CN100398644C - A heat-resistant tyrosine protein phosphatase and its separation and purification method - Google Patents

Abstract

The invention discloses a heat-resistant tyrosine protein phosphatase induced and produced from the metarhizium anisopliae strain CQMa102 and a separation and purification method thereof. It is prepared by adopting a special induction medium to induce and cultivate the Metarhizium anisopliae to form the enzyme, and then isolate and purify it. The obtained enzyme protein is glycoprotein, which can specifically hydrolyze the phosphate group on tyrosine in the protein, has the characteristics of high activity and high substrate specificity, molecular weight of 82.5kDa, isoelectric point of 9.5, and good thermal stability , the enzyme activity can be kept unchanged for 4 hours at 70°C; N-ethylmaleimide, molybdate, vanadate and divalent zinc ions can specifically inhibit the activity of the enzyme, while Okada Acid and fluoride had no obvious effect on its enzyme activity. Can be used as a biochemical reagent.

Description

A heat-resistant tyrosine protein phosphatase and its separation and purification method

technical field

The invention relates to a tyrosine protein phosphatase, in particular to a heat-resistant tyrosine protein phosphatase induced from the metarhizium anisopliae strain CQMa102 and a separation and purification method thereof.

Background technique

Tyrosine protein phosphatase is an enzyme belonging to a large family of protein phosphatases, which can specifically hydrolyze the phosphate group on tyrosine in protein. In organisms, together with tyrosine phosphorylation kinase, it controls the phosphorylation and dephosphorylation status of protein tyrosine sites. This control effect is of great significance to regulate many important life activities such as cell growth, differentiation, metabolism, intercellular communication, cell migration, gene transcription, ion channel activity, immune response and survival. Therefore, it is of great theoretical significance to further study and understand the characteristics of tyrosine protein phosphatase. It is an extremely urgent problem to find a simple method to obtain the highest recovery rate to obtain heat-resistant tyrosine protein phosphatase.

Contents of the invention

The object of the present invention is to provide a heat-resistant tyrosine protein phosphatase of entomogenic fungi.

Another object of the present invention is to provide a separation and purification method for the above-mentioned enzyme.

The object of the present invention is achieved like this: a kind of thermostable tyrosine protein phosphatase, it can specifically hydrolyze the phosphate group on the tyrosine in the protein under the condition of pH5.5, 75 ℃, and has following physical Chemical properties:

(1) Molecular weight: about 82.5kDa on sodium dodecylsulfonate denaturing polyacrylamide gel electrophoresis (SDS-PAGE);

(2) Isoelectric point: about 9.5 on 5% native polyacrylamide gel isoelectric focusing electrophoresis (IEF);

(3) Optimum reaction temperature and optimum reaction pH: All proteins containing phosphate groups on tyrosine are the substrates of the enzyme protein, the optimum reaction temperature is 75±1°C, and the optimum pH is 5.5 ±0.5;

(4) Thermal stability: Incubation of the enzyme protein at 70°C for 240 minutes has no significant effect on the enzyme activity. When the incubation temperature is 80°C, the enzyme activity decreases linearly with the extension of the incubation time. rapid loss;

(5) Inhibitors: N-ethylmaleimide, molybdate, vanadate and divalent zinc ions are all specific inhibitors of this enzyme protein;

(6) Kinetic properties of the enzyme: the Michaelis constant K _m of the enzyme protein is 1.273±0.083mM, and the maximum enzymatic reaction speed V _max is 8.72±0.12μmol.min ^-1 .mg ^-1 ;

Wherein said enzyme is extracted from Metarhizium anisopliae (Metarhizium anisopliae) Metarhizium anisopliae CQMa102 (Metarhizium anisopliae var.acridum CQMa102), and the preservation number is CGMCC No.0877.

Another object of the present invention, the above-mentioned tyrosine protein phosphatase is produced in the following way: the Metarhizium anisopliae strain CQMa102 can be induced and cultured in the induction medium to produce the enzyme, and then purified.

The phosphorus source used in the induction culture is casein, which can effectively induce the production of the tyrosine protein phosphatase; in addition, adding inorganic nitrogen as a nitrogen source can effectively inhibit the production of protease, which is beneficial to the purification; According to the specificity of the isoelectric point of the tyrosine protein phosphatase, the tyrosine protein phosphatase is separated and purified by two-step ion exchange chromatography.

The medium for the above-mentioned induction culture is specifically composed of glucose 20±1g/l, casein 4-6g/l, inorganic nitrogen: ammonium nitrate or ammonium sulfate 2±0.2g/l, potassium chloride 0.5±0.1g/l, five Magnesium sulfate water 0.5±0.05g/l, 2-(N-morpholine)-ethylsulfonic acid 10±0.5g/l, trace element mixed solution 10±0.5ml/l, adjust the pH value to 5.90～6.10, of which The trace element mixed solution consists of ferrous sulfate heptahydrate 0.22-0.25%, zinc sulfate heptahydrate 0.95-1.05%, sodium molybdate dihydrate 0.01-0.02%, copper sulfate pentahydrate 0.01-0.02%, manganese chloride tetrahydrate 0.01- 0.02% composition, in weight percentage; the chromatographic column used in the ion exchange chromatography adopts the High QSepharoes anion exchange medium column and the 25S Sepharoes cation exchange medium column which can be purchased directly on the market respectively.

Induction culture conditions for the above enzymes: collect conidia of CQMa102 strain on 1/4 SDA plate medium, and prepare 5×10 ⁷ ～5.5×10 ⁷ spores/ml bacterial suspension; inoculate 1ml per 100ml liquid induction medium, and inoculate Shake culture at 150 rpm on an open shaker for 78 hours, and the culture temperature is 26-27°C.

Separation and purification conditions for the above enzymes: after the induction culture is completed, remove the hyphae in the culture medium, collect the filtrate for thorough dialysis and centrifugation, and control its conductivity at 0.26-0.36ms/cm ² , prepare the crude enzyme solution to be purified before loading it on the column ; The HighQ anion-exchange chromatography is used to completely adsorb non-target proteins; the 25S cation-exchange chromatography column is used to adsorb the tyrosine protein phosphatase, using a linear elution gradient of 0.0-0.2M sodium chloride The bound tyrosine protein phosphatase is eluted, and the active part is collected to obtain purified tyrosine protein phosphatase.

More specifically, in the above purification, the preparation of the crude enzyme solution to be purified means that after the induction culture is completed, the culture solution is filtered with gauze to remove mycelia, and the filtrate is collected and put into a dialysis bag with a molecular cut-off of 5000 Da. The deionized water was dialyzed at 4-8°C for 36-40h, and the water was changed 6-8 times during the period to completely remove the salt in the culture medium. The solution was centrifuged at 13,500rpm and 4°C for 20min; Adjust the pH value of the dialysate to 9.5, control the conductivity to 0.26-0.36ms/cm ² , and obtain the crude enzyme solution to be purified before loading into the column;

The above-mentioned first step of ion chromatography is that the 40ml High Q Sepharoes anion-exchange medium column is first equilibrated with 200ml tris hydroxymethyl aminomethane equilibrium buffer (15mM, pH9.5, conductance 0.21～0.26ms/cm ² ), and the above crude Enzyme liquid is loaded on the column at a flow rate of 3.0ml/min, and the liquid passing through the column is collected for the next step of purification;

The above-mentioned second step of ion chromatography refers to the column liquid collected after High Q Sepharoes anion exchange medium column chromatography, adjust the pH to 8.0 with hydrochloric acid, put it into a dialysis bag, and dialyze in 15L distilled water at 4°C for 5~ 8h, control the conductivity at 0.25～0.35ms/cm ² , put on a 5ml 25 S Sepharoes cation exchange medium column, this chromatographic column is first equilibrated with 50ml tris buffer solution (10mM, pH8.0), and put on the column The flow rate is 2.5-3.0ml/min; after the column is loaded, equilibrate with 60ml tris buffer (10mM, pH8.0) at 2.0ml/min; use 80ml 0.0-0.25M sodium chloride linear elution gradient Elute bound tyrosine protein phosphatase.

The present invention makes full use of the high isoelectric point of the enzyme protein and the characteristics of low interaction ability with the ion exchange medium, first adopts 40ml High Q anion exchange as the first step of purification, and controls the conductivity of the upper column liquid at 0.26-0.36ms/ ^cm2 , adjust the pH of the upper column to 9.5 to remove non-target proteins by adsorption as much as possible; the second step of purification uses 5ml 25S cation exchange and sets the pH of the upper column to 8.0 to collect the target protein as firmly as possible. The separation and purification method of the invention has a high recovery rate, and the obtained tyrosine protein phosphatase enzyme protein has a purity of more than 95%, has the characteristics of high activity and high substrate specificity, and can be used as a biochemical reagent.

Description of drawings

Figure 1: 25 S Sepharoes (Bio-Rad) cation chromatography elution diagram;

Figure 2: The molecular weight and isoelectric point of the purified enzyme protein;

Figure 3: Substrate specificity analysis;

Figure 4: Effects of different protein phosphatase inhibitors on the activity of purified tyrosine protein phosphatases;

Figure 5: Effects of different metal ions on the activity of purified tyrosine protein phosphatase;

Figure 6: Effect of temperature on tyrosine protein phosphatase activity;

Fig. 7. pH is to the influence of tyrosine protein phosphatase activity;

Figure 8. Thermostability of tyrosine protein phosphatase;

Figure 9. Lineweaver-Burk equation determination of purified tyrosine protein phosphatase K _m and V _max .

In Figure 2, 1 is enzyme protein; 2 is molecular weight marker; 4 is isoelectric focusing silver staining of pure enzyme protein; 5 is isoelectric focusing enzyme staining of pure enzyme protein

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and implementation examples, but the present invention is not limited thereto.

Example

Activation of Spores of Metarhizium anisopliae Strain CQMa102

The Metarhizium anisopliae strain CQMa102 (Metarhizium anisopliae vsr.acridum CQMa102) in this example was isolated and purified from the diseased Bamboo locust anisopliae. It was isolated and purified by the Genetic Engineering Center of Chongqing University and preserved by the Chinese Institute of Microbiology. The preservation number is CGMCC No.0877, and its conidia are stored in 30% glycerin solution and stored in a -80°C ultra-low temperature refrigerator for a long time. Inoculate the CQMa102 strain CQMa102 of Metarhizium anisopliae stored at -80°C on 1/4 SDA plate medium for activation. The medium used is composed of 10-11g/l glucose, 2.3-2.7g/l peptone, 4.8-5.2g/l yeast The extract is composed of 19-21g/l agar, and its pH value is adjusted to 6.0. The strain is grown in the dark at 26-28°C for 7-14 days until sporulation, and then stored in a refrigerator at 4°C for use.

Enzyme production induction culture conditions

The medium for the induction culture consists of glucose 20±1g/l, casein 4～6g/l, ammonium nitrate or ammonium sulfate 2±0.2g/l, potassium chloride 0.5±0.1g/l, magnesium sulfate pentahydrate 0.5 ±0.05g/l, 2-(N-morpholine)-ethylsulfonic acid 10±0.5g/l, mixed solution of trace elements (ferrous sulfate heptahydrate 0.22～0.25%, zinc sulfate heptahydrate 0.95～1.05%, 0.01-0.02% sodium molybdate dihydrate, 0.01-0.02% copper sulfate pentahydrate, 0.01-0.02% manganese chloride tetrahydrate (by weight percentage) 10 ± 0.5ml/l, adjust the pH value to 5.90-6.10. The specific preparation method: firstly dissolve the casein in 0.1M sodium hydroxide, adjust the pH to 8.5 with 0.05M hydrochloric acid for later use; weigh according to the formula of the induction medium, and adjust the pH to 5.90-6.10. Finally, the culture medium was divided into 250ml Erlenmeyer flasks, with 100ml culture solution in each bottle. The medium is sterilized for later use.

Collect conidia of CQMa102 strain on 1/4 SDA plate medium, and prepare 5×10 ⁷ ～5.5×10 ⁷ spores/ml bacterial suspension with sterile 0.05% Tween 80 solution; inoculate 1 ml per 100 ml induction culture solution. The culture solution was shaken and cultivated on an open shaker at 150 rpm for 78 hours, and the culture temperature was 26-27°C.

Acid phosphatase isoenzyme detection

Refer to Wang Jiazheng (2001) for isoelectric focusing electrophoresis, and use T=5%, C=3 wide pH range ampholyte (pH 3～9.5). The electrophoresis equipment is Mini-Protean apparatus (Bio-Rad)% vertical plate polyacrylamide isoelectric focusing electrophoresis (IEF-PAGE). The samples contained 10% glycerol. The catholyte is 2mM NaOH solution; the anolyte is 5mM acetic acid solution. Using step-by-step electrophoresis, first electrophoresis at 100V voltage for 1h, and then electrophoresis at 250V voltage for 2h.

Gel Staining Gels after isoelectric focusing were stained by the method published by Nagy et al. The specific method is to put the gel into 100ml staining solution, incubate at room temperature for 20 minutes, then transfer the gel to 7% acetic acid solution to stop the staining. The staining solution was dissolved in 50mM 2-(N-morpholine)-ethylsulfonic acid, pH 5.5, by dissolving 150mg sodium α-naphthyl phosphate and 50mg Granule RR.

Tyrosine protein phosphatase activity assay

Tyrosine protein phosphatase activity was determined by the amount of inorganic phosphorus released by the reaction of the enzyme with phosphotyrosine. Take a certain volume of enzyme solution (xμl) and 20μl of 20mM phosphotyrosine solution, react in 20mM 2-(N-morpholine)-ethanesulfonic acid (pH5.5) at 70°C for 10min, the total volume of the reaction system 100 μl. Add 100 μl of 20% trichloroacetic acid to terminate the reaction, then add 100 μl of bovine serum albumin (10 mg/ml), and centrifuge at 13,500 rpm for 20 minutes. Take 50 μl of supernatant, add 50 μl of water, and then add 50 μl of chromogenic solution. The chromogenic solution contains 3.6M sulfuric acid, 0.5% ammonium molybdate, and 2% vitamin C. In a water bath at 37°C for 30 minutes, the absorbance was measured at 700 nm. A standard curve was prepared using potassium dihydrogen phosphate as a standard. Enzyme activity is defined as: at 70°C, the amount of enzyme required to release 1 μmol of inorganic phosphorus per minute is an enzyme activity unit (U).

In order to facilitate sample processing, in this example, each batch of culture was inoculated with 2L of culture solution, and after 78 hours of culture, the obtained filtrate had a volume of about 1.8L after dialysis treatment.

Purification of tyrosine protein phosphatase

After the cultivation, the culture solution was filtered with gauze to remove the hyphae, and the filtrate was collected and put into a dialysis bag with a molecular cut-off of 5000 Da. Dialyzed with 10 times the volume of deionized water at 4-8°C for 36-40 hours, changing 6-8 hours during the period. Thoroughly remove the salt in the culture medium, centrifuge the solution at 13,500rpm, 4°C for 20min; then adjust the pH value of the dialysate to 9.5 with tris, and control the conductivity to 0.26-0.36ms/cm ² . The crude enzyme solution to be purified before being loaded on the column is obtained.

All chromatography in the present invention was carried out on Duo-flow high pressure chromatography system (Bio-Rad). The first step of ion chromatography refers to that the 40ml High Q Sepharoes anion exchange medium column is first equilibrated with 200ml trimethylaminomethane equilibrium buffer (15mM, pH9.5, conductance 0.21～0.26ms/cm ² ), and the above-mentioned to-be-purified The crude enzyme liquid was put on the column at a flow rate of 3.0ml/min, and the liquid passing through the column was collected for the next step of purification.

The second step of ion chromatography refers to the column liquid collected after High Q Sepharoes anion exchange medium column chromatography, adjust the pH to 8.0 with hydrochloric acid, put it into a dialysis bag, and dialyze in 15L distilled water at 4°C for 5 to 8 hours , control the conductivity at 0.25～0.35ms/cm ² , put on 5ml 25 S Sepharoes cation exchange medium column, this chromatographic column is first equilibrated with 50ml tris buffer solution (10mM, pH8.0), and put on the column The flow rate is 2.5-3.0ml/min; after the column is loaded, equilibrate with 60ml tris buffer (10mM, pH8.0) at 2.0ml/min; use 80ml 0.0-0.25M sodium chloride linear elution gradient Bound tyrosine protein phosphatases are eluted (see Figure 1).

In this case, combined with the isoenzyme analysis data, according to the specificity of the isoelectric point of the target protein, 40ml High QSepharoes (Bio-Rad) anion exchange chromatography was first selected. The non-target protein in the sample is adsorbed to the medium, so as to achieve the purpose of quickly removing the non-target protein. The pH of the upper column is set to 9.5, and the conductivity is controlled at 0.26-0.36ms/ ^cm2 to allow the non-target protein to adsorb well. To the column, through this step of chromatography, the non-target protein in the sample is removed, which is beneficial to the next step of purification.

After the first step of anion exchange chromatography, the protein in the sample is very small, and 5ml of 25 S Sepharoes cation exchange medium is further used. The diameter of 25 S Sepharoes cation medium is 25 μm. Due to the small column and fine medium, the resolution is higher and the pH 8.0, the conductance is controlled at 0.25-0.35ms/cm ² , the chromatographic column is first equilibrated with 50ml tris buffer solution (10mM, pH8.0), and the flow rate of the upper column is 2.5-3.0ml/min; After completion, equilibrate with 60ml tris buffer solution (10mM, pH8.0) at 2.0ml/min; use 80ml 0.0-0.25M sodium chloride linear elution gradient to elute the bound tyrosine protein phosphatase, A single elution peak was obtained, which is the tyrosine protein phosphatase activity peak.

The inventors identified the biochemical characteristics of the purified tyrosine protein phosphatase in this example:

1. Molecular weight and isoelectric point

The molecular weight was determined by 12% SDS-PAGE according to the method of Laemmli, and the protein molecular weight marker was a low molecular weight marker (Pharmacia). Isoelectric points were determined on IEF-PAGE.

Referring to Fig. 2, it can be seen that the molecular weight of this tyrosine protein phosphatase is about 82.5kDa on sodium dodecylsulfonate denaturing polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis; ) The upper isoelectric point is about 9.5.

2. Substrate specificity analysis

The hydrolysis of phosphotyrosine, phosphoserine and phosphothreonine by purified tyrosine protein phosphatase was measured respectively. The determination method is the same as the above-mentioned tyrosine protein phosphatase enzyme activity detection. Only the substrates were replaced by phosphoserine and phosphothreonine. Each substrate was repeated three times (including blank and experimental treatment).

Referring to Figure 3, phosphotyrosine is the specific substrate of this protease.

3. Effects of inhibitors and metal ions on the activity of tyrosine protein phosphatase

After mixing different concentrations and types of inhibitors and metal ions with the purified enzyme protein, and incubating at room temperature for 30 minutes, the enzyme activity was measured according to the above-mentioned tyrosine protein phosphatase enzyme activity detection method, and water was used as a control to calculate its relative enzyme activity. See Figures 4 and 5.

4. Determination of optimum temperature and pH of tyrosine protein phosphatase reaction

Set a temperature gradient at intervals of 5°C from 30°C to 90°C, react in water baths with different temperature gradients for 10 minutes according to the above-mentioned assay method for tyrosine protein phosphatase activity, and repeat three times for each temperature gradient. See Figure 6.

Set a gradient every 0.5 pH from pH 3.0 to 9.5, among which 3.0 to 5.0 uses acetate buffer, 5.5 to 6.5 uses MES buffer, 7.0 to 7.5 uses HEPES buffer, and 8.0 to 9.5 uses Tris-HCl as buffer , according to the above-mentioned tyrosine protein phosphatase enzyme activity assay method in different pH value buffer solution. Each pH gradient was performed in triplicate. See Figure 7.

Conclusion: The optimum reaction temperature of tyrosine protein phosphatase is 75℃, and the optimum reaction pH value is 5.5.

5. Determination of thermal stability of tyrosine protein phosphatase

The purified tyrosine protein phosphatase was bathed in water for 10 min, 30 min, 60 min, 120 min and 240 min at 50, 60, 70, 80 and 90°C, respectively, and then the enzyme activity was determined according to the above-mentioned tyrosine protein phosphatase enzyme activity assay method. Each treatment was repeated three times.

Referring to Figure 8, it can be seen that incubation of this tyrosine protein phosphatase at 70°C for 240 minutes has no effect on the enzyme activity. When the incubation temperature is 80°C, the enzyme activity decreases linearly with the extension of the incubation time. When the temperature reaches 90°C, the enzyme activity Activity is lost rapidly.

6. Determination of Enzyme Kinetic Properties

The final concentration of phosphotyrosine was set to 0, 0.08, 0.16, 0.32, 0.48, 0.64, 0.80, 0.96, and 1.44mM, and phosphotyrosine with different concentration gradients were mixed with 25ng of purified tyrosine protein phosphatase, according to the above Enzyme activity was determined by tyrosine protein phosphatase enzyme activity assay (n=3). Purified tyrosine protein phosphatase K _m and V _max were calculated using the Lineweaver-Burk equation. (Figure 9)

See Figure 9: the characteristic Michaelis constant K _m of this tyrosine protein phosphatase is 1.273±0.083mM, and the maximum enzymatic reaction velocity V _max is 8.72±0.12μmol.min ^-1 .mg ^-1 .

Claims (7)

1. A heat-resistant tyrosine protein phosphatase, which can specifically hydrolyze the phosphate group on the tyrosine in the protein at pH5.5 and 75°C, and has the following physicochemical properties: (1) Molecular weight: about 82.5kDa on denaturing polyacrylamide gel electrophoresis with sodium dodecyl sulfate; (2) Isoelectric point: about 9.5 on 5% natural polyacrylamide gel isoelectric focusing electrophoresis; (3) Optimum reaction temperature and optimum reaction pH: All proteins containing phosphate groups on tyrosine are the substrates of the enzyme protein, the optimum reaction temperature is 75±1°C, and the optimum pH is 5.5 ±0.5; (4) Thermal stability: Incubation of the enzyme protein at 70°C for 240 minutes has no significant effect on the enzyme activity. When the incubation temperature is 80°C, the enzyme activity decreases linearly with the extension of the incubation time. rapid loss; (5) Inhibitors: N-ethylmaleimide, molybdate, vanadate and divalent zinc ions are all specific inhibitors of this enzyme protein; (6) Kinetic properties of the enzyme: the Michaelis constant K _m of the enzyme protein is 1.273±0.083mM, and the maximum enzymatic reaction speed V _max is 8.72±0.12μmol.min ^-1 .mg ^-1 ; Wherein said enzyme is extracted from Metarhizium anisopliae (Metarhizium anisopliae) Metarhizium anisopliae CQMa102 (Metarhizium anisopliae var.acridum CQMa102), and the preservation number is CGMCC No.0877. 2. A method for separation and purification of tyrosine protein phosphatase as claimed in claim 1, said method comprising: cultivating in the induction medium the microorganism capable of inducing the formation of said enzyme to form said enzyme, and separation and purification The obtained enzyme; the microorganism is Metarhizium anisopliae (Metarhizium anisopliae) CQMa102 (Metarhizium anisopliae var.acridum CQMa102), and the preservation number is CGMCC No.0877; The medium for the induction culture consists of glucose 20±1g/l, casein 4～6g/l, ammonium nitrate or ammonium sulfate 2±0.2g/l, potassium chloride 0.5±0.1g/l, magnesium sulfate pentahydrate 0.5 ±0.05g/l, 2-(N-morpholine)-ethylsulfonic acid 10±0.5g/l, trace element mixed solution 10±0.5ml/l, adjust the pH range to 5.90～6.10; The element mixed solution is composed of ferrous sulfate heptahydrate 0.22%~0.25%, zinc sulfate heptahydrate 0.95%~1.05%, sodium molybdate dihydrate 0.01%~0.02%, copper sulfate pentahydrate 0.01%~0.02%, chloride tetrahydrate Manganese is composed of 0.01% to 0.02%, calculated by weight percentage. 3. the separation and purification method of tyrosine protein phosphatase as claimed in claim 2, is characterized in that: in described separation and purification method, described enzyme is carried out isozyme detection and enzyme activity detection, to determine the purpose The isoelectric point of the protein is then purified. 4. the separation and purification method of tyrosine protein phosphatase as claimed in claim 2 is characterized in that: the phosphorus source of described induction medium adopts casein, and nitrogen source adopts inorganic nitrogen; The preparation of the enzyme solution and the separation and purification of the tyrosine protein phosphatase by two steps of ion exchange chromatography. 5. the separation and purification method of tyrosine protein phosphatase as claimed in claim 4, is characterized in that: the chromatographic column that described ion exchange chromatography adopts is respectively High Q Sepharoes anion exchange medium post and 25S Sepharoes cation exchange medium column. 6. the separation and purification method of tyrosine protein phosphatase as claimed in claim 5, is characterized in that: The activation of the microorganism Metarhizium anisopliae strain CQMa102 is inoculated on 1/4 SDA plate medium for activation, and the medium used is composed of 10～11g/l glucose, 2.3～2.7g/l peptone, 4.8～5.2g/l yeast The extract is composed of 19-21g/l agar, and its pH value is adjusted to 6.0. The strain is grown in the dark at 26-28°C for 7-14 days until sporulation, and then stored in a refrigerator at 4°C for use; The induction culture: collect conidia of CQMa102 strain on 1/4 SDA plate medium, and prepare 5×10 ⁷ ～5.5×10 ⁷ spores/ml bacterial suspension; inoculate 1 ml per 100 ml of liquid induction medium, and inoculate Shaking at 150rpm on a shaker for 78 hours at a temperature of 26-27°C; The separation and purification: after the induction culture is finished, the hyphae in the culture solution are removed, the filtrate is collected for thorough dialysis and centrifugation, and its conductivity is controlled at 0.26-0.36ms/cm ² , and the crude enzyme solution to be purified before being put on the column is prepared; The High Q anion-exchange chromatography is used to completely adsorb non-target proteins; the 25S cation-exchange chromatography column is used to adsorb the tyrosine protein phosphatase, and the 0.0-0.2M sodium chloride linear elution gradient is used to wash The active part of the decombined tyrosine protein phosphatase is collected to obtain purified tyrosine protein phosphatase. 7. The separation and purification method of tyrosine protein phosphatase as claimed in claim 4, is characterized in that: the preparation of the crude enzyme liquid to be purified in the described purification refers to after the cultivation finishes, and the culture solution is filtered with gauze to remove mycelium, Collect the filtrate and put it into a dialysis bag. The molecular cut-off is 5000Da. Dialyze with 10 times the volume of deionized water at 4-8°C for 36-40 hours. During this period, change the water 6-8 times to completely remove the salt in the culture medium. Centrifuge at 13,500rpm and 4°C for 20min; then adjust the pH value of the dialysate to 9.5 with Tris, and control the conductivity to 0.26-0.36ms/cm ² to obtain the crude enzyme solution to be purified before loading on the column; The first step in the two-step ion-exchange chromatography is to use High Q Sepharoes anion-exchange medium column, first use 15mM Tris, pH 9.5, and an equilibrium buffer with a conductivity of 0.21-0.26ms/ ^cm2 Equilibrate the medium, put the crude enzyme liquid to be purified on the column, the flow rate of the upper column is 3.0ml/min, collect the liquid through the column, and use it for the next step of purification; The second step of ion chromatography is to use the High Q Sepharoes anion exchange medium column. After the chromatography, the collected through-column liquid is adjusted to pH 8.0 with hydrochloric acid, put into a dialysis bag, and dialyzed in distilled water at 4°C for 5 to 8 hours. , control the conductivity at 0.25～0.35ms/cm ² , and put it on a 25S Sepharoes cation exchange medium column. This chromatography column is first equilibrated with 10mM Tris, pH8.0 buffer, and then loaded on the column, and the flow rate of the column is 2.5 ~ 3.0ml/min; after loading the column, equilibrate with Tris, pH 8.0 buffer at a speed of 2.0ml/min; in Tris, pH 8.0 buffer with The combined tyrosine protein phosphatase was eluted in a linear elution gradient of 0.0-0.25M sodium chloride.

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