JPS5973526A - Fractional purification of protein existing in mammalian brain - Google Patents

Abstract

PURPOSE:To obtain the objective protein by treating a liquid containing various proteins and obtained by the extraction of the brain of mammals with a specific water-insoluble carrier, collecting the adsorbed or unadsorbed fraction, and carrying out the fractional purification of the fraction. CONSTITUTION:A liquid containing various proteins (e.g. enolase, calmodulin, S-100 protein, glia fibrillary protein, etc.) is made to contact with CH3-(CH2)n- (water-insoluble carrier) (n is 3-7) or NH2-(CH2)m-(water-insoluble carrier) (m is 4-8). Various proteins adsorbed to the water-insoluble carrier are subjected to the fractional eluation with a salt such as sodium chloride, an hydrophobic solvent such as alcohol, a surface active agent, an amine, etc. Various protein components of the brain of a mammal can be separated and purified at the same time, in high yield.

Description

[Detailed Description of the Invention] The present invention relates to a method for fractionating and purifying proteins contained in the brain of mammals, and its purpose is to separate each protein from each other and obtain each of them with high purity. It is in.

It is known that the mammalian brain is rich in neural tissue-specific proteins, physiologically active peptides, and the like. Therefore, mammalian brain is extremely useful as a material for separating and purifying these proteins or peptides. Many of these brain components have potential applications in medicine due to their physiological activity, or are important as research materials. Examples of brain components include enolase, calmodyulin, and S.
-100 protein, dahlia fiber protein, etc. Enolase is a glycolytic enzyme that catalyzes the reaction of 2-phosphoglycerate and phosphoenolpyruvate. The enolase molecule has a dimeric structure and consists of two types of subunits with a molecular weight of approximately 50,000, and depending on the combination of subunits, α
α, αγ, and Tγ types exist in the brain. These enolases are known to be increased in the serum or tissues of patients with cancer, particularly undifferentiated cancer. Calmodeulin is widely distributed in the animal and plant kingdoms and is involved in various calcium-dependent enzymes, such as cyclic nucleotide phosph.
odiesterase.

Adenylate cyclase, Myostn
light chain kinase+Phosph
orylase b kinase, Glycoge
n 5ynthBsekinase, (Ca” −
Mg”) 8TPase, NAD”Kinase
The molecular weight is approximately 17.0, which activates calcium-dependent substances such as
It is a calcium receptor protein of 00, and is involved in a wide range of in vivo regulatory mechanisms through the activation of these enzymes.

In animals, it is known to be present in high concentrations in the cerebrum and endomaru. S'-100 protein is also one of the neural tissue-specific proteins and is present in high concentration in the brain. Molecular weight is approximately 20
．． 000 and consists of a dimer of two types of subunits 2
Isomers of the species (αβ, ββ) exist. Although the physiological activity of this protein is currently unknown, it has been reported that the protein increases in the cerebrospinal fluid of the brain due to diseases such as neuroblastoma. Dahlia fibrillary protein is present in nervous tissues, particularly Dahlia cells, and has been extensively studied in the field of neurohistochemistry.

Conventionally, purification methods specific to individual proteins have been reported for purifying these brain proteins. For enolase, K. Kato et al. J. Bioch.
-emistry, vol. 117, pages 1587-1594, 1980, regarding carmodeulin, Te
o,T,S,Jang,T,t(,+J.

Biological Chemistry+ 248
Volume, 588 pages.

In 1973, regarding the S-100 protein, T, l5ob
e et at.

Biochimica et Biophysica
Acta+ Volume 494, Pages 222-232, 19
1977, etc. However, there are no reports on purification methods for separating these proteins from mammalian brains. The brain of a mammal is a limited resource, and in order to make effective use of this resource, it is desirable to separate and purify as many components from the brain as possible at the same time.

The present inventors have conducted intensive studies in consideration of the above-mentioned current situation, and have found that a solution containing these proteins and peptides, which are components of the mammalian brain, is transferred to a CH3(CH2)n-water-insoluble carrier (however, n
= 3 to 7) or NH2-(CH2)II+- to a water-insoluble carrier (m-4 to 8), and the protein and peptide are adsorbed or passed through the carrier, and then the adsorbed proteins and peptides are adsorbed. We have discovered a purification method in which these proteins or peptides are eluted from a carrier, separated from each other, and purified simultaneously.

The adsorption carrier used in the present invention has the general formula CH3-(CH
2) Alkylamine represented by n-NH2 (however, n-3 to 7) or α,ω-diaminoalkay represented by the general formula NH2-(CH2)m-NH2 (however, m=4 to B) is dissolved in water. Obtained by binding to an insoluble carrier. Further, specific examples of the alkylamine or α,ω-diaminoalkane used herein include butylamine, pentylamine, hexylamine, heptylamine, octylamine, 1,4-diaminobutane, 1,5-diaminopenkune, Examples include 1,6-diamithexane, 1,7-diaminohebutane, and 1,8-diaminooctane. The water-insoluble carrier is preferably an inert, sparse, insoluble, and hydrophilic insoluble carrier, such as agarose (registered trademark Sepharose, manufactured by Fine Chemical Co., Ltd.), dextran (registered trademark Sephadesox, manufactured by Pharmacia Fine Chemical Co., Ltd.), (manufactured by Bio-Rad), cellulose powder, polyacrylamide gel (registered trademark Biogel: manufactured by Bio-Rad), etc. are used. The binding reaction is accomplished by first activating a water-insoluble carrier with bromic cyanide, sodium periodate, epichlorohydrin, etc., and adding thereto a buffer containing an alkylamine or an α,ω-diaminoalkane.

The material used in the present invention may be a mammalian brain extract or an extract that has been purified to some extent, such as one that has been subjected to treatments such as salting out and solvent fractionation. These materials are C
H3-(CH2)n-water-insoluble carrier (however, n = 3~
7) or NH2-(CH2)lI+-water-insoluble carrier, provided that m = 4 to B)
The pH can be selected within the range of 4 to 0, at which each component is stable, but pH 6 to 9 is particularly preferable to separate enzymes such as enolase with good yield. In addition, the components adsorbed on the water-insoluble carrier can be eluted using salts such as common salt, hydrophobic solvents such as alcohol, surfactants, amines (
(including amino acids) can be used alone or in combination.

The purified specimens of each component obtained here are highly purified, but if a more highly purified specimen is required, it may be used in combination with the adsorption carrier used in the present invention, or
Alternatively, conventional purification means can be applied. Common purification means include ion exchange chromatography, hydrogen bond chromatography, gel filtration, affinity chromatography, salting out, fractionation with organic solvents, adsorption chromatography, isoelectric focusing, electrophoresis, and the like.

As described above, according to the present invention, each protein component of the mammalian brain can be separated and produced at a high yield. Next, test examples and examples of the present invention will be shown.

Test Example 1 After washing 1156100 g of beef size, 50 mM phosphate buffer (
Add 400me (pH 6,0), crush the cerebrum in a mixer, filter the suspension of the crushed cerebrum, and make 420 ml of filtrate! I got it. The filtrates 5h+f were each passed through a column (5-) filled with a water-insoluble adsorption carrier equilibrated with the same grade IH liquid. Next, after washing the column with the same buffer solution 50-, 0.1M and 0.
The same buffer solution containing 5M sodium chloride (NaCl) was applied to each 50-column. Type of water-insoluble adsorption carrier used, unadsorbed fraction when using each adsorption carrier,
Enolase, Calmodeulin, S in 0.1M sodium chloride elution fraction and 0.5M sodium chloride elution fraction
The yields of -100 protein and Dahlia fibrous protein from the filtrate are shown in Table 1, Table 2, Table 3, and Table 4, respectively. If these proteins and peptides are not eluted even though 0.5M sodium chloride is used, IM)
Elution was possible using a hydrochloric acid buffer (p) 17.0) or a 40% propatool solution containing 1M sodium chloride. Assays for each protein and peptide were performed by enzyme immunoassay using specific antibodies. However, the enzymatic activity of only enolase was measured using 2-phosphoglyceric acid as a substrate.

Table-1 Enolase Table-23-100 Protein Table-3 Calmodulin Table-4 Dahlia Fibrous Protein Example 1 After washing 300 g of bovine brain, 50 mM phosphate buffer (pH 6)
.

The obtained filtrate is passed through a butyl-Sepharose column (5 (7')) equilibrated with the same buffer solution, and various proteins contained in the bovine cerebrum are adsorbed onto or passed through the column, and then the various proteins adsorbed on the column are was fractionated and dissolved using a concentration gradient of sodium chloride.
- After washing with sodium chloride (NaC) using the same buffer,
Elute with a concentration gradient of 1) to a final concentration of 0.4M,
γ-type enolase, γT-type enolase, S-100 protein, calmodyulin, and dahlia fibrous protein could be clearly fractionated. Note that αα-type enolase was eluted as an unadsorbed amount. αγ type, γγ type, and αα of enolase
Type discrimination was investigated using antibodies against α and γ subunits.・The elution pattern is shown in Figure 1.

Example 2 After washing 300 g of bovine brain, 50 mM phosphate buffer (pl+
6.0 >1200 mJ' was added to crush the cerebrum in a mixer, and the resulting suspension of crushed cerebrum was filtered to obtain a filtrate of 1380 mJ'.
I got -.

The obtained filtrate is passed through a butyl-Sepharose column (50+ne) equilibrated with the same buffer, and the various proteins contained in the bovine cerebrum are adsorbed onto or passed through the column, and then the various proteins adsorbed on the column are fractionated and eluted. did. That is, after washing the column with the same buffer solution 1000-
Elution was performed with 200 ml of the same buffer solution containing 140 mM, 210 mM, and 400 mM sodium chloride, respectively. Table 5 shows the yields of enolase, S-100 protein, calmodyulin, and dahlia fibrous protein from the filtrate in the unadsorbed fraction and each sodium chloride concentration eluate. Regarding enolase, the αα type was added to the unadsorbed fraction, the αγ type was added to the 100mM NaCl elution fraction, and the γγ type was added to the 210mM NaCl elution fraction.
M was eluted in the NaCl fraction.

Regarding the eluted protein, the protein yield (
As is clear from A 2fOy1m ), it is purified 10 to 40 times, and it can be seen that each component is separated from each other and simultaneously purified effectively.

1 Table-5 Lower margin) 2 Example 3 After washing 300 g of bovine brain, 50 mM phosphate buffer (pl+
6.0) Add 1200 ml' and crush the cerebrum with a mixer, filter the resulting suspension of crushed cerebrum, and make a filtrate of 1300 ml.
I got an F.

The obtained filtrate was applied to a hexyl sepharose column (50 ml) equilibrated with the same buffer.
After washing with 10100O' solution, sodium chloride (NaCI
) to a final concentration of 0.5 M, and α
γ-type enolase, γT-type enolase, S-100 protein, calmodyulin, and dahlia fibrous protein could be clearly fractionated. Note that αα-type enolase was eluted as an unadsorbed fraction. The elution pattern is shown in Figure 2.

Example 4 The 100mM NaCl elution fraction obtained in Example 2 was fractionated with ammonium sulfate, and the resulting precipitate was dissolved. This solution is 1.8M
Toyopearl HW-55 equilibrated with ammonium sulfate solution (pH 7)
column (15mf), from 1.8M to 1.0M
The active fraction of αγ-type enolase was collected by elution with a concentration gradient of ammonium sulfate. Both fractions were dialyzed against 20 mM phosphate buffer (pH 7) and then applied to an aminohexyl Sepharose column (15 ml') equilibrated with the same buffer. After washing the column with 20mM phosphate buffer, OmM
The active fraction was collected by elution with a concentration gradient of 350 mM NaCl. Both fractions were passed through a Sepharose 6B column for gel filtration, and about 10 μm of a single αγ-type enolase was obtained by disk electrophoresis.

Example 5 Ammonium sulfate was added to the 140 mM NaCl elution fraction obtained in Example 2 to a saturation concentration of 0.35, and the resulting impure protein was removed by centrifugation. The obtained centrifugation supernatant was dialyzed using 20mM phosphate buffer.

A column (5-) packed with aminohexyl-Sepharose prepared by binding 1,6-diamithexane to Sepharose was equilibrated using the same buffer.

The S-100 fraction after dialysis was applied to an equilibrated aminohexyl-Sepharose column, and 100ml of the same buffer was added.
After washing the column with NaCl, the s-too protein adsorbed on the column was eluted using a sodium chloride (NaCl) concentration gradient up to a final concentration of 0.4 M. After concentrating the obtained S-100 protein fraction, Sephadex G-75 (
Gel filtration was performed using a gel filtrate (manufactured by Pharmacia Fine Chemicals) to obtain 13.0 μl of electrophoretically single, highly pure S-100 protein.

Example 6 The 210+nM NaCl elution fraction obtained in Example 2 was subjected to ammonium sulfate fractionation, and the resulting precipitate was added to a 1.5 M ammonium sulfate solution (pH
7> was melted. This solution was diluted with 1.5 M ammonium sulfate solution (pH
Toyopearl HW-55 column (1) equilibrated with
5me), and the active fraction of rr-type enolase was collected using a concentration gradient of 1.5M to 1.0M ammonium sulfate. Both fractions were dialyzed against 20 mM phosphate buffer (pH 6) and then applied to an aminohexyl-Sepharose column (15+nl') equilibrated with the same buffer. 200 columns
After washing with 2On+M phosphate buffer containing mM NaCl, it was eluted with a concentration gradient of 200mM to 350mM NaCl, and active fractions were collected. Both fractions were passed through a Sepharose 6B column and subjected to gel filtration, and approximately 15 μm of single rr-type enolase was obtained by disk 5 electrophoresis. The activity of the obtained γγ enolase was 31 U/■.

Example 7 The 400mM NaCl elution fraction obtained in Example 2 was
Dialysis was performed using 0mM phosphate buffer. A column (5-) packed with aminohexyl-Sepharose was equilibrated using the same buffer. The calmodyulin fraction after dialysis was applied to an equilibrated aminohexyl-Sepharose column.
After washing the column with 100 mf of the same buffer, calmodulin adsorbed on the column was eluted with a concentration gradient of sodium chloride (NaCl) up to a final concentration of 0.4 M using the same buffer. After concentrating the obtained calmodyulin fraction to 0.5M
Sephadex G-75 equilibrated with the same buffer containing sodium chloride (manufactured by Pharmacia Fine Chemicals)
By performing gel filtration using a gel, it was possible to obtain 20 μg of electrophoretically single, highly pure calmodulin. The activity of the obtained calmodulin was determined by the method of T. S. Tea et al. (J.

Biological Chemistry, Volume 248, Page 588, 19736), it was 12,000 U/■.

Example 8 αα-type enolase was purified from the unadsorbed fraction of butyl-Sepharose obtained in Example 2.

The unadsorbed fraction of butyl-Sepharose was fractionated with ammonium sulfate, and the resulting precipitate was dissolved in a 1.7M ammonium sulfate solution (pl+7) and equilibrated with an ammonium sulfate solution of the same concentration.Toyopearl HW-55
column. After washing the column, from 1.7M to 1.2
The active fraction was collected by elution with a concentration gradient of M ammonium sulfate. Both fractions were dialyzed against 20mM phosphate buffer (pH 7) and applied to a hexyl-Sepharose column equilibrated with the same buffer.
Only miscellaneous proteins were adsorbed. The hexyl-Sepharose column was washed, and the unadsorbed fraction was fractionated using a Sepharose 6B column.
I got 0■. The activity of the αα type enolase obtained was 58U.
It was /■.

Example 9 The butyl-Sepharose 2107 mM saline eluate obtained in Example 2 was dialyzed against 20 mM phosphate buffer (pH 6) and applied to an aminohexyl-Sepharose column equilibrated with the same buffer. After washing the column, from 0.1M to 0
．． Dahlia fibrous protein and γγ-type enolase were separated by elution with a concentration gradient of sodium chloride up to 5 M.

The Tγ-type enolase was further purified according to Example 6, and about 15 μm of an electrophoretically homogeneous specimen was obtained.

On the other hand, both portions of the dahlia fibrous protein were salted out with ammonium sulfate and filtered through Sepharose 6B and Sephadex G-150 to obtain 20 μm of electrophoretically single dahlia fibrous protein.

Example 10 Bovine cerebrum was extracted in the same manner as in Example 2, and the extract was applied to a column of aminohexyl-Sepharose (NH2-(CH2)6-Sepharose) equilibrated with 50 mM phosphate buffer (pH 6>). After washing the column with a buffer containing 1M NaCl, it was eluted with a concentration gradient of 0.1M to 0.5M NaCl.

The elution order was the same as that for butyl-Sepharose, with αγ type enolase, S-100 protein, Dahlia fibrillary 8 protein, TT type enolase, and calmodyulin eluted in this order. αα type enolase was not adsorbed.

When we collected both parts containing each protein, we found 3900 U of αα-type enolase, 1200 U of αγ-type enolase, 18 μg of glial fibrous protein (converted to pure product), 1500 U of γγ-type enolase, and 14 μg of S-100 protein (purified product). ) and 160,000 U of calmodyulin were obtained separately.

Example 11 The same bovine cerebrum extract as above was passed through aminooctyl-Sepharose (NH2(CH2)8-Sepharose) and eluted with a concentration gradient of salt from 0 to 0.5 M. Almost the same results as in Example 9 were obtained. was gotten.

Example 12 Bovine cerebral extract (20mM) was added to Lis-HCl buffer (pl+7
), and after washing the column with a buffer containing 0.5M NaCl, a concentration gradient (from 20mM to L
eluted up to M). αα-type enolase was eluted first, and then αγ-type enolase, S-100 protein, glial fibrillary protein, γγ-type enolase, and calmodyulin were mutually separated and eluted in the same order as shown in Figure 2. .

[Brief explanation of drawings]

Figure 1 shows various proteins contained in bovine cerebrum extract.
It shows the elution pattern obtained by fractionation using a Sepharose column. Figure 2 shows the elution pattern obtained by fractionating various proteins contained in a bovine cerebrum extract using a hexyl-Sepharose column. a-----αγ-type enolase b----, S-100 protein c-----Glial fibrillary protein a'--・--1-γT-type enolase d-----Uniforce lumodulin Patent applicant: Amano Pharmaceutical Co., Ltd. 0

Claims (1)

[Claims] (11) Various protein-containing liquids obtained by extraction from mammalian brains are combined with CH3(CH2)n-water-insoluble carrier (where n =
3~? ) or NH2-(CH2)m-water-insoluble carrier (where m = 4 to 8), the various proteins are adsorbed onto or passed through the water-insoluble carrier, and then the various proteins adsorbed on the water-insoluble carrier are separated. A method for fractionating and purifying proteins contained in mammalian brains, characterized by elution. (2) A protein contained in the brain of a mammal according to claim 1, wherein the protein adsorbed to the water-insoluble carrier is αγ-type enolase, S-100 protein, Dahlia fibrous protein, Tγ-type enolase, or calmodyulin. fractional purification method. (3) The method for fractionating and purifying proteins contained in mammalian brains according to claim 1, wherein the protein that passes through the water-insoluble carrier is αα-type enolase.