed in advance by centrifugation or membrane filtration are treated according to the following steps.

(A) -1: Contaminant proteins are precipitated by adjusting the pH of the culture. The pH is such that contaminant proteins contained in the culture specifically precipitate,
Adjust so that the protein having F activity does not precipitate. When Bacillus brevis HPD31 is used as a host bacterium, the main contaminating protein is a cell surface protein produced by the strain outside the cells (HW
P; J. Bacteriol., 172 , 1312-1320 (1990)), and the pH is in the range of 3.0 to 4.5, more preferably 3.5 to 3.9.
By adjusting to within the range, specific precipitation can be achieved. As the acid used for adjusting the pH, an acid such as hydrochloric acid, nitric acid, and sulfuric acid may be used.

(A) -2: In the step of removing contaminating proteins precipitated in the step (a) -1, the precipitate can be removed from the system by centrifugation or membrane filtration. When removing by membrane filtration, a filtration membrane having a pore size in which proteins having EGF activity are filtered and contaminating proteins including precipitated HWP are concentrated. For example, if microfiltration is performed using a filtration membrane having a pore size of 0.45 μm, the precipitate is concentrated on the membrane and can be removed. At this time, if the precipitate is further washed with a buffer having a pH of 3.0 to 4.5, the protein having EGF activity can be more efficiently collected in the filtrate. The buffer used at this time may be a buffer that does not cause the protein having EGF activity to lose its activity, such as an acetate buffer or a phosphate buffer. In this way, contaminating proteins are removed from the Bacillus brevis culture, and a protein solution having enhanced EGF activity can be obtained.

(B) -1: A protein having EGF activity is specifically precipitated by adding a salt to the filtrate collected in the step (a) -2. The salt to be added may be any salt which has good cohesiveness of the protein having EGF activity and does not lose the activity, and for example, ammonium sulfate, sodium sulfate, potassium phosphate, magnesium sulfate, sodium citrate, sodium chloride and the like are preferable. Can be used. The salt concentration may be a concentration at which a protein having EGF activity is precipitated, and may be a concentration of 9% or more. For example, when sodium chloride is used, the concentration may be 9% or more, more preferably 12% or more.

(B) -2: E precipitated in step (b) -1
To recover a protein having GF activity, (a)-
As in the case of 2, the precipitate can be collected by centrifugation or membrane filtration. When collecting by membrane filtration, a membrane having a pore size in which the precipitate is concentrated on the membrane may be used. For example, the precipitate can be concentrated and recovered by performing microfiltration using a filtration membrane having a pore size of 0.45 μm.
At this time, the precipitate can be washed with a buffer solution having the same salt concentration as the added salt concentration to wash away impurities. The protein having EGF activity thus recovered is dissolved in a buffer, and then desalted by dialysis or membrane filtration. When desalting is performed by membrane filtration, for example, filtration is performed using an ultrafiltration membrane having a molecular weight cutoff of 5,000, and at the stage when the concentration is advanced, an appropriate amount of pure water is added to sufficiently remove salts. Good. In addition, desalting by membrane filtration can simultaneously concentrate the protein having the desired EGF activity, so that the liquid volume can be made compact, which is effective in the subsequent freeze-drying. As described above, a protein having EGF activity purified to high purity can be obtained.

According to the recovery and purification method of the present invention, a protein having EGF activity can be very easily purified from a Bacillus brevis culture to a purity of 90 to 95% or more with a recovery of 85% or more. The protein having EGF activity obtained by the method of the present invention can be applied to various uses, but when it is necessary to further increase the purity,
A higher-purity protein having EGF activity can be obtained by a known method such as chromatography.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the examples are illustrative and the present invention is not limited thereto.

[0024]

Embodiment 1 Bacillus brevis HP926 (FERM)
BP-5382).
HT-EGF high secretion production plasmid pHT1 prepared from
Bacillus brevis HPD31 (FERMBP-1089) was electroporated (H. Takagi et al. Agric. Biol. C.) with 10 EGF (Japanese Patent Laid-Open No. 6-133782).
hem., 53 , 3099-3100 (1989)).
Was obtained. The strain obtained here was used for peptone 4%, yeast extract 0.5%, MgSO ₄ 5 mM,
MnSO ₄ 1 mM, FeSO ₄ 36 μM, glucose 2
%, Agar 1.5%, pH 7.2 at 30 ° C. overnight. This was mixed with 1 L of a liquid medium having the same composition as above.
And subjected to shaking culture at 30 ° C. overnight to obtain a pre-culture solution.

After 17 L of a production medium having the same composition as that of the preculture was dispensed into a 30 L jar fermenter, sterilized at 120 ° C. for 15 minutes, cooled, inoculated with 200 ml of the preculture, stirred at 200 rpm, aerated at 1 vvm, and cooled. Cultivation was carried out at 3 ° C. for 3 days.

The amount of h-EGF in the culture was determined by HPLC (column: C18-100A, diameter 4 mm × length 250 mm, buffer: 0.1% trifluoroacetic acid (TFA) / H ₂ ).
O, 0.1% TFA / 50% acetonitrile, linear gradient, detection: UV 276 nm), and peak area when HPLC was performed under the same conditions using commercially available h-EGF (manufactured by Funakoshi Co., Ltd.) as a standard product. As a result of obtaining the production amount in comparison with, 1.0 g / L of h-EGF was produced in the culture.

The 17 L of the culture was used to remove bacterial cells using a 0.45 μm filtration membrane (Pellicon cassette system (manufactured by Millipore)), and the culture supernatant containing h-EGF in the filtration fraction was recovered. The pH of the culture supernatant was adjusted to 3. using 6N hydrochloric acid.
8 to precipitate contaminating proteins including HWP,
Filtration was performed using the same filtration membrane as described above, and h-EGF was collected in the filtration fraction. In addition, contaminating proteins on the membrane
After washing with 1 L of an mM acetate buffer (pH 3.8), h-EGF remaining on the membrane was collected in a filtration fraction.

Next, NaC was added to the filtered fraction containing h-EGF.
1 was added to a concentration of 12% to precipitate h-EGF. This was filtered using a 0.45 μm filtration membrane (Pellicon cassette system (Millipore)) to obtain h-EG.
F was collected on the membrane. The recovered h-EGF was 12% N
After washing and filtering with 1 L of 20 mM acetate buffer (pH 3.8) containing aCl to remove impurities in the filtrate, the h-EGF fraction on the membrane was concentrated to 200 ml. The thus recovered h-EGF was added to a 0.1 M Tris buffer (pH 8.
0).

This h-EGF-containing solution was subjected to a molecular weight cutoff of 5,
000 ultrafiltration membrane (Pellicon cassette Biomax5 (manufactured by Millipore)) was used for ultrafiltration to desalinate. 50
After concentration to about 0 ml, the mixture was filtered while 10 L of pure water was gradually added to completely remove salts. The recovery rate and purity of the finally recovered h-EGF were determined, and as a result, the recovery rate of h-EGF from the culture was 90% and the purity was 95%.

[0030]

Example 2 Plasmid pHT-ABC carrying a gene encoding the amino acid sequence of a protein having EGF activity shown in SEQ ID NO: 7 (Japanese Patent Application No. 8-123970)
In, Bacillus Brevis HPD31 (FERM BP-
1089) by electroporation ((H. Takagi e
Agric. Biol. Chem., 53 , 3099-3100 (1989)) and a protein ABC having EGF activity.
Was obtained.

The strain obtained here was prepared by adding 4% of peptone, 0.5% of yeast extract, 5 mM of MgSO ₄ , 1 mM of MnSO ₄ ,
FeSO ₄ 36 μM, glucose 2%, agar 1.5%,
The cells were cultured overnight at 30 ° C. on a pH 7.2 agar medium. 500 ml of 100 ml of liquid medium of the same composition as above
The mixture was dispensed into an Erlenmeyer flask, sterilized at 120 ° C. for 15 minutes, inoculated after cooling, and cultured with shaking at 30 ° C. overnight to obtain a pre-culture solution.

After dispensing 1 L of a production medium having the same composition as that of the preculture to each of two 2 L jar fermenters,
The mixture was sterilized for 5 minutes, cooled, inoculated with 20 ml of the pre-culture liquid, and cultured at 30 rpm at a stirring speed of 200 rpm at a ventilation rate of 1 vvm for 3 days.

The amount of protein ABC in the culture was determined by HPL
C (column: C18-100A, diameter 4 mm x length 250)
mm, buffer: 0.1% trifluoroacetic acid (TF
_{A) / H 2 O, 0.1} % TFA / 50% acetonitrile, linear gradient, detection: analyzed by UV276nm), subjected to HPLC under the same conditions a commercial h-EGF and (Funakoshi Co., Ltd.) as a standard As a result of estimating the production amount in comparison with the peak area at the time of
Protein was being produced.

The pH of this culture was adjusted using 6N hydrochloric acid.
8 to precipitate contaminating proteins including HWP,
Filtration membrane (Mini-tan cassette system (Millipore))
The protein ABC was collected in the filtration fraction by filtration. 20 mM of contaminating proteins and cells on the membrane
Wash with 500 ml of acetate buffer (pH 3.8),
A small amount of the protein ABC remaining on the membrane was recovered in the filtration fraction.

Next, ammonium sulfate was added to the filtered fraction containing h-EGF to a concentration of 15%.
Was precipitated. This was filtered using a 0.45 μm filtration membrane (Minitan Cassette System (manufactured by Millipore)).
The protein ABC was collected on the membrane. The collected protein ABC was washed with 500 ml of a 20 mM acetate buffer (pH 3.8) containing 15% ammonium sulfate to remove impurities, and then 200 ml of the protein ABC fraction on the membrane was removed.
Concentrated. The thus recovered protein ABC was dissolved in a 0.1 M Tris buffer (pH 8.0).

The protein ABC-containing solution was applied to an ultrafiltration membrane having a molecular weight cutoff of 5,000 (Pellicon cassette Biomax5).
(Millipore)) for desalting. After concentration to about 500 ml, the mixture was filtered while 10 L of pure water was gradually added to completely remove salts. The purity and recovery of the finally recovered protein ABC having EGF activity were determined, and as a result, the recovery of protein ABC having EGF activity from the culture was estimated at 85%, with a purity of 93%.

[0037]

Industrial Applicability The present invention provides a method for recovering and purifying a protein having EGF activity from a culture very simply and efficiently, and is particularly useful for recovering and purifying the protein on an industrial scale. It is valid.

[0038]

Sequence Listing The amino acid sequences of human EGF, mouse EGF, and rat EGF are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the four types of related proteins are shown in SEQ ID NO: 4, respectively.
The results are shown in 5, 6, and 7. Tables 1 to 7 below show the respective sequences shown in SEQ ID NOs: 1 to 7.

[0039]

[Table 1]

[0040]

[Table 2]

[0041]

[Table 3]

[0042]

[Table 4]

[0043]

[Table 5]

[0044]

[Table 6]

[0045]

[Table 7]

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 21/02 C12N 15/00 ZNAA // (C12N 1/21 C12R 1:08) (C12N 15/09 ZNA C12R 1:08) (C12P 21/02 C12R 1:08)

Claims (4)

[Claims] 1. A culture containing a protein having epidermal growth factor activity produced by cultivating Bacillus brevis as a host bacterium, comprising: (a) adjusting the pH thereof to obtain a contaminant protein contained in the culture; Specifically, and removing the precipitated protein and cells to obtain a clear fraction; (b) adding a salt to the obtained clear fraction to precipitate a protein having epidermal growth factor activity. Recovering the precipitated protein fraction and separating and removing coexisting contaminant components. 2. In the step (a), the pH is adjusted to 3.0 to 3.0.
4.5, more preferably 3.5 to 3.9,
The method for recovery and purification according to claim 1, characterized in that: 3. In the step (b), as a salt, at least one selected from sodium chloride, ammonium sulfate, sodium sulfate, potassium phosphate, magnesium sulfate and sodium citrate is used, and the concentration of the salt is 9%.
The recovery and purification method according to claim 1 or 2, wherein the content is more preferably 12% or more. 4. The protein having epidermal growth factor activity is human epidermal growth factor shown in SEQ ID NO: 1, mouse epidermal growth factor shown in SEQ ID NO. 2, and rat epidermal growth factor shown in SEQ ID NO. 4. The method according to claim 1, wherein the method is at least one of the proteins represented by SEQ ID NOs: 4 to 7.