CN106011099A - Separation and purification method of insect transglutaminase - Google Patents

Abstract

The invention relates to a method for separating and purifying insect Transglutaminase. The separation and purification method includes (1) the step of preparing insect transglutaminase; (2) using ammonium sulfate to separate the insect transglutaminase obtained in step (1) by precipitation to obtain a preliminary target protein (3) the step of obtaining the target protein through glucose gel column and ion exchange column chromatography in turn for the preliminary target protein; or, the step of obtaining the target protein through ion exchange column and glucose gel column chromatography in turn for the preliminary target protein step. The invention successfully separates and purifies insect transglutaminase for the first time, which lays a foundation for the research on the physiological regulation of pests with transglutaminase as the target and the research and development of new pest control or insecticidal methods with transglutaminase as the target Base.

Description

Method for separating and purifying insect transglutaminase

technical field

The invention relates to a method for separating and purifying insect Transglutaminase.

Background technique

Transglutaminase (Transglutaminase, EC 2.3.2.13) can catalyze the acyl transfer reaction between the γ-formamide group of the glutamine peptide bond and the ε-amino group of the lysine peptide bond to form ε-(γ -glutamyl) acid isopeptide bond. It can specifically recognize glutamine-like protein substrates, and has low specificity for amine-like acyl receptors. The ε-amino group of the lysine-peptide bond or the primary amine of low-molecular polymerization can be used as the reactive acyl group receptor. It has excellent catalytic properties such as catalyzing the cross-linking reaction between protein molecules or within the molecule, the connection between amino acids and proteins, and the hydrolysis of glutamine groups in protein molecules.

In mammals, transglutaminase is involved in processes such as coagulation, immune defense, cell differentiation and apoptosis, inflammatory autoimmunity and neurodegenerative diseases. It is used for the repair of external trauma, fractures and cartilage lesions, as well as the treatment of certain intestinal diseases and autoimmune antigens, and is also used for many major diseases (such as: cataract, arteriosclerosis, Alzheimer's, Huntington's disease , Parkinson's disease, etc.) regulation process. At present, transglutaminase has extremely wide application value in the fields of food, cosmetics and medicine.

Research on transglutaminase found that transglutaminase exists in microorganisms, plants, invertebrates, amphibians, fish, birds and other species. However, transglutaminases from different sources not only have great differences in enzymatic properties such as: molecular weight, isoelectric point, optimum pH value, optimum temperature, etc., but also have great differences in protein structure, amino acid sequence, spatial structure, activity, etc. There are also differences. Among them, the molecular weight of transglutaminase derived from microorganisms is generally small, mostly 40 kDa, which is about half of the molecular weight of mammalian transglutaminase. The common point of transglutaminases from different sources is that the active center of the protein has Cys residues, and the catalytic triad of the enzyme is highly conserved.

Under natural conditions, transglutaminase easily forms irreversible polymers, which are difficult to isolate and purify. In addition, maintaining the stability of transglutaminase and establishing a highly sensitive enzyme activity detection method are also major difficulties to be solved in the process of separating and purifying transglutaminase.

Although transglutaminase has been found to be widely distributed in insect muscles and cells, the characteristics of insect transglutaminase have been reported less, and currently mainly focus on Drosophila melanogaster and Anopheles gambiae ) and other insect-borne transglutaminases. It has been reported that transglutaminase in Drosophila is involved in the formation of plasma coagulation, knocking out the transglutaminase gene in Drosophila can significantly reduce the expression level of transglutaminase in Drosophila, and larvae are more susceptible to Infection with exogenous pathogenic fungi and bacteria significantly increases the infectious mortality of larvae; in Anopheles gambiae, transglutaminase may be involved in the regulation of reproductive and immune physiology.

The limitation of understanding of insect transglutaminase has greatly restricted the research on the physiological regulation of pests and the development of new pest control or insecticidal methods with transglutaminase as the target. However, so far, the isolation and purification process of insect transglutaminase has not been reported in the literature.

Contents of the invention

In order to solve the above-mentioned pest physiological regulation research and the research and development of new pest management methods with transglutaminase as the target, and further deepen the difficult problem of understanding insect transglutaminase, the object of the present invention is to: provide an insect cereal A method for separation and purification of aminotransferase.

The separation and purification method of insect transglutaminase of the present invention, it comprises the steps:

(1) the step of preparing insect transglutaminase;

(2) using ammonium sulfate to separate the insect transglutaminase obtained in step (1) by precipitation to obtain a preliminary target protein; and,

(3) the step of obtaining the target protein through sequential chromatography on glucose gel column and ion exchange column for the preliminary target protein; or, the step of obtaining the target protein through sequential chromatography on ion exchange column and glucose gel column for the preliminary target protein;

Wherein, the insects are pests of Lepidoptera, Orthoptera, Coleoptera, Diptera, Homoptera or Diptera, and the separation range of the glucose gel column is 3,000-150,000.

Description of drawings

Figure 1 Specific activity of transglutaminase in samples after ammonium sulfate precipitation with different saturation degrees.

Figure 2 Sephadex G-75 sephadex separation of transglutaminase.

Figure 3 Sephadex G-100 sephadex separation of transglutaminase.

The Tris-HCl buffer solution of Fig. 4 pH7.8 is to the ion-exchange chromatography elution separation of transglutaminase;

In the figure: each lane corresponds to 4 protein samples with transglutaminase activity.

Fig. 5 Tris-HCl buffer solution at pH 8.5 for ion-exchange chromatography elution separation of transglutaminase.

The gel electrophoresis pattern of Fig. 6 transglutaminase purified by method 1;

In the figure: M—standard protein; TG—target protein.

The gel electrophoresis pattern of Fig. 7 transglutaminase purified by method 2;

In the figure: M—standard protein; 1—crude enzyme solution; 2—enzyme solution after ammonium sulfate precipitation; 3—enzyme solution after the first ion exchange precipitation; 4—enzyme solution after the second ion exchange precipitation; 5 - Enzyme solution after gel chromatography.

detailed description

In a preferred technical solution of the present invention, ammonium sulfate with 55% saturation is used to separate the insect transglutaminase obtained in step (1) by precipitation to obtain a preliminary target protein.

In another preferred technical solution of the present invention, in step (3), when the primary target protein is first subjected to ion-exchange column chromatography and then glucose gel column chromatography, it needs to be subjected to ion-exchange column chromatography twice, Then through glucose gel column chromatography;

In yet another preferred technical solution of the present invention, the glucose gel column used in step (3) is a Sephadex G-100 gel column (separation range 4,000-150,000).

In yet another preferred technical solution of the present invention, the ion exchange column used in step (3) is a DEAE-cellulose-52 ion exchange column, and the eluent used is: the pH value is 7.0～9.0 (more preferably 7.8～8.5) . Tris-HCl buffer solution with a concentration of 15mM-25mM, and the Tris-HCl buffer solution contains 0-1.0M NaCl (more preferably 0.2M-0.8M).

The inventors of the present invention successfully separated and purified insect transglutaminase for the first time, which is the basis for the research on the physiological regulation of pests with transglutaminase as the target and the new pest control or insecticidal means with transglutaminase as the target R&D laid the groundwork.

The present invention is described further below in conjunction with embodiment, and its purpose is only to understand content of the present invention better. Therefore, the examples given do not limit the protection scope of the present invention.

Example 1

Preparation of Insect Transglutaminase

experimental method:

(1) Several fourth-instar armyworm larvae of uniform size were taken, starved for 4 hours, and washed with 20 mM Tris-HCl buffer solution (pH 8.0).

(2) According to the volume ratio of 3:1 (v:v), (the volume ratio of buffer: larvae is 3:1), the larvae were placed in 20mM Tris-HCl buffer (pH8.0), under ice bath conditions, After standing still for 30min, fully homogenate.

(3) After the worms were fully broken, the homogenate was centrifuged (10,000 g, 4° C.) for 20 min, the supernatant was taken for freeze-drying, and the dry powder was collected to be used as an enzyme source of transglutaminase.

(4) Determination of the activity of transglutaminase. Take 100 μl of benzylcarbonyl oxide-L-glutamine glycine (N _α -CBZ-GIn-Gly) substrate solution preheated to 37°C (preparation method: weigh 100 mg of N _α -CBZ-GIn-Gly solution In 2mL of 0.2mol/L NaOH solution, after completely dissolving, add 4mL of 0.2M Tris-HCl (pH 6.0) buffer solution, 2mL of 0.1M hydroxylamine solution and 2mL of 0.01M reduced glutathione solution, and adjust pH is 6.0), add 50 μl enzyme solution, react at 37°C water bath temperature for 10 minutes, add 100 μl TCA color development solution (made from 3M HCl, 12% trichloroacetic acid, 5% ferric chloride hexahydrate according to 1:1:1, etc. volume mixed) to terminate the reaction, centrifuge at room temperature (8,000g) for 5 minutes, take the supernatant and place it in a microplate reader at a wavelength of 525nm to read the absorbance value (OD ₅₂₅ ), and calculate the glutamine transamination according to the standard curve Enzyme specific activity. The substrate solution without adding N _α -CBZ-GIn-Gly was used as the control.

Experimental results:

The results showed (Table 1) that there was transglutaminase in the armyworm, and the total activity of transglutaminase gradually increased with the increase of protein content in the enzyme source sample, but the activity of transglutaminase The lower specific activity indicated that the content of transglutaminase in armyworms was lower.

Table 1 Specific activity of transglutaminase in parasite enzyme source samples

Example 2

Ammonium Sulfate Precipitation Separation of Insect Transglutaminase

experimental method:

(1) Slowly add solid ammonium sulfate to the armyworm homogenate in an ice bath until the saturation is 25%, 35%, 45%, 55%, 65%, 75%, and 85%, and stir evenly with a glass rod After the ammonium sulfate solid was completely dissolved, continue to stir for 30 minutes, and after standing overnight at 4°C, centrifuge (10,000g, 4°C) for 30 minutes to collect the supernatant and precipitate.

(2) Dissolve the precipitate in 20mM Tris-HCl buffer solution (pH 8.0), transfer it into dialysis bags like the supernatant, place it in 4°C running buffer and dialyze overnight, and check the 1% BaCl solution to confirm the dialysis The dialysis was stopped when there was no ammonium sulfate in the solution, and the enzyme solution was taken for freeze-drying.

(3) Determination of the specific activity of transglutaminase.

Experimental results:

The result shows (Fig. 1), reaches 55% in the saturation degree of ammonium sulfate solution, and the transglutaminase specific activity reaches maximum in the precipitation of sample, and in the supernatant of sample, transglutaminase specific activity significantly reduces, shows Precipitating transglutaminase from samples with 55% saturation ammonium sulfate works best.

Example 3

Sephadex G-75 Sephadex Gel Separation of Insect Transglutaminase

experimental method:

(1) Take 50g of Sephadex G-75 dextran gel (separation range 3,000-80,000) into 2L of deionized water, stir evenly, let stand at room temperature to swell overnight, then swell in boiling water for 4 hours, cool down and set aside.

(2) The swollen gel was loaded into a chromatography column (1.6×100 cm) at one time, and 20 mM Tris-HCl (pH 8.0) buffer solution of 3 times the volume of the column bed was added for equilibration.

(3) When the level of the equilibrium solution and the gel is about 1cm away, take the enzyme solution to load the sample. After the enzyme solution has completely entered the gel surface, add 20mM Tris-HCl (pH 8.0) buffer to elute, and adjust the flow rate to 0.5mL/min, collect the effluent components, measure the absorption value of each component at a wavelength of 280nm,

(4) Measure the specific activity of transglutaminase in each fraction sample.

(5) The samples were subjected to polyacrylamide gel electrophoresis (SDS-PAGE). Using 12% separating gel (3.36mlddH ₂ O, 2.5ml 1.5M Tris-HCl (pH8.8), 100μl 10% SDS, 4ml 30% polyacrylamide, 25μl 10% ammonium persulfate, 15μl TEMED) and 4% Stacking gel (2.4ml ddH ₂ O, 1.0ml 0.5M Tris-HCl (pH6.8), 40μl 10% SDS, 0.53ml 30% polyacrylamide, 15μl 10% ammonium persulfate, 10μl TEMED). The voltage of the stacking gel was 80V, and the voltage of the separating gel was 120V. The gel was stained with 0.1% Coomassie Brilliant Blue R250 staining solution, and the stained gel was decolorized with a decolorizing solution (water:methanol:glacial acetic acid 8:1:1).

Experimental results:

The results showed that transglutaminase activity was concentrated in tubes 13-20 in Sephadex G-75 gel chromatography (Fig. 2A). In the SDS-PAGE pattern, the molecular weight of transglutaminase is about 63.31kDa, showing that transglutaminase protein is within the separation range of Sephadex G-75, but after Sephadex G-75 gel chromatography, The target protein sample still contained more miscellaneous proteins (Figure 2B).

Example 4

Sephadex G-100 Sephadex Separation of Insect Transglutaminase

experimental method:

(1) Take 50g of Sephadex G-100 dextran gel (separation range: 4,000-150,000) into 2L of deionized water, stir well, let stand at room temperature to swell overnight, then place in boiling water to swell for 4 hours, cool down and set aside.

(2) The swollen gel was loaded into a chromatography column (1.6×100 cm) at one time, and 20 mM Tris-HCl (pH 8.0) buffer solution of 3 times the volume of the column bed was added for equilibration.

(3) When the level of the equilibrium solution and the gel is about 1cm away, take the enzyme solution to load the sample. After the enzyme solution has completely entered the gel surface, add 20mM Tris-HCl (pH 8.0) buffer to elute, and adjust the flow rate to 0.5mL/min, collect the effluent components, measure the absorption value of each component at a wavelength of 280nm,

(4) Measure the specific activity of transglutaminase in each fraction sample.

Experimental results:

The results showed (Figure 3) that in Sephadex G-100 gel chromatography, transglutaminase activity was concentrated in tubes 7-11, indicating that the transglutaminase protein was within the separation range of Sephadex G-100 , and the peak shape of protein elution is better than that of Sephadex G-75 elution peak.

Example 5

Ion Exchange Chromatography Separation of Transglutaminase in Tris-HCl Buffer at pH 7.8

experimental method:

(1) Dissolve 30g of DEAE-cellulose-52 in 2L of deionized water, stir well, let stand at room temperature to swell overnight, remove the deionized water by suction filtration, soak in 0.5M NaOH solution for 30min, and remove the upper layer by suction filtration solution, washed with deionized water until neutral. Then put it in 0.5M HCl solution for 30 minutes, filter it with suction and wash it with water until neutral, and finally put it in 0.5M NaOH solution for 30 minutes, filter it with suction and wash it with water until it becomes neutral.

(2) The swollen DEAE-cellulose-52 was loaded into a chromatographic column (3.9×20 cm) at one time, and 20 mM Tris-HCl buffer solution (pH 7.8) of 3 times the column bed volume was added for equilibration.

(3) When the level of the equilibrium solution and the gel liquid is about 1cm away, take the enzyme solution to load the sample. After the enzyme solution has completely entered the gel surface, use 20mM Tris-HCl buffer (pH7.8) containing 0-1.0M NaCl ) to carry out gradient elution, adjust the flow rate to 0.5mL/min, collect the effluent components, and measure the absorption value of each component at a wavelength of 280nm.

(4) Measure the specific activity of transglutaminase in each component.

(5) The samples containing transglutaminase activity were subjected to polyacrylamide gel electrophoresis.

Experimental results:

The results show that when the 20mM Tris-HCl eluent of pH 7.8 is used for elution, ion exchange column chromatography can detect four protein absorption peaks with transglutaminase activity and closely adjacent to each other. Not obvious (Fig. 4A). The polyacrylamide gel electrophoresis patterns showed that there were still many miscellaneous proteins in these four protein samples with transglutaminase activity, and the bands of miscellaneous proteins were relatively close to the bands of transglutaminase (Fig. 4B).

Example 6

Ion Exchange Chromatography Separation of Transglutaminase in Tris-HCl Buffer at pH 8.5

experimental method:

(1) Take 30 g of DEAE-cellulose-52 and carry out swelling treatment according to the method in Example 5.

(2) Pack the swollen DEAE-cellulose-52 into a chromatography column (3.9×20 cm) at one time, and add 20 mM Tris-HCl buffer solution (pH 8.5) of 3 times the column bed volume for equilibration.

(3) When the level of the equilibrium solution and the gel liquid is about 1cm away, take the enzyme solution to load the sample. After the enzyme solution has completely entered the gel surface, use 20mM Tris-HCl buffer (pH8.5) containing 0-1.0M NaCl ) to carry out gradient elution, adjust the flow rate to 0.5mL/min, collect the effluent components, and measure the absorption value of each component at a wavelength of 280nm.

(4) Measure the specific activity of transglutaminase in each component.

(5) The samples containing transglutaminase activity were subjected to polyacrylamide gel electrophoresis.

Experimental results:

The results showed that when 20 mM Tris-HCl eluent at pH 8.5 was used for elution, ion exchange column chromatography could detect a protein absorption peak with transglutaminase activity ( FIG. 5A ). The polyacrylamide gel electrophoresis pattern showed that in the protein sample with transglutaminase activity, the band of transglutaminase was far away from the bands of other miscellaneous proteins (Fig. 5B).

Embodiment 7 (purification of insect transglutaminase)

Method 1 specifically includes the following steps:

(1) Prepare the enzyme source of armyworm transglutaminase according to the method in Example 1.

(2) Use ammonium sulfate with a saturation of 55% for protein precipitation, and put the target protein into a dialysis bag for dialysis overnight.

(3) Sephadex G-100 gel column is used for chromatography, and every 15mL eluent is collected in one tube,

(4) Adopt DEAE-cellulose-52 ion exchange column chromatography (D2.6×20cm) and pH8.5 elution buffer

Experimental results:

The results showed that after Sepadex G-100 gel chromatography, the target protein sample containing transglutaminase activity was initially separated, and the transglutaminase protein was purified by 5.683 times. After the target protein is concentrated, it undergoes DEAE-cellulose-52 ion exchange chromatography, most of the protein is eluted without being adsorbed, and the transglutaminase protein is eluted by NaCl eluent with an ion concentration of about 0.5M The collected transglutaminase protein was purified by 48.36 times, and the final recovery rate was 12.69%. In the polyacrylamide gel electrophoresis profile, the target protein only showed a clear band of transglutaminase protein with a molecular weight of about 63.87kDa (Figure 6), which reached the N-terminal sequencing purity of the protein. The disadvantage is that the protein loss is large and the recovery rate of the target protein is low.

Method 2 specifically includes the following steps:

(1) Prepare the enzyme source of armyworm transglutaminase according to the method in Example 1.

(2) Use ammonium sulfate with a saturation of 55% for protein precipitation, and put the target protein into a dialysis bag for dialysis overnight.

(3) Chromatography was performed on a DEAE-cellulose-52 ion-exchange column, and the eluent was 20 mM Tris-HCl (containing 0-1.0 M NaCl) buffer at pH 7.8.

(4) Chromatography was performed on a DEAE-cellulose-52 ion-exchange column, and the eluent was Tris-HCl (containing 0-1.0 M NaCl) buffer at pH 8.25.

(5) Sephadex G-100 gel column is used for chromatography to collect the target protein.

Experimental results:

The results show that, in the first ion exchange column chromatography, after elution with 20mM Tris-HCl (containing 0-1.0M NaCl) buffer solution at pH 7.8, the eluent in 0.3M-0.8M NaCl solution Under the above conditions, the protein containing transglutaminase activity is desorbed and eluted to a greater extent, and a larger protein absorption peak appears. In the second ion exchange column chromatography, the target protein was eluted with 20mM Tris-HCl (containing 0-1.0M NaCl) buffer at pH 8.25, and the protein containing transglutaminase activity was eluted by 0.3M～1.0M NaCl. The eluent was desorbed and eluted with 0.6 M NaCl solution. After further chromatography on a Sephadex G-100 gel column, the collected transglutaminase protein was purified by 40.68 times, and the final recovery rate was 18.62%. However, in the polyacrylamide gel electrophoresis pattern, the molecular weight of the target protein is about 63.17, and the impurity protein with a molecular weight of about 56.68 is not removed. The advantage and disadvantage are that the recovery rate of the target protein is high, but there is a small amount of impurity protein.

Claims (8)

1. a method for isolated and purified insecticide glutamine transaminage, it comprises the steps:

(1) step of insecticide glutamine transaminage is prepared；

(2) use ammonium sulfate, separated by the insecticide glutamine transaminage of step (1) gained by coprecipitation mode, obtain preliminary The step of target protein；With,

(3) preliminary aim albumen is successively through glucose gel post and ion-exchange chromatography, obtains the step of target protein；Or, Preliminary aim albumen through ion exchange column and glucose gel column chromatography, obtains the step of target protein successively；

Wherein, described insecticide is Lepidoptera, Orthoptera, coleoptera, Diptera, Homoptera or Diptera pest, described glucose Gel column separating ranges is 3,000～150,000.

2. the method for claim 1, it is characterised in that in step (2), ammonium sulfate used is the sulphuric acid of 55% saturation Ammonium.

3. method as claimed in claim 1 or 2, it is characterised in that in step (3), preliminary aim albumen is first through two secondary ions Exchange column chromatographs, then through glucose gel column chromatography, obtains target protein.

4. method as claimed in claim 3, it is characterised in that in step (3), glucose gel post used is Sephadex G-100 gel column.

5. method as claimed in claim 3, it is characterised in that in step (3), ion exchange column used be DEAE-cellulose- 52 ion exchange column, eluent used is: the Tris-HCl buffer that pH value is 7.0～9.0, concentration is 15mM～25mM, and 0～1.0M NaCl is contained at described Tris-HCl buffer.

6. method as claimed in claim 5, it is characterised in that wherein said pH value is 7.8～8.5.

7. method as claimed in claim 5, it is characterised in that wherein said NaCl concentration is 0.2M～0.8M.

8. the method as described in any one in claim 1～7, it is characterised in that wherein insecticide glutamine transaminage by The preparation method comprised the steps prepares:

(1) take uniform four age the some heads of armyworm larvae, Nature enemy is after 4 hours, with pH value be 8.0, concentration be 20mM Tris-HCl buffer solution for cleaning polypide；

(2) by pH value be 8.0, concentration be Tris-HCl buffer and the cleaned polypide of 20mM, be 3: 1 mixing by volume, Obtain mixture, under condition of ice bath, gained mixture is stood at least 30 minutes, is fully homogenized, obtains homogenate；

(3) gained homogenate in step (2) is centrifuged, takes supernatant and carry out lyophilization, collect dry powder, turn as glutamine The enzyme source of amine enzyme.