JPS58159419A - Separating and obtaining method of alpha1-acidic glycoprotein - Google Patents

Abstract

PURPOSE:To obtain the titled protein having the stabilizing action on a proteinic solution, e.g. casein or erythrocytes, etc. at room temperature, by adding acetone to a solution containing an alpha1-acidic glycoprotein obtained from a blood plasma. CONSTITUTION:Acetone of 4.5-8pH in >=35% concentration is added to a solution containing an alpha1-acidic glycoprotein (AGP) obtained from a blood plasma. The resultant mixture is then allowed to stand at room temperature for 15min- 2hr to deposit the AGP fully. The formed precipitate is then separated by the ultracentrifugation, filtration, etc. If the purity of the separated precipitate is high, the precipitate is dried by the vacuum drying, etc. to give the AGP product. The blood plasma may be not only the human blood plasma but also the blood plasma of an animal, e.g. a rabbit, horse, sheep, pig, etc.

Description

[Detailed Description of the Invention] ) from plasma at room temperature and in high yield.

AGP is one of the proteins contained in plasma, and is known to have a stabilizing effect on protein solutions such as casein, red blood cells, and white blood cells (Japanese Unexamined Patent Application Publication No. 1989-1993).
3 7 1. 8 No. 7). Conventionally, AGP was isolated by treating plasma proteins with trichloroacetic acid, followed by ethanol fractionation and ammonium sulfate fractionation, but this method recognizes the instability of sialyl chains in trichloroacetic acid treatment. YO (1) Since then, it has not been used much. and,
Subsequently, a method was developed in which plasma was diluted and ammonium sulfate precipitated under a specific pH, but this method was insufficient in terms of yield, purity, and operability, and currently only the ethanol fractionation method developed by Cohn et al. is being utilized. The method of Cohn et al. adjusts the ethanol concentration, salt concentration, etc. at a low temperature below 0°C and low salt concentration, and further produces precipitation one after another by utilizing the interaction between proteins and zinc ions, barium ions, etc. Among the various improved methods developed since then, method 10 has been particularly praised for the separation of AGP. And AGP is under this 10th law.
AGP is separated and obtained from the fraction through a further three-step fractionation process, but this method has the drawbacks of low yield and the need to operate at low temperatures below 0°C throughout. .

The inventors of the present invention have carried out intensive research to resolve these drawbacks, and as a result, by adding acetone precipitation to the process of separating and purifying AGP from plasma, AGP can be extracted (2) with high yield and by operation at room temperature. Get scales and find out,
Based on this, the present invention has been completed.

That is, the present invention relates to a method for separating and obtaining AGP, which is characterized in that when AGP is separated and obtained from an AGP-containing solution obtained from plasma, acetone is added to the solution.

The plasma may be not only human plasma but also animal plasma such as rabbit, horse, sheep, or pig.

It goes without saying that AGP-containing solutions obtained from plasma are not limited to those obtained directly from plasma;
Contains a containing solution. Examples of such purification methods include the various methods of Cohn et al. that perform ethanol fractionation at low temperatures, as well as the method of Winzler et al. that incorporates ammonium sulfate fractionation;
The method developed by Michon and Bourr'il Ion, the method of Weimer et al., the method of Schmid et al. incorporating an adsorption method on an ion exchange resin, DE
AE-cellulose) (method of Ardwicke et al., Winzle method using CalM-xymethyl cellulose)
Examples include the method of R et al.

Moreover, the purification step is not a problem, and the method of the present invention can be applied to the AGP-containing solution in any step of the purification method described above. However, since the method of the present invention is effective in separating and removing albumin, it is particularly effective for AGP-containing solutions in which albumin is the main impurity. An example of such a solution is Co
Mention may be made of the hn fraction ■ and the Cohn fraction ■. The concentration of AGP in the AGP-containing solution is preferably about 20 to 3007119/I.

The present invention is characterized in that acetone is added to the solution. What is added is acetone and other ketones such as methyl ether ketone are AG
It is unsuitable because it denatures P. The concentration of acetone depends on the concentration of AGP, but is preferably about 35% or more, and about 45 to 70% is particularly suitable from the viewpoint of AGP recovery rate and the amount of acetone used. In particular, if the AGP-containing solution contains a considerable amount of albumin, acetola
If the precipitate precipitated at an agricultural level of 35 or below, especially around 20 to 30 degrees, is once separated and acetone is further added to the remaining supernatant, albumin can be easily removed and ACP can be obtained in high purity. . This is based on the new finding obtained by the present inventors that the solubility of albumin in acetonate is much lower than that of AGP.

The pH of the solution to which acetone is added is preferably about 45 to 8. When pH exceeds 8, denaturation of AGP begins, while P
This is because if H is less than 4.5, AGP will also co-precipitate in one precipitate of albumin etc., resulting in poor AGP yield.

The temperature of the solution may be room temperature, and there is no need to prepare the solution at a low temperature for 11 days as in the method of Cohn et al.

After adding acetone, the mixture is allowed to stand at room temperature for about 15 minutes to 2 hours to sufficiently precipitate AGP, and the precipitate is separated by a known method such as centrifugation or filtration. If the purity of the separated precipitate is good, it can be dried as it is by drying under reduced pressure, freeze drying, etc. to make a product. On the other hand, if the purity is insufficient, water may be added to dissolve the precipitate. The product may be further purified by means such as dialysis, cell filtration, etc.

(5) Unlike conventional methods, the method of the present invention can be operated at room temperature, and the yield of AGP can be increased.

If the method of the present invention is applied to threads containing albumin as an impure protein, albumin is first selectively precipitated and removed, and then AGP is precipitated, thereby producing AGP in a simple manner with high purity and high yield. can be obtained. For example, referring to the method of Cohn et al., conventionally,
By applying the method of the present invention, AGP that was previously obtained from the precipitate of fraction Ⅰ with a yield of 40% and a purity of 60% can be obtained from the supernatant obtained by separating the precipitate of fraction V with a yield of 80% and a purity of 90%.
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Next, the results of examining the effects on the precipitation rate of AGP and serum albumin by changing the PH and acetone concentration are shown.

As for the experimental method, Cohn Frac/Yon V Ichihiko serum was used as a raw material, and after adjusting the pH to a predetermined value, a predetermined amount of acetone was added at room temperature, and after stirring, the mixture was allowed to stand at room temperature for 30 minutes. The resulting precipitate was centrifuged, the resulting precipitate was dissolved in water, and the recovery rate was determined by determining the percentage of AGP and (6) clear albumen/by immunodiffusion method. The results obtained are shown in the table below.

Table I Recovery rate of AGP (7) Table 2 Recovery rate of serum albumin as an impurity A+1
1-11 Examples will be shown below. It should be noted that all percentages representing the concentration of acetate in this specification are based on volume. The purity of the AGP used in the following examples was determined to be 5+ using high performance liquid chromatography in combination with cell permeation chromatography and disk electrophoresis.

Example 1 Cohn fraction V supernatant 51 containing AGP 182m9/e was adjusted to pal 45 with IN HCt, acetone was added to 30%, stirred, and then incubated at room temperature for 30 minutes (8). did. The deposited precipitate was removed by centrifugation at 5,000 rpm, and 1 L of the resulting clear liquid was stirred with acetone added to 60 parts, and left to stand at room temperature for 30 minutes.

The deposited precipitate was centrifuged at 5000 rpm, and the precipitate was collected. This precipitate was dissolved in 50 ml of water, dialyzed against water overnight, and then freeze-dried. The obtained freeze-dried product was 830
m9, and the AGP purity was 90. Recovery rate from Cohn Fracun Yong V supernatant was 82%.

Example 2 Cohn containing AGP 169 m9/,,e
Add acetone to 5A of Fraction V supernatant (pH 5,4).
The mixture was added so as to have a concentration of 0, and after stirring, it was allowed to stand at room temperature for 30 minutes.

The deposited precipitate was removed by centrifugation at 5000 rpm,
Acetone was added to the obtained supernatant liquid to a thickness of 60 φ, and after stirring, the mixture was allowed to stand at room temperature for 30 minutes. The deposited precipitate was centrifuged at 500 rpm and collected. This precipitate was dissolved in a 1K bath of 50 ml of water, dialyzed against water overnight, and then freeze-dried. The lyophilized product was dissolved in water] and Ome to dissolve the old o-
Gel filtration was performed using gelplO (manufactured by Bio-Rad). The resulting AGP fraction (9) contained 800 mg of AGP, and the AGP protein content of this fraction was 95. Recovery rate from Cohn fraction V supernatant Oo%
.

Patent applicant: Fuji 1 Organ Pharmaceutical Co., Ltd. Representative Patent Attorney Masahiro Tanaka

Claims (1)

[Claims] αl from a solution containing α1-acidic glycoprotein obtained from plasma
- A method for separating and obtaining α1-acidic glycoprotein, which comprises adding acetone to the solution when separating and obtaining acidic glycoprotein.