KR920000098B1 - Method for producing urokinase - Google Patents

Abstract

A method for the production of urokinase comprises precipitating urokinase from human urine using a reagent contg. zinc. chloride or copper sulfate,eluting the precipitated urokinase using EDTA soln., and purifying it by passing through benzaidin-cepharose affinity column. Pref. the concentration of zinc chloride and copper sulfate is each 1-2 mM. The above column can be replaced by single colony antibody-cepharose column, and EDTA can be replaced by glycine and citrate soln.

Description

Production method of urokinase

1 shows the absorbance and activity of the fraction obtained by column chromatography in Example 1,

2 is a graph showing the precipitation of the protein according to the zinc chloride concentration by the protein quantitative method,

3 is a graph showing the correlation between protein and urokinase activity in the precipitate according to zinc chloride concentration,

4 is a graph showing the weight of precipitated solids of 1000 liters of urine according to zinc chloride concentration.

The present invention relates to a method for the production of highly purified purified eurokinase from human urine on an industrial scale. More specifically, the present invention relates to a method for producing urokinase, characterized by precipitating urokinase from urine containing a large amount of urine in humans containing urokinase, based on zinc chloride or copper sulfate.

Urokinase is a kind of enzyme present in human urine of about 30 to 80 µg / l. It converts plasminogen into active plasmin to dissolve clot clots in blood vessels. Let's do it. Purified urokinase has been used for decades for the treatment of myocardial infarction, stroke, etc. due to vascular thrombosis.

Conventionally, high concentration method is recognized as the most important process in the first process when purifying urokinase from human urine. Also, barium sulfate, silica gel, bentonite, zeolite, hydroxylapatite, cellulose phosphate and controlled pore glass are used as adsorbents. [Controlled pore glass (CPT-10)], etc. are used, but most of the processes are complicated, low recovery rate, there is a lot of restrictions on the industrial use because there is a high risk of denaturation due to elution. In addition, precipitation methods of proteins using heavy metals as adsorbents have been reported since the 1920s, but rarely used for industrial purposes. Recently, Zawarski et al. (PG Zawarski and GSGill, Anal. Biochem. 173,440 (1988)) used zinc to recover recombinant proteins, but this report partially precipitated the recombinant proteins secreted in a small amount of culture. It was only to let.

The present inventors have extensively researched to solve the various problems described above. As a result of using zinc chloride as a precipitant, recovering urokinase with a suitable eluate, and directly affinity chromatography, it is possible to quickly and easily human urine. The present invention has been found to be able to produce urokinase with high yield.

It is an object of the present invention to provide a process for the production of highly purified purified urokinase.

Hereinafter, the present invention will be described in detail. The pH of the human urine is adjusted to 7.5-8.0 with an aqueous alkali solution, and the solution is left to precipitate most solids in the urine. The supernatant is filtered, and the pH is adjusted with an acid solution such as hydrochloric acid to sufficiently dissolve zinc chloride therein. Adjust to 4 ~ 6 and add so that the final concentration of zinc chloride is 1 ~ 2mM. At this time, the zinc chloride added is present in an aqueous solution by combining histidine residues contained in human urine. When pH is adjusted to neutral with an aqueous sodium hydroxide solution, zinc ions form a complex with each other and precipitate. Precipitate the protein. As shown in FIGS. 2 to 3, urokinase selectively precipitates at a low zinc chloride concentration of 1-2 mM, whereas albumin, which accounts for 80-95% of human urine, occurs at 10 mM or more. Precipitation of urokinase by zinc chloride has a purification effect as well as a concentration effect. As described above, the precipitate is formed by centrifugation to recover the precipitate, and then dissolved by a high concentration of EDTA (Ethyleue Diamine Tetraacetate Disodium).

At this time, the EDTA solution used as the eluent is used between pH 7.0 and 8.0 and can be obtained at a recovery rate of 95% or more by using sufficient stirring four times the volume of the precipitate obtained by treatment with zinc chloride.

Recently, it has been reported that it can be eluted with a column light (manufactured by Fujigaku Co., Ltd.) or acrylonitrile in a yield of about 90% (see Patent Publication No. 77-49), but these materials are expensive and eluted. Because strong alkali is used for this purpose, there is a risk of denatured urokinase activity. EDTA used in the present invention does not affect the activity of urokinase at all, the activity of urokinase is increased by about 20 times compared to urine, and the purity shows a value of about 1000 IU per mg of total protein.

The above-mentioned precipitation and partial purification processes can be performed within one hour in one reactor, so the process is quick and simple, economical and high recovery and purity.

The urokinase eluted by the EDTA solution is present in an aqueous solution, but when left at low temperature, solids such as urea, which have no effect on urokinase activity, are reprecipitated. This is either centrifuged to remove the precipitate or the supernatant is carefully tilted and hung on an affinity column. Sepharose combined with a single antibody and Sepharose combined with benzaidine are used for the affinity column. Both Sepharose is excellent in terms of purity and yield, but the immunoaffinity column using a single antibody when processing more than 10,000 liters of human urine for commercial purposes requires dozens of grams of monoantibody when used on various scales, such as on an industrial scale. Although it is not technically and cost-effective, and it is inconvenient to be separated and included in the product even if a single antibody is solidified on a support, the final purification process by benzydine-sepharose affinity column chromatography is performed. Particular preference is given to performing.

Benzaidine-Sepharose has been reported to have affinity with several serine proteases in addition to urokinase, but in the present invention, it was confirmed that these serine proteases were selectively removed depending on the concentration of zinc chloride.

Through the above purification process using benzydine-sepharose, the inactivation of urokinase is very high, about 123,000 IU / mg total protein, and high purity urokinase having a molecular weight of 53,000 is obtained. Here, the measurement of specific activity is the amount of protein obtained as a result of quantitative determination of the titer using a known urokinase activity assay (Astrup et al. Arch, Biochem. Biophys. 40, 346 (1952)) (Bradford (1976) Anal, Biochem. 72, 248).

Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is not limited to these examples.

Example 1

[Setting of Zinc Chloride Concentration]

1000 liters of a fresh urine solution of 5N sodium hydroxide was added thereto, adjusted to pH 8.5, and left for 1 hour. The precipitate was discarded by centrifugation and 5N hydrochloric acid was added to the obtained supernatant and adjusted to pH 4.5. Zinc chloride was added so that the final concentration of zinc chloride in each 5 ml of the solution separated from this solution was 0 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, and the pH of the solution was adjusted to 7 with 5 N aqueous sodium hydroxide solution, and then 30 Precipitates and filtrates were left to stand for a minute to determine the protein content according to the protein determination method. The results are shown in Table 1 and FIG. 2.

TABLE 1

Example 2

[Weight of precipitate formed according to zinc chloride concentration]

The final concentration of zinc chloride was obtained in the same manner as in Example 1 except that the final concentration was 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, and obtained by continuous centrifugation (flow rate: 8 liters / minute), and the weight thereof. Are shown in Tables 2 and 4 below. As a result, at 3mM or more, the amount of solid precipitate was so large that the volume of the eluate rapidly increased and difficulty occurred in the next process, so 2mM or less was appropriate.

TABLE 2

Example 3

5N aqueous sodium hydroxide solution was added to 100 liters of fresh human urine to adjust the pH to 8.5, and left for 1 hour to form a precipitate. To the supernatant obtained by centrifugation, 5N hydrochloric acid was added to adjust the pH to 4.5, and zinc chloride was added so that the final concentration of zinc chloride was 2 mM. Again 5N aqueous sodium hydroxide solution was added to adjust the pH of the solution to 7 and left for 30 minutes to form a precipitate. The supernatant was removed by continuous centrifugation, and the precipitate was recovered, eluted with 4 times EDTA, and left at 4 ° C. for 1 hour, whereupon another solid was reprecipitated. The supernatant was recovered and passed through a benzydine-sepharose column (5 cm x 20 cm) and eluted with 0.1 M glycine-HCl (pH 3.0). The obtained fractions were measured for absorbance at 280 nm using an absorber (manufactured by Pharmacia), and showed high absorbance and specific activity between fractions 70-100. This is shown in FIG. The solid line in FIG. 1 shows the protein concentration, and the dotted line shows the urokinase activity.

The quantity and yield of the urokinase produced by the said Example are shown in Table 3 below.

TABLE 3

Example 4

100 liters of fresh urine were collected by adding aprotinin, a protease inhibitor, and then, in each step, aprotinin was added at a concentration of 10KIU / ml to purify a single chain prourokinase. It was found that 80% of the purified urokinase during aprotinin treatment corresponds to a molecular weight of 53,000 in a single chain even under reducing conditions. In order to purify pure single-chain urokinase, the protein of molecular weight of 53,000 Daltons of single chain was purified through EPLC Superose 12 column after DTT (dithiothreitol) treatment.

The results are shown in Table 4 below.

TABLE 4

Example 5

[Comparison Experiment Results of Protein Precipitation and Eurokinase Activity According to Zinc Chloride Concentration]

Except that the final concentration of zinc chloride to 0mM, 1mM, 5mM, 10mM, 25mM, 50mM was carried out in the same manner as in Example 3, and the protein weight and urokinase activity in the precipitate according to the zinc chloride concentration change Table 5 and FIG. 3 below.

TABLE 5

Example 6

100 liters of fresh urine were prepared in the same manner as in Example 3, and purified using a single cell antibody-Sepharose instead of benzydine-Sepharose. The results were almost the same as in Example 3.

Example 7

100 liters of fresh urine were treated with zinc chloride in the same manner as in Example 3 to precipitate urokinase activity, followed by 1M glycine (pH 9.0) instead of EDTA as eluent. 1M glycine (pH 9.0) solution was treated with four times the weight of the precipitate to elute urokinase, which showed the same effect as EDTA.

Example 8

In Example 7, urokinase was eluted from the precipitate using 1M citrate (pH 9.0) instead of 1M glycine (pH 9.0), and the dissolution rate was almost the same as that of the EDTA solution or the 1M glycine solution.

Example 9

5N sodium hydroxide aqueous solution was added to 100 liters of fresh urine, adjusted to pH 8.5, and it was left to stand at room temperature for 2 hours. After only the supernatant was transferred to a new vessel, copper sulfate was added to a final concentration of 2 mM and stirred for 30 minutes. The precipitate was recovered only using a centrifuge and eluted with 4 times EDTA solution.

The recovery rate of the obtained urokinase was about 90%, and the complete purification process could be performed in the same manner as in Example 3.

The results are shown in Table 6 below.

TABLE 6

As shown in the above-described examples and the like, the method of the present invention provides a method capable of mass production of highly purified purified urokinase from human urine and also obtains a rapid and economical method.

Claims (7)

After precipitating urokinase from human urine with zinc chloride or copper sulfate as the main component, the precipitated urokinase is eluted with EDTA solution according to a known method, and purified by benzidine-sepharose affinity column. Production method of urokinase. The method for producing urokinase according to claim 1, wherein the concentration of zinc chloride is 1-2 mM. The method for producing free kinases according to claim 1, wherein the concentration of copper sulfate is 1-2 mM. The method for producing urokinase according to claim 1, wherein a single cell antibody-sepharose column is used in place of a benzydine-sepharose affinity column. 2. A process for producing urokinase according to claim 1, characterized by eluting urokinase activity in the precipitate with a solution of glycine and citrate instead of EDTA. The method of claim 1, wherein the pro-urokinase other than urokinase is purified and produced. The method for producing urokinase according to claim 1, wherein the concentration of copper sulfate instead of zinc chloride is 1-2 mM.

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