JPH01294689A - Production of proteinous growth factor - Google Patents

Abstract

PURPOSE:To obtain the subject growth factor having high specific activity in high purity and yield by homogenizing an organism-constitution element in a lower alcohol, mixing the obtained precipitate with an extraction liquid, a lower alcohol and a halogenated hydrocarbon and extracting the objective factor. CONSTITUTION:An organism-constitution element is homogenized in a lower alcohol (preferably methanol), the precipitate is separated with a filter paper and the filtrate is mixed with 1 pt.vol. of an extraction liquid selected according to the kind of the objective growth factor, 4 pts.vol. of a lower alcohol (preferably methanol) and about 1 pt.vol. of a halogenated hydrocarbon (preferably chloroform). The produced precipitate is separated by centrifugal separation and the halogenated hydrocarbon is removed from the separated precipitate. The resultant precipitate is extracted with an extraction liquid and purified by salting-out, ion-exchange chromatography, etc., to obtain the objective growth factor.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for producing proteinaceous growth factors. More specifically, the present invention relates to a method for producing proteinaceous growth factors by isolating and purifying growth factors consisting of proteins present in living organisms.

<Prior art and problems to be solved by the invention> It is known that various growth factors exist in tissues, organs, body fluids, etc. that are biological components, and a large number of substances have been isolated and reported so far. . Some of these growth factors are known to consist of proteins, such as heparin-binding growth factor (Heparin Bindlng Growth F
actor % aka fibroblast growth prisoner Plblob
last Growth Factor) ,) Transforming Growth Factor (Transforming G
rowth Factor), platelet-derived growth factor (P
latelet-derlved GrowthFae
tOr), epidermal growth factor (Epldermal Gr)
owth Pac-tor), etc. Applications of the above-mentioned growth factors have been studied in various fields based on their physiological activities. Taking heparin-binding growth factors as an example, they have been shown to have vascular endothelial cell growth promoting activity, In the body, it has effects such as promoting angiogenesis, healing wounds, and reducing inflammation at inflammatory sites, and in vitro, it causes vascular endothelial cells to attach and proliferate on the inner surface of tubular polymeric materials. It is useful as a cell growth promoter in the production of the resulting non-thrombotic artificial blood vessel. Therefore, it is expected to be used as therapeutic medicines, biochemical reagents, etc. (for example, ANALYTIAL BIOCHEMISTRY
, Vol, 154.1-14.1986, Protein Nucleic Acid Enzyme, Vol, 33.373-881.1988
etc.).

Conventional methods for isolating and purifying proteinaceous growth factors from biological components include extraction, salting out, centrifugation, dialysis, ion exchange chromatography, and gel filtration chromatography, which are common protein isolation and purification methods. , affinity chromatography, etc. are used. For example, biological tissue is homogenized with a neutral buffer, and the supernatant is subjected to ion exchange chromatography, gel filtration chromatography,
A method for purifying by subjecting to affinity chromatography, etc., homogenizing biological tissue in an aqueous ammonium sulfate solution, redissolving the precipitate, dialysis, and purifying by subjecting to ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc. (See the above-mentioned documents, etc.).

However, these methods also extract proteins from other species, resulting in a large amount of contaminant proteins and a decrease in specific activity, as well as a lack of operability as the amount of extraction solution increases, and furthermore, the yield of the desired growth factor is low. There was a problem.

As a result of intensive research into methods for isolating and purifying proteinaceous growth factors from biological components, the inventors discovered a simple method for obtaining proteinaceous growth factors with high specific activity and high purity, and have developed the present invention. completed.

Therefore, an object of the present invention is to provide a novel method for producing proteinaceous growth factors.

<Means for Solving the Problems> The production method of the present invention, which has been made to achieve the above object, is a method for collecting proteinaceous growth factors from biological components, at least (ω) Step of homogenizing in lower alcohol and separating the precipitate; (b) Step of extracting the proteinaceous growth factor from the precipitate obtained in the rice field with an extract solution; (c) Step of extracting the protein growth factor from the precipitate obtained in the above song. A step of mixing the extract with a lower alcohol and a halogenated hydrocarbon and separating the precipitate; (Small) A step of extracting proteinaceous growth factors from the precipitate obtained in step (c) above using the extract; Includes It is characterized by this.

The production method of the present invention can be widely adopted as a method for collecting proteinaceous growth factors from biological constituents, such as living tissues, organs, body fluids, etc., but preferably the growth factors are lower alcohols and carbonized halides. The present invention is preferably applied to the production of hydrogen-stable substances, such as heparin-binding growth factor, transforming growth factor, platelet-derived growth factor, and epidermal growth factor.

As the starting material for the production method of the present invention, any biological component can be used as long as it contains the desired growth factor. For example, in the case of heparin-binding growth factors, cerebrum, Hypothalamus, pituitary gland, heart, retina, eyes, kidneys, adrenal glands, placenta, corpus luteum, cartilage, tumor cells, etc., platelets for transforming growth factors and platelet-derived growth factors, and jaws for epidermal growth factors. An example is the lower gland.

Furthermore, these biological constituents are preferably as fresh as possible, and after being extracted from a living body, they are preferably stored at a low temperature, preferably at about -35°C, in order to suppress protein decomposition.

Next, an example of the manufacturing method of the present invention will be specifically explained. First, biological components containing the target growth factor are homogenized in lower alcohol (e.g., methanol, ethanol, propatool, etc.), and then the precipitate is separated ((ω
process). The amount of lower alcohol to be used for the biological component is preferably about 4 g of lower alcohol (preferably methanol) per 1 kg of the biological component. Homogenization can be carried out using a conventional homogenizer, such as a Waring blender or a Polytron homogenizer.
Genizer) etc. are used. The temperature and time of this homogenization operation are not particularly limited, but are usually between room temperature and
The process is completed in about 1 to 15 minutes, preferably about 3 to 5 minutes under cooling. Further, the precipitate can be separated by a conventional method such as using a filter paper or centrifugation.

Next, an extract solution that dissolves the target protein growth factor is added to the obtained filtrate to extract the protein growth factor, and then the extract solution is separated (step). The extract solution is appropriately selected depending on the type of target growth factor, for example,
For heparin-binding growth factors, 5 mM E D
T A 50 containing 5 m M benzamidine, 1 m M phenylmethylsulfonyl fluoride, 1 μt/xl leupeptin and 1 μt/wl pepstatin A
(mM) Lis-HCl (pH 7, O) buffer, etc. are exemplified. The amount of extract to be used is appropriately selected depending on the type of extract, solubility of the target growth factor, etc., but usually, the amount of precipitate 1
It is used in an amount of about 2 parts by weight. This extraction operation is preferably carried out using a homogenizer, and the homogenizer exemplified in the above (ω step) is used as the homogenizer, and the homogenization conditions are substantially the same as the above (ω step). The extraction is completed by stirring at a temperature of about 4°C (preferably about 4°C) for about 1 to 24 hours, preferably overnight.The extraction liquid can be separated by the method exemplified in step (a) above. However, it is preferably carried out by centrifugation.

The extract thus obtained is then treated with a lower alcohol and a halogenated hydrocarbon (e.g. chloroform,
methylene chloride, dichloroethane, etc.), and the resulting precipitate is separated (step (c)).

The extract, lower alcohol, and halogenated hydrocarbon can be mixed by an appropriate method such as stirring or shaking. At this time, methanol is used as the lower alcohol,
It is preferable to use chloroform as the halogenated hydrocarbon, and the amount used is preferably about 4 volumes and 18 volumes, respectively, per 1 volume of the extract.

Separation of the precipitate produced by mixing the extract, the lower alcohol, and the halogenated hydrocarbon can be carried out by centrifugation, although the method exemplified in the above (ω step) may be used.

The precipitate separated above is washed with lower alcohol to remove halogenated hydrocarbons as necessary, and then the proteinaceous growth factors are extracted using the above extract, and then the extract is separated ( By step (d)), a solution containing the desired proteinaceous growth factor is obtained (this extract is referred to as a chloroform sample for convenience). This operation can be carried out in substantially the same manner as in step (b) above, and the operating conditions include the conditions shown in step (b) above.

The chloroform sample obtained in this way can be subjected to salting out, dialysis, centrifugation, ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc., depending on the physical properties and properties of the target growth factor.
A highly purified proteinaceous growth factor can be obtained by further purification using a suitable combination of conventional methods such as freeze-drying.

To explain this using the example of a heparin-binding growth factor, NaCl was added to the chloroform sample obtained above.
and 3-[(cholamidopropyl)dimethylammonio]
-1-propanesulfonate (3-[(chola
sldopropyl) dig+ethylaa+5o
nlo-propanesuhonate (hereinafter abbreviated as CIIAPS) at 0.5M and 0.5M, respectively.
After dissolving the solution to 1% and creating conditions similar to the equilibration solution described below, add the equilibration solution (e.g. 10mM) to the solution.
Hydrochloric acid pH 7, 4, 0, 5M NaCl and 0.1% CHA
It is adsorbed through a heparin-Sepharose column equilibrated with PS (containing PS). After washing the column with equilibration solution to remove unadsorbed proteins, approximately 1.5-2.0 M Na
By elution with an eluent containing C9 (for example, containing 10 m M Tris-HCl, 2.OMNaCj! and 0.1% CHAPS), a high specific activity, high purity heparin-binding growth factor solution can be obtained. I can do it. Further, in some cases, a small amount of low molecular weight protein may be mixed in the growth factor obtained by the above-mentioned heparin affinity chromatography treatment. In this case, contaminating low molecular weight proteins can be removed by further purifying the heparin affinity chromatography-treated product by high performance liquid reverse phase chromatography using a C4 column.

<Operations and Effects of the Invention> According to the production method according to the present invention, first, since biological components are homogenized in lower alcohol, a considerable amount of protein is denatured and it is difficult to increase the specific activity of growth factors. In addition, since the proteolytic enzymes are inactivated, the decomposition of growth factors is suppressed, and since the contaminant proteins are denatured, the amount of solubilized protein is small, so the amount of extract liquid in the next extraction step is reduced. This makes the operation easier.

Furthermore, since the extract containing growth factors is treated with lower alcohols and halogenated hydrocarbons, protein denaturation progresses further and the specific activity of growth factors can be increased. Inactive proteolytic enzymes can be further inactivated to avoid decomposition of growth factors, and since the degree of denaturation of contaminant proteins is increased, the amount of extract can be reduced, and subsequent centrifugation and affinity Chromatography operations etc. become easier. Therefore, the production method of the present invention has the unique effects of being able to obtain high yields of proteinaceous growth factors with high specific activity and high purity, as well as being excellent in workability.

<Examples> Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited to these Examples.

Example 1 Production of heparin-binding growth factor from bovine heart Hearts were transported from a slaughterhouse on ice within several hours after slaughter and stored at -35°C until use. 1kg beef heart
Add methanol 4II to Waring Blend
It was homogenized with an er. Filter paper was placed in a Buchner funnel for filtration. The extract solution (50mM
) Lis-HCl pH 7, 0, 5mM EDTA, 5mM benzamidine, 1mM phenylmethylsulfonyl fluoride, 1μtr/1110 Ipeptin, 1μt/
Add 2g of xl pepstatin A), Varlng B
Homogenized with render. After that, extraction was carried out overnight at 4°C with stirring, followed by centrifugation (10,000g
, 20 minutes, 4°C) to obtain a supernatant. 4 volumes of methanol were added to 1 volume of supernatant and stirred, and further 1 volume of chloroform was added and stirred. The mixture was allowed to stand at room temperature for 30 to 60 minutes, and the resulting precipitate sank to the bottom of the container. The supernatant, which has almost no precipitate, is removed by decantation.
After reducing the total volume, the precipitate was collected by centrifugation under the same conditions as above. 200 yl of the above extract was added to the obtained precipitate, and the mixture was stirred overnight at 4°C for extraction. This was centrifuged under the same conditions as above to obtain a supernatant.

200 chloroform samples obtained by the above method
0.5M NaC9 and 0.1% CHAPS were dissolved in equilibration solution (10mM
Tris-HCl pH 7, 4, 0, 5M NaCjl, 0.
The mixture was passed through a column of heparin-Sepharose (volume 40xl, manufactured by Pharmacia) equilibrated with 1% CIAPS). After passing all the samples, add 400 ml of the above equilibration solution.
11 to remove proteins that did not bind to heparin. Thereafter, the growth factors bound to the column were eluted with an eluent (10mM Lis-HCl, 2.0M NaCN, 0.1% CHAPS), and approximately 31% of the growth factors were bound to the column. A fraction was taken, subjected to SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis (SDS-PAGE), growth factors were detected by silver staining, and the fractions containing the growth factors were saved. .

By the above operation, the molecular weight was 18.0 on 5DS-PAGE.
1.24 μl of bovine heart-derived heparin-binding growth factor showing only one 00 dalton was obtained.

In addition, if the above-mentioned heparin affinity chromatography-treated product contains a contaminant protein with a molecular weight of about 117.500, the low-molecular-weight protein can be purified by high-performance liquid reverse phase chromatography under the following conditions and operating method. can be removed.

■) Chromatography conditions Column 204 column (4J x 250mm) Eluent: (A) solution: 0.1% trifluoroacetic acid (TFA) solution (B) solution: 90% containing 0.1% TFA Acetonitrile solution A solution prepared by mixing the above solutions (A) and (B) with varying acetonitrile concentrations was used.

Flow rate: 111/min Detection method: Absorbance at 214 nm 11) Operation method High performance liquid reverse phase chromatography using a C4 column equilibrated the above heparin affinity chromatography treated solution with 18% acetonitrile solution containing 0.1% TFA. The mixture was eluted using eluents in which the acetonitrile concentration was successively changed from 18%.

As a result, the bovine heart-derived heparin-binding growth factor was eluted at an acetonitrile concentration of 38%, but the mixed low-molecular proteins were eluted before the growth factor, making it possible to completely separate the two.

Example 2 Production of heparin-binding growth factor from bovine cerebrum 1 kg
Extraction and heparin affinity chromatography were performed in the same manner as in Example 1. Then,
In the same manner as in Example 1, the mixture was subjected to high performance liquid reverse phase chromatography using a 04 column, and the eluates (A) and (B) were appropriately mixed and eluted with an eluate with varying acetonitrile concentrations. However, the acetonitrile concentration was 43
%, 1.1 mg of heparin-binding growth factor (molecular weight: 18,000 Daltons) was obtained from both the fractions.

The obtained heparin-binding growth factor was tested for the proliferation-promoting effect of heparin on human vascular endothelial cells (Sc
ience, Vol, 222.623-825.19
83), it is presumed that the heparin-binding growth factor corresponds to the existing bovine cerebrum-derived heparin-binding growth factor type 1.

The isolated yield of heparin-binding growth factor type 1 derived from bovine cerebrum that has been reported so far is:
400 kg (Biochemistry, Vol.
, 23°6295-6299.1984) and 36 u
g (Proc,, Natl,.

Acad, Sci, USA, Vol, 81.
357-381.1984). However, the above income
The report of i1400 μg/kg was obtained only by purification using heparin Sepharose, and considering the manufacturing method, it is presumed that impurities were mixed in. Compared to the yields reported in these reports, the method of the present invention allows bovine cerebrum-derived heparin-binding growth factor type 1 to be obtained with high purity and high yield.

Claims (1)

[Scope of Claims] 1. A method for producing a proteinaceous growth factor, which comprises at least the following steps (a) to (d) in a method for collecting a proteinaceous growth factor from biological components. (a) A step of homogenizing biological components in a lower alcohol and separating the precipitate; (b) A step of extracting protein growth factors from the precipitate obtained in step (a) above using an extract; (c ) mixing the extract obtained in step (b) above with a lower alcohol and a halogenated hydrocarbon and separating the precipitate; (d) extracting from the precipitate obtained in step (c) above; Process of extracting proteinaceous growth factors. 2. The method for producing a proteinaceous growth factor according to claim 1, wherein the lower alcohol in steps (a) and (c) is methanol. 3. The method for producing a proteinaceous growth factor according to claim 1 or 2, wherein the halogenated hydrocarbon in step (c) is chloroform.