RU1554377C - Method of purifying alkaline phosphatase - Google Patents

Abstract

The invention relates to biochemistry, and in particular to methods for the purification of alkaline phosphatase of the small intestine of seals. The purpose of the invention is to increase the activity of the enzyme. The enzyme-containing solution is chromatographed on concanavalin A immobilized on a copolymer of hydroxyethyl methacrylate and ztilendimethacrylate. Sorption is carried out with a solution of 20 mM Tris-HCI pH 7.5 buffer containing 0.4-0.6 M sodium chloride, 1 mM calcium chloride, 1 mM manganese chloride, and 1--20 mM magnesium chloride. Elution of the target product is carried out with the same buffer system, optionally containing 50 mM-methyl-O-mannopyranoids, with a linear speed of 30-100 cm / h. The method allows the production of alkaline phosphatase with a specific activity of 3000-3500 u / mg protein. sl c

Description

The invention relates to the production of purified alkaline phosphatase of animal origin, which is used in genetic engineering, immunohistochemical procedures and enzyme immunoassay, scientific research.

The purpose of the invention is to increase the activity of an alkaline phosphatase preparation.

The invention is carried out as follows.

120 mg of a commercial preparation of alkaline phosphatase seals with a specific activity of 58-60 units. per 1 mg of protein, dissolved in 20 mm Tris-HCI, pH 7.4, containing 1-20 mm MgCb, 1 mm CaC, 1 mm

MnCl2, 0.5 M NaCI. The enzyme solution is applied to a column of concanavalin A immobilized on a copolymer of hydroxyethyl methacrylate and ethylene dimethyl methacrylate, equilibrated with the same buffer. After washing the column, the enzyme is eluted with a start buffer additionally containing 50 mM a-methyl-O-mannopyranoside. All chromatographic procedures were carried out at 4 ° C. The elution rate is 30-100 cm / h. The active fractions are combined and the enzymatic activity of p-nitrophenylphosphate and protein concentration are determined according to Lowry. The method allows the preparation of alkaline phosphatase seals with specific

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31-ginnog 11, ju. 3300 ZBOO RD / MG protein. The use of hydroxyethyl methacrylzene and ethylene dimethyl crystallite copolymer in polymer carrier allows one to increase the degree of separation of the mixture, probably due to additional differences in the strength of hydrophobic interactions of the carrier matrix with the proteins present in the separated mixture and, at the same time, significantly increase the linear elution rate, which reduces the duration of the chromatographic cycle and the duration of exposure to inactivating factors.

The upper limit of the elution rate of 100 cm / h is due to a decrease in the degree of purification of the preparation, while the lower limit (30 cm / h) is due to a decrease in the amount of processed raw materials due to an increase in the duration of the chromatographic cycle.

Non-specific binding can be prevented in the presence of 0.4-0.6 NaCI. With a decrease in NaCI concentration below 0.4 M, a decrease in the specific activity of the target product is observed. An increase in the concentration of NaCI in the buffer solution does not lead to any noticeable improvement in the quality of alkaline phosphatase.

The addition of 1–20 m№ McjCh to the zlate system during desorption promotes the stable tetrameric form of alkaline phosphatase seals. The upper limit of the concentration of magnesium chloride of 20 mM is due to a decrease in the selectivity of separation and a decrease in the activity of the reproductive product, the lower limit 1 is due to a decrease in the stability and activity of the enzyme. The activity of the target product to ZZSO-3500 u / mg beam.

EXAMPLE 1 120 mg of a commercial preparation of alkaline phosphatase seals with a fugitive activity of 58 u / mg protein are dissolved in 40 ml of 20 mM Tris-HCl, pH 7.4, containing 10 mM MgCh, 1 mM MnCl2. 1 mM CaCl2. 0.5 M NaCl (buffer A). The enzyme solution was applied to the column with concavalin A immobilized on a copolymer of hydroxyethylene methacrylate and ethylene dimethacrylate (26x94 mm), equilibrated with buffer A, and washed with the same buffer to A280. equal to 0.01-0.03. The enzyme is eluted with start buffer containing 50 mM a-methyl-O-mannopyranoside. Chromatography was performed at 4 ° C. The elution rate is 50 cm / h. Fractions containing enzymatic activity are pooled and dialyzed against buffer A without MpCb and CeCl3. The activity of the obtained alkaline phosphatase preparation is equal to 3500 units / mg protein.

Example 2. 15 mg of a commercial alkaline phosphatase seal preparation with a specific activity of 58 u / mg protein was dissolved in 5 ml of 20 mM Tris-HCI, pH 7.4.

containing 1 mm MgCl2, 1 mm MnCl2, 1 mm 0.5 M NaCl (buffer B). The enzyme solution is applied to a column of concanavalin A immobilized on a copolymer of hydroxyethyl methacrylate and ethylene dimethacrylate

(9x91 mm), balanced with buffer B and washed with the same buffer to A280 0.01-0.03. The enzyme is eluted with a start buffer containing 50 mM methyl-0-mannopyranoside. Chromatography was performed at 4 ° C.

Elution rate 30 cm / h. Fractions containing enzymatic activity are combined and dialyzed against buffer A without MnCl2 and CaCl2. The activity of the obtained alkaline phosphatase preparation is 3300

u / mg protein.

Example 3. 120 mg of a commercial preparation of alkaline phosphatase seals with a specific activity of 58 units / mg protein was dissolved in 40 ml of 20 mM Tris-HCI, pH 7.4,

containing 20 mM MgCte, 1 mM MnCl2, 1 mM, 0.5 M NaC (buffer C). The enzyme solution was applied to a column of concanavalin A immobilized on a copolymer of hydroxyethyl methacrylate and ethylene dimethacrylate (26x94 mm), equilibrated with buffer C, and washed with the same buffer to A280 of 0.01-0.03. The enzyme is eluted with a start buffer containing 50 mM methyl O-mannopyranoside. Chromatography was performed at 4 ° C. Elution rate 100 cm / h. Fractions containing enzymatic activity were pooled and dialyzed against buffer A without and CaCl2. The activity of the drug

alkaline phosphatase is equal to 3370 u / mg protein.

Example 4. 15 mg of a commercial preparation of alkaline phosphatase seals with a specific activity of 60 u / mg

protein, dissolved in 5 ml of 20 mM Tris-HCI, pH 7.4, containing 0.5 mM MgCl2.1 mM MnClz, 1 mM CaCl2. 0.5 M NaCI (buffer D) - The enzyme solution is applied to a column with concanavalin A immobilized on

a copolymer of hydroxyethyl methacrylate and ethylene dimethacrylate (9x91 mm), equilibrated with buffer G, and washed with the same buffer to A280 equal to 0.01-0.03. The enzyme is eluted with start buffer containing 50 mM

a-methyl-O-mannopyranoside. Chromatography was performed at 4 ° C. Elution rate 25 cm / h. Fractions containing enzymatic activity are pooled and dialyzed against a buffer without MnCl2 and CaCl2. The activity of the obtained alkaline phosphatase preparation is 2500 units / mg protein.

EXAMPLE 5 15 mg of a 9-commercial alkaline phosphatase seal preparation with a specific activity of 55 u / mg protein is dissolved in 5 ml of 20 mM Tris-HCl, pH 7.4, containing 25 mM MgCl2. 1 mM MnCl2. 1 mM CaCl2. 0.5 M NaCI (buffer D). The enzyme solution was applied to a column of concanavalin A immobilized on a copolymer of hydroxyethyl methacrylate and ethylene dimethacrylate (9x91 mm), equilibrated with buffer D and washed with the same buffer to A280 equal to 0.01-0.03. The enzyme is eluted with start buffer containing 50 mM a-methyl-O-mannopyranoside. Chromatography was performed at 4 ° C. Elution rate 105 cm / h. Fractions containing enzymatic activity are pooled and dialyzed against buffer D without MnCte and CaCte. The activity of the obtained alkaline phosphatase preparation is equal to 2450 units / mg protein.

EXAMPLE 6 An alkaline phosphatase preparation of seals is purified under the conditions of the prototype method by chromatography on concavalin A immobilized on sepharose. A buffer system is used for sorption of the enzyme and washing: 20 mM Tris-HCI pH 7.5, 1 mM CaCl2, 1 mM MnCl2. 0.9% NaCI and 0.05% Triton X-100. The desorption and elution of the enzyme is carried out in the same buffer system, additionally containing -methyl-D-mannopyranoside Р concentration or zero to 0.2 M. Linear speed;) lucidity 7.5 cm / h. An alkaline phosphatase preparation with a specific activity of 1500 u / mg protein is obtained.

The method allows the preparation of an active alkaline phosphatase preparation of seals while shortening the production cycle.

Claims (1)

SUMMARY OF THE INVENTION A method for purifying alkaline phosphatase, which involves chromatography of an enzyme preparation from animal tissues on concanavalin A immobilized on a polymer matrix by sorption in a buffer system containing 20 mM Tris-hydrochloric acid buffer, pH 7.5. sodium chloride, 1 mM calcium chloride and 1 mM manganese chloride, and eluting the target product with the same buffer system additionally containing 50 mM omethyl-0-mannopyranoside, characterized in that, in order to increase the activity of the enzyme, an alkaline enzyme preparation is used phosphatase from the small intestine of seals, as a polymer matrix take a copolymer of hydroxyethyl methacrylate and ethylene dimethacrylate. An additional 1-20 mM of magnesium chloride is added to the buffer system for sorption of the enzyme, and sodium chloride is used at a concentration of 0.4-0.6 M, while elution is carried out at a linear speed of 30-100 cm / h.