Preparation method of uricase of natural mammals

Abstract

The invention discloses a preparation method of uricase of natural mammals. The method comprises the following steps: (1) pretreatment: after homogenizing and crushing fresh mammal livers, washing by using a buffering liquid with the pH value between 7.2 to 8.8, dissolving washing precipitations by using a buffering liquid with the pH value between 9.7 to 10.5 to obtain a uricase primary extraction liquid; (2) coarse purification: carrying out ammonium sulfate fractional precipitation and dissolution on the primary extraction liquid, and performing anion exchange chromatography purification on a compounded solution to obtain a uricase coarse purified product solution; (3) fine purification: performing affinity chromatography purification on the uricase coarse purified product solution to obtain a uricase purified product solution of which the purity is higher than 97 percent. The preparation method does not comprise the deactivation and degeneration of the uricase, is short in preparation period and high in yield; the obtained product is high in enzyme specific activity and high in purity, and can be used for the structure function, physicochemical property and immunogenicity researches of the uricase of natural or recombinant mammal.

Description

technical field

[0001] The invention relates to a method for preparing natural mammalian uricase, which belongs to the technical field of bioengineering and enzyme preparation. Background technique

[0002] Uricase (EC1.7.3.3., Uricase) is an oxidase that exists in the process of purine metabolism. In the presence of oxygen, it can catalyze uric acid to generate hydrogen peroxide and 5-hydroxyisouric acid. Spontaneously transform into allantoin and emit carbon dioxide. Uricase protein is widely present in microorganisms (Bacillus, Aspergillus) (Bayol A, Capdevielle J, Malazzi P, et al.Biotechnol Appl Biochem.2002.36 (Pt1): 21-31.), plants (legumes) (Lucas K, Boland MJ, Schubert KR.Arch Biochem Biophys.1983.226(1):190-197.) and animals (mammals, amphibians) (Varela-Echavarria A, Montes de Oca-Luna R, Barrera-Saldana HA.FASEB J.1988.2(15):3092-3096.). The results of crystal diffraction showed that the active uricase was a homotetramer formed by the non-covalent association ...

Examples

Embodiment 1

[0023] Embodiment 1. Preparation of high-purity natural mouse uricase protein

[0024] (1) Weigh 10 g of fresh mouse liver, homogenize it with a homogenizer, mix it with 150 mL of washing buffer (25 mM tris-hydrochloric acid buffer, pH 7.8), and stir at room temperature for 1.5- Centrifuge after 3h, and wash the centrifuged pellet repeatedly with the above-mentioned washing buffer for 2-4 times;

[0025] (2) Mix the precipitate after liver washing with 750mL dissolution buffer (50mM carbonate buffer, pH 10.2), stir at room temperature overnight and then centrifuge. The supernatant after centrifugation is the initial uricase extract;

[0026] (3) Add 75mL saturated ammonium sulfate solution to 750mL uricase initial extraction solution, centrifuge after standing overnight, redissolve the precipitate with 250mL reconstitution buffer (50mM carbonate buffer, pH10.2) overnight, and centrifuge the supernatant Carry out anion chromatography purification;

[0027] (4) Take the QAE Se...

Embodiment 2

[0029] Embodiment 2. Preparation of high-purity natural rat uricase protein

[0030] (1) Weigh 20 g of fresh rat liver, homogenize it with a homogenizer, mix it with 300 mL of washing buffer (25 mM tris-hydrochloric acid buffer, pH 7.8), and stir at room temperature for 1.5- Centrifuge after 3h, and wash the centrifuged pellet repeatedly with the above-mentioned washing buffer for 2-4 times;

[0031] (2) Mix the precipitate after liver washing with 1.5L dissolution buffer (50mM carbonate buffer, pH 10.2), stir at room temperature overnight, and then centrifuge. The supernatant after centrifugation is the initial uricase extract;

[0032] (3) Add 150mL of saturated ammonium sulfate solution to 1.5L of uricase initial extract, centrifuge after resting overnight, redissolve the precipitate with 500mL reconstitution buffer (50mM carbonate buffer, pH 10.2) overnight, and put on Purified by anion chromatography;

[0033] (4) Take the QAE Sepharose Fast Flow filler, put it into the...

Embodiment 3

[0035] Embodiment 3. Preparation of high-purity natural porcine uricase protein

[0036] (1) Weigh 100g of fresh pig liver, homogenize it with a homogenizer, mix it with 1.5L washing buffer (20mM phosphate buffer, pH8.2), stir at room temperature for 1.5-3h and then centrifuge, centrifuge and precipitate with the above Repeatedly wash 2-4 times with washing buffer;

[0037] (2) Mix the precipitate after liver washing with 7.5L of dissolving buffer (50mM carbonate buffer, pH 10.2), stir overnight at room temperature and then centrifuge. The supernatant after centrifugation is the initial uricase extract;

[0038] (3) Add 750mL of saturated ammonium sulfate solution to 7.5L of uricase initial extract, centrifuge after resting overnight, redissolve the precipitate with 2.5L of reconstitution buffer (50mM carbonate buffer, pH10.2) overnight, and centrifuge The supernatant was purified by anion chromatography;

[0039] (4) Take the QAE Sepharose Fast Flow filler, put it into the ...

technical field

[0001] The invention relates to a method for preparing natural mammalian uricase, which belongs to the technical field of bioengineering and enzyme preparation. Background technique

[0002] Uricase (EC1.7.3.3., Uricase) is an oxidase that exists in the process of purine metabolism. In the presence of oxygen, it can catalyze uric acid to generate hydrogen peroxide and 5-hydroxyisouric acid. Spontaneously transform into allantoin and emit carbon dioxide. Uricase protein is widely present in microorganisms (Bacillus, Aspergillus) (Bayol A, Capdevielle J, Malazzi P, et al.Biotechnol Appl Biochem.2002.36 (Pt1): 21-31.), plants (legumes) (Lucas K, Boland MJ, Schubert KR.Arch Biochem Biophys.1983.226(1):190-197.) and animals (mammals, amphibians) (Varela-Echavarria A, Montes de Oca-Luna R, Barrera-Saldana HA.FASEB J.1988.2(15):3092-3096.). The results of crystal diffraction showed that the active uricase was a homotetramer formed by the non-covalent association ...

Examples

Embodiment 1

[0023] Embodiment 1. Preparation of high-purity natural mouse uricase protein

[0024] (1) Weigh 10 g of fresh mouse liver, homogenize it with a homogenizer, mix it with 150 mL of washing buffer (25 mM tris-hydrochloric acid buffer, pH 7.8), and stir at room temperature for 1.5- Centrifuge after 3h, and wash the centrifuged pellet repeatedly with the above-mentioned washing buffer for 2-4 times;

[0025] (2) Mix the precipitate after liver washing with 750mL dissolution buffer (50mM carbonate buffer, pH 10.2), stir at room temperature overnight and then centrifuge. The supernatant after centrifugation is the initial uricase extract;

[0026] (3) Add 75mL saturated ammonium sulfate solution to 750mL uricase initial extraction solution, centrifuge after standing overnight, redissolve the precipitate with 250mL reconstitution buffer (50mM carbonate buffer, pH10.2) overnight, and centrifuge the supernatant Carry out anion chromatography purification;

[0027] (4) Take the QAE Se...

Embodiment 2

[0029] Embodiment 2. Preparation of high-purity natural rat uricase protein

[0030] (1) Weigh 20 g of fresh rat liver, homogenize it with a homogenizer, mix it with 300 mL of washing buffer (25 mM tris-hydrochloric acid buffer, pH 7.8), and stir at room temperature for 1.5- Centrifuge after 3h, and wash the centrifuged pellet repeatedly with the above-mentioned washing buffer for 2-4 times;

[0031] (2) Mix the precipitate after liver washing with 1.5L dissolution buffer (50mM carbonate buffer, pH 10.2), stir at room temperature overnight, and then centrifuge. The supernatant after centrifugation is the initial uricase extract;

[0032] (3) Add 150mL of saturated ammonium sulfate solution to 1.5L of uricase initial extract, centrifuge after resting overnight, redissolve the precipitate with 500mL reconstitution buffer (50mM carbonate buffer, pH 10.2) overnight, and put on Purified by anion chromatography;

[0033] (4) Take the QAE Sepharose Fast Flow filler, put it into the...

Embodiment 3

[0035] Embodiment 3. Preparation of high-purity natural porcine uricase protein

[0036] (1) Weigh 100g of fresh pig liver, homogenize it with a homogenizer, mix it with 1.5L washing buffer (20mM phosphate buffer, pH8.2), stir at room temperature for 1.5-3h and then centrifuge, centrifuge and precipitate with the above Repeatedly wash 2-4 times with washing buffer;

[0037] (2) Mix the precipitate after liver washing with 7.5L of dissolving buffer (50mM carbonate buffer, pH 10.2), stir overnight at room temperature and then centrifuge. The supernatant after centrifugation is the initial uricase extract;

[0038] (3) Add 750mL of saturated ammonium sulfate solution to 7.5L of uricase initial extract, centrifuge after resting overnight, redissolve the precipitate with 2.5L of reconstitution buffer (50mM carbonate buffer, pH10.2) overnight, and centrifuge The supernatant was purified by anion chromatography;

[0039] (4) Take the QAE Sepharose Fast Flow filler, put it into the ...

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