KR100738022B1 - Methods of purification of human vWF from milk of transgenic pigs - Google Patents

Abstract

 The present invention relates to a method for purifying human von Willebrand factor protein through milk pretreatment of transgenic pigs, and more particularly, centrifugation, cryoprecipitation, ammonium sulfate precipitation of swine milk of transgenic animals. and a method for purifying fast, economical and high-purity human von Willebrand factor protein by performing pretreatment through sulfate precipitation and sequentially performing purification steps such as protein A chromatography, heparin chromatography, and SP chromatography. .

Transgenic animals, milk, protein, purification, cryoprecipitation, ammonium sulfate precipitation, protein A chromatography, heparin chromatography, SP chromatography

Description

Purification method of human von Willebrand factor protein through milk pretreatment of transgenic pigs {Methods of purification of human vWF from milk of transgenic pigs}

1 is cryoprecipitation and ammonium sulfate precipitaion (ASP), which is a step 1 milk pretreatment step of the purification process of the present invention, and through this process, human von Willebrand factor protein (human vWF) and many others. Removal of impure protein (casein) shows polyacrylamide gel electrophoresis. [MW: Molecular weight label; Lane 1: fat removal milk; Lane 2: supernatant after low temperature precipitation; Lane 3: precipitate after low temperature precipitation, lane 4: supernatant after precipitation of ammonium sulfate; Lane 5: precipitate after ammonium sulfate precipitation]

Figure 2 shows the results of polyacrylamide gel electrophoresis by removing the unnecessary fat (lipid) in the purification process using a membrane having a pore size of 0.45 ㎛ of the sample shown in FIG.

Figure 3 shows a protein A chromatography process, which is a one-step purification process, and shows the result of polyacrylamide gel electrophoresis by removing a large amount of pig-derived antibody protein through this process.

Figure 4 shows the chromatogram of heparin chromatography, a two-step purification process, and shows the results of polyacrylamide gel electrophoresis of purified von Willebrand factor protein (human vWF) purified through the purification process [MW: Molecular weight labeling; Lane S: precipitate after ammonium sulfate precipitation; Lane FT: fractions 1-14 after heparin chromatography passage; Lane elution: fractions 1 to 7 of the heparin chromatography eluate].

Figure 5 shows the SP chromatography process, which is a three-step purification process, shows the polyacrylamide gel electrophoresis of the von Willebrand factor protein separated through the purification process.

6 a) and b) confirm the recovery of von Willebrand factor in each purification step by the enzyme immunoassay method.

Figure 7 illustrates the purification of von Willebrand factor from the milk of transgenic animals provided by the present invention.

The present invention relates to a method for purifying human von Willebrand factor protein through milk pretreatment of transgenic pigs, and more specifically, centrifugation, cryoprecipitation, ammonium sulfate precipitation of milk of transgenic animals. The present invention relates to a method for purifying fast, economical and high-purity human von Willebrand factors by performing pretreatment through precipitation and sequentially performing purification steps such as protein A chromatography, heparin chromatography, and SP chromatography.

Common congenital bleeding diseases include VWD (von Willebrand disease) and hemophilia. Unlike hemophilia, VWD has a similar incidence in men and women. VWD, unlike Hemophilia A or B, is a blood clotting disease that causes bleeding in soft tissues, especially mucosa. The so-called VWD is a disease caused by deficiency or functioning of von Willebrand factor. To date, this factor has been administered to patients only in unsatisfactory quality and quantities through Factor VII preparations that also contain vWF. To date, a method of purifying vWF from donated blood is widely used. However, there is an urgent need to establish a purification method using high purity and economic method of human von Willebrand factor protein from milk of transgenic pigs made by genetic engineering for reasons of low purification efficiency and expensive raw materials (plasma). Thus, the present invention provides a purification method for commercial production of von Willebrand factor protein in milk of transformed pigs.

The purification methods of von Willebrand factor protein known to date are mostly performed by plasma chromatography. For example, ion exchange chromatography, lectin binding chromatography, gel filtration chromatography, affinity adsorption chromatography, etc. have. There is no method for purification of von Willebrand factor protein and its commercial production in transgenic animal milk.

On the other hand, the study on the purification method of human protein from the milk of transgenic animals, the method of purifying lactoferrin (alpha-1 proteinase inhibitor), fibrinogen (fibrinogen) and the like [US Patent No. 5,919,913, U.S. Patent No. 6,194,553, but there are problems in the complex purification process and purification efficiency.

Accordingly, the present inventors have conducted research to develop a method for purifying proteins from milk of a simplified transgenic animal. The milk pre-treatment process and chromatographic method have been established, and through this, the present invention has been completed by not only simplifying the overall purification process by separating proteins of high purity, but also establishing a low-cost, high-efficiency protein purification method.

Accordingly, an object of the present invention is to provide a method for purifying human von Willebrand protein of a simple, economical, high purity through the milk pre-treatment method of transgenic pigs.

The present invention is characterized by a method for purifying human von Willebrand protein through the pretreatment method of von Willebrand factor in milk of transgenic pigs.

Referring to the present invention in more detail as follows.

The present invention is pre-treated with milk through the centrifugation, cryoprecipitation, ammonium sulfate precipitation, milk of transgenic animals, and purification step such as protein A chromatography, heparin chromatography, SP chromatography The present invention relates to a method for purifying von Willebrand factor protein of rapid, economical and high purity by performing the purification steps sequentially.

The von Willebrand factor protein purification method is characterized by centrifugation for fat removal and cryoprecipitation and ammonium sulfate precipitation to remove a large amount of impurities other than von Willebrand factor protein in milk pretreatment. Precipitation is used, and von Willebrand factor protein purification includes protein A chromatography, heparin chromatography, and SP chromatography.

In addition to the characteristic purification step, filter filtration for fine fat removal, and dialysis step for exchange of buffer.

In the method for purifying von Willebrand factor protein of the present invention, a special environment called milk must be considered. Therefore, the milk pretreatment process must be performed in one step, and the von Willebrand factor protein is hardly lost through centrifugation of the milk, and the excess fat in the milk can be easily and quickly removed.

At this time, centrifugation is preferably performed for 20 to 30 minutes at 4 to 8 ℃ at 8,000 to 10,000 rpm. Only the supernatant obtained by centrifugation in this way is taken, and then cryoprecipitation is performed. To explain the low temperature precipitation method, EDTA 5-50 mM was added to the supernatant obtained above, frozen at -80 to -50 ° C for 12 to 16 hours, dissolved at 4 to 8 ° C for 24 to 36 hours, and then centrifuged. Eliminate impure protein. Then, ammonium sulphate precipitation is carried out, which is added slowly saturated ammonium sulphate to the final 35% ammonium sulphate in the supernatant obtained after low temperature precipitation, and stored at 0-8 ° C for 0.5-1 hour, followed by centrifugation. The supernatant is filtered to remove large amounts of impurity protein primarily. At this time, the amount of ammonium sulfate is 20 ~ 35% according to the characteristics of the von Willebrand factor protein, the loss of the most von Willebrand factor protein is determined by the amount of 35% ammonium sulfate is the smallest. In this milk pretreatment process, it is essential to remove fat and large amounts of impurity proteins, reduce purification time and cost, reduce the number of steps of the entire purification method, minimize unnecessary losses, and concentrate human von Willebrand factor protein during purification. Thus, convenience may be given to the execution of subsequent steps. As mentioned above, pre-treatment of milk on industrial scale is essential in terms of column reuse, efficiency and cost.

Purification step Chromatography step Initially, protein A chromatography was used to remove the buffer exchange and a large amount of antibody protein, and there was little loss of von Willebrand factor protein, the purification target protein. Then dialysate in sufficient amount (about 100-fold) with Tris buffer (20 mM Tris-Hcl, pH 7.4). Heparin chromatography is then performed, in the case of von Willebrand factor protein, protein fractions having a concentration of 15-25% sodium chloride on the chromatogram are collected and combined.

The eluted fractions were then diluted with Tris buffer (20 mM Tris-Hcl, pH 7.4) in a 1: 1 volume ratio, followed by SP chromatography, and 1M sodium chloride-containing Tris buffer (20 mM Tris-Hcl, pH 7.4). Eluting from) and fractionating.

This purification method can reduce the purification time and cost compared to the conventional purification method, and can purify the high-purity von Willebrand factor protein can be mass-produced to transform the transgenic animal into a human therapeutic protein and other It can be usefully used in the fields of industrial protein production, separation, purification.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Purification of Human Von Willebrand Factor (human vWF)

1) Centrifugation

80 ml of milk collected from transgenic pigs (provided by the Institute of Animal Science and Technology) was centrifuged (supra 22K, hanil) at 8000 rpm for 20 minutes at 4 ° C. Then, the aqueous layer was collected, except for the maintenance solids of the top layer and the precipitates of the bottom layer [FIG. 1].

2) cryoprecipitation

The obtained product from 1) was added to a final concentration of 5 mM EDTA, frozen at -70 ° C for at least 12 hours, and then completely dissolved at 4 ° C (at least 24 hours).

After centrifugation at 4 ℃ at 8000 rpm for 20 minutes and only the supernatant was taken. Through this, a large amount of impurity protein, casein, etc. can be removed [FIG. 1].

3) ammonium sulfate precipitation and filtration

To the obtained product obtained in 2) the final concentration of saturated ammonium sulfate (ammonium sulfate) solution was slowly added to a total concentration (v / v) of 35%, and stored at 4 ℃ mixed for 2 hours. After centrifugation at 4000 rpm for 20 minutes at 4 ℃, only the supernatant was taken and filtered with a 0.22 micron Nalgene filter device (Fig. 2).

4) Dialysis and Protein A Chromatography

Sulfuric acid in the eluate was added to the activated dialysis membrane (SIGMA, MWCO 12K), and the resultant obtained from the above 3) was sealed and dialyzed three times at an interval of 6 hours against a 100-fold 20 mM Tris buffer, pH 7.4. The concentration of ammonium was sufficiently dialyzed. Thus buffer was exchanged through dialysis. The dialyzed solution was collected and passed through a 5 ml protein A column before use of the next column, heparin column. Samples were passed through a Protein A column after sufficient equilibration to a volume of 25 ml with 20 mM Tris buffer, pH 7.4 solution. Samples passed through the Protein A column were used for the next step heparin column [Fig. 3].

5) Heparin Chromatography

Heparin chromatography was performed at room temperature using a fast protein liquid chromatograpy (FPLC) system (Pharmacia). 20 ml of Heparin Sepharose 6 Fast Flow (Pharmacia) resin were charged into an empty column (XK 16/20 column). It was then equilibrated with a 20 mM Tris buffer, pH 7.4 solution at a flow rate of 1 ml / min. Samples were added to the column at 1 ml / min using a P 50 pump [Pharmacia]. The column was then washed with equilibration buffer until the absorbance at 280 mm was stabilized. 20 ml Tris buffer, pH 7.4, containing 0.05 M NaCl was washed with 5-fold column volume (280 ml) to elute loosely bound protein to collect 1 ml fractions. After washing 20 mM Tris buffer at pH 7.4 with 0.05 M NaCl with 3 column volumes (60 mL), elution buffer was increased to 10 M column volume (200 mL) and 20 mM Tris buffer above 1.0 M NaCl. .

After monitoring the total protein by absorbance at 280 nm, the salt concentration was checked for elution of von Willebrand factor from 0.15 to 0.25M, which was analyzed by eluent fraction by SDS-PAGE [FIG. 4].

6) SP Chromatography

SP chromatography was performed using a fast protein liquid chromatograpy (FPLC) system (Pharmacia) at room temperature. 20 ml of Heparin Sepharose 6 Fast Flow (Pharmacia) resin were charged into an empty column (XK 16/20 column). It was then equilibrated with a 20 mM Tris buffer, pH 7.4 solution at a flow rate of 1 ml / min. Samples were diluted to a volume of 1: 1 with 20 mM Tris buffer and added to the column at 1 ml / min using a P 50 pump [Pharmacia]. The column was then washed with equilibration buffer until the absorbance at 280 mm was stabilized. A 1 ml fraction was collected by eluting loosely bound protein by washing 20 mM Tris buffer at pH 7.4 with 0.05 M NaCl with a 5-fold column volume (200 mL). 20 mM Tris buffer at pH 7.4 containing 0.05 M NaCl was washed with three column volumes (60 mL), then elution buffer was eluted with at least five column volumes (100 mL) of 20 mM Tris buffer in 1.0 M NaCl. Fractions were made in 1 ml [Fig. 5].

Through this process, von Willebrand factor protein of high purity showing 90% or more purity can be isolated, and the purification efficiency of each step von Willebrand factor protein is purified by enzyme immunoassay kit (human vWF-ELISA, american diagnostica inc.). The absorbance measured at 405 nm is shown in Table 1 below.

Purification Efficiency of Step-by-Step Purification of Transgenic Pork Milk vWF division vWF concentration (ug / ml) Volume (ml) Purification Efficiency (%) Low temperature precipitation Total milk 357 73 100 precipitate 97.5 20 70.5 Supernatant 306 60 7.5 Ammonium Sulfate Precipitation Total milk 306 60 70.5 40% ASP precipitate 318 70 0 40% ASP Supernatant 0.1 80 85.3 Protein A Chromatography After passing through the column 246 80 75.4 After filter After 0.45um filter 246 75 70.7 dialysis After dialysis 86 120 39.5 Heparin chromatography Start 86 120 39.5 FT 3.6 250 3.4 Elution sum 27.2 240 25 SP chromatography Start 27.2 240 25 FT 0.5 210 0.4 wash 8.7 38 1.3 Elution sum 58.5 46 12.4 Final purification efficiency                       12.4%

Experimental Example 1: Purity analysis using polyacrylamide gel electrophoresis (SDS-PAGE)

Purity for the final SP eluate was determined using polyacrylamide gel electrophoresis.

SDS-PAGE gel (10%) was placed on an electrophoresis device (Xcell II Mini-Cell) and filled with SDS running buffer. 30 μl was mixed with 3 μl of sample buffer, heated at 95 ° C. for 5 minutes, centrifuged at 10,000 rpm for 3 to 4 minutes, and 25 μl were injected into the well with a molecular weight standard [MBI fementas]. After injection, electrophoresis was performed at a voltage of 130 V, and when the development was completed, the gel was removed and stained with coomassie (coomassie stain R-250). SDS-PAGE results are shown in b) of FIG. 5 and the final purification of von Willebrand factor protein showed greater than 90% purity.

As described above, the method for purifying von Willebrand factor protein from the milk of transgenic pigs according to the present invention is a pre-processing process consisting of centrifugation, low temperature precipitation, ammonium sulfate precipitation and chromatographic steps. Compared with the purification method, the purification time and cost can be reduced, and von Willebrand factor protein can be purified in high purity and economically.

Claims (1)

1) centrifuging the milk of the transgenic animal at 8000 to 10000 rpm at 4 to 8 ° C. for 20 to 30 minutes to take only the supernatant; 2) adding EDTA to the supernatant and freeze-preserving for 12-16 hours at -80 to -50 ° C and melting for 24 to 36 hours at 4 to 8 ° C, followed by low temperature precipitation through centrifugation; 3) 20 to 35% of ammonium sulfate was slowly added to the supernatant obtained after the low temperature precipitation, and the mixture was stored at 0 to 8 ° C. for 0.5 to 1 hour, and then centrifuged to filter the supernatant. Removing with; 4) fractionating the obtained product of step 3 by performing protein A chromatography and heparin chromatography; And 5) Purifying human von Willebrand factor (vWF) protein by diluting the fraction with 20 mM Tris buffer at a ratio of 1: 1 volume and performing SP (Sulphopropyl) chromatography. Method for purifying high-purity human von Willebrand factor protein from the milk of the transgenic animal, characterized in that consisting of.

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