Production method of ubiquitin-like protein protease
阅读说明：本技术 一种类泛素蛋白蛋白酶的生产方法 (Production method of ubiquitin-like protein protease ) 是由 倪斌 蔡玥 常婧洋 黄小星 于 2021-08-17 设计创作，主要内容包括：本发明提供一种类泛素蛋白蛋白酶的生产方法,涉及基因工程技术领域。本发明的SUMO蛋白酶的生产方法,包括：1)构建SUMO蛋白酶表达载体；2)将所述SUMO蛋白酶表达载体转化至宿主菌,筛选获得基因工程菌；3)对所述基因工程菌进行培养,从培养液中提取SUMO蛋白酶。本发明的SUMO蛋白酶表达载体的表达量高,SUMO蛋白酶的提取方法简便,适合工业化大规模生产；特别是,本发明方法生产的SUMO蛋白酶的活性高,在用于切割去除融合标签以获得目的蛋白等方面具有良好的应用前景。(The invention provides a method for producing ubiquitin-like protein protease, relating to the technical field of genetic engineering. The method for producing SUMO protease of the present invention comprises: 1) constructing an SUMO protease expression vector; 2) transforming the SUMO protease expression vector to host bacteria, and screening to obtain genetically engineered bacteria; 3) culturing the genetic engineering bacteria, and extracting SUMO protease from the culture solution. The expression quantity of the SUMO protease expression vector is high, and the extraction method of the SUMO protease is simple and convenient and is suitable for industrial large-scale production; particularly, the SUMO protease produced by the method has high activity, and has good application prospect in the aspects of cutting and removing the fusion tag to obtain the target protein and the like.)
1. A production method of SUMO protease is characterized by comprising the following steps:
1) constructing an SUMO protease expression vector;
2) transforming the SUMO protease expression vector to host bacteria, and screening to obtain genetically engineered bacteria;
3) culturing the genetically engineered bacteria, and extracting SUMO protease from a culture solution;
in the step 1), the SUMO protease expression vector is pET30 a-stag-Bsulpstar;
in the step 2), the host bacteria are Bacillus subtilis;
further, step 2) comprises:
uniformly mixing the plasmid pET30a-stag-Bsulpstar with competent cells, then carrying out ice bath, water bath heat shock and ice bath, and then carrying out recovery culture in a culture medium;
and coating the culture solution after recovery culture on a solid culture medium containing kanamycin and chloramphenicol for culture, and selecting a monoclonal and screening a positive transformant after culture to obtain the genetically engineered bacteria.
2. The method of claim 1, wherein the concentration of kanamycin and chloramphenicol in the solid medium is 45-55 μ g/mL and 30-40 μ g/mL.
3. The method of claim 1, wherein the fermentation medium in step 3) is selected from the group consisting of peptone (18-22 g), yeast extract (8-12 g), and Na (Na) per liter of fermentation medium2HPO4·7H2O 6-7g，KH2PO4 3-4g，NH4Cl 2-3g，Na2SO4 0.5-1.0g，MgSO4 0.4-0.6g, 4-6mL of glycerol, 0.4-0.6g of glucose, 1.8-2.2g of lactose and FeCl3 0.001-0.005g。
4. The method of claim 1, wherein the culturing in step 3) is performed under the following conditions: the inoculation amount is 0.5-1.5%, the culture temperature is 28-32 ℃, the stirring speed is 200-.
5. The method of claim 1, wherein the step 3) of extracting SUMO protease from the culture medium comprises:
collecting thallus from the culture liquid, suspending the thallus in a lysis buffer solution, and ultrasonically lysing cells in an ice bath; wherein, the cell ultrasonic lysis conditions are as follows: the power is 25%, the ultrasonic treatment is stopped for 3-5 seconds after 2-3 seconds to be used as a cycle, and 360 cycles of 340-;
centrifuging the cracking solution, and filtering the supernatant by using a microporous filter membrane to obtain a crude enzyme solution;
further, the production method of the SUMO protease also comprises the steps of purifying the crude enzyme liquid;
specifically, the purification comprises:
carrying out column chromatography on the crude enzyme solution by adopting a nickel column;
dialyzing the effluent of the column chromatography.
6. A strain of SUMO protease, with the preservation number of CGMCC No.22263, is proposed to be classified and named as Bacillus subtilis.
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a production method of SUMO protease.
Background
The SUMO protease is cysteine protease with higher activity, is derived from yeast coded MIP1 gene, can specifically recognize the SUMO (Small Ubiquitin-Like Modifier) with the tertiary structure of UBL protein, and depends on correct protein conformation; that is, SUMO protease is not able to cleave the target protein when only the complete sequence is present without the correct structure. SUMO proteases are currently the most cleavage-specific proteases and can function over a wide range of operating temperatures and PH ranges (PH 7-9).
The target protein can be expressed by fusion with ubiquitin-like protein (sumo) so as to improve the solubility of the target protein; therefore, the high-activity SUMO protease can be used for cutting off the fusion tag to obtain the target protein. Currently, the biological activity of SUMO protease in the market is generally low, and in addition, the conventional SUMO protease production method is generally complicated in steps and high in cost. Therefore, it is highly desirable to improve the expression level and activity of SUMO protease and to reduce the production cost.
Chinese patent application with publication number CN 108774634A discloses a production method of recombinant SUMO protease in Escherichia coli, which comprises the following steps: s1, construction of a PET-21b-SUMO protease expression plasmid; s2, performing induced expression and identification on SUMO protease; s3, and purification and identification of SUMO protease. The method realizes the expression of the recombinant SUMO protease in the escherichia coli by applying an escherichia coli expression system and designing an expression vector. However, the method has limited improvement of the expression level and activity of SUMO protease, and is not suitable for industrial mass production.
In view of this, the invention has been particularly developed.
Disclosure of Invention
The invention provides a production method of SUMO protease, and when the SUMO protease is produced by the method, the expression level and activity of the SUMO protease are obviously improved.
The invention provides a production method of SUMO protease, which is carried out according to the following steps:
1) constructing an SUMO protease expression vector;
2) transforming the SUMO protease expression vector to host bacteria, and screening to obtain genetically engineered bacteria;
3) culturing the genetic engineering bacteria, and extracting SUMO protease from the culture solution.
In the step 1), the SUMO protease expression vector is pET30 a-stag-Bsulpstar.
In the step 2), the host bacteria are Bacillus subtilis.
Further, step 2) comprises:
uniformly mixing the plasmid pET30a-stag-Bsulpstar with competent cells, then carrying out ice bath, water bath heat shock and ice bath, and then carrying out recovery culture in a culture medium;
and coating the culture solution after recovery culture on a solid culture medium containing kanamycin and chloramphenicol for culture, and selecting a monoclonal and screening a positive transformant after culture to obtain the genetically engineered bacteria.
Has been preserved in China general microbiological culture Collection center (CGMCC) at 30/4/2021, with the following addresses: no. 3 of Xilu No. 1 of Beijing, Chaoyang, the institute for microbiology of Chinese academy of sciences, with the collection number of CGMCC No.22263, and the suggested classification name is Bacillus subtilis.
Further, the concentration of kanamycin in the solid culture medium is 45-55 mug/mL, and the concentration of chloramphenicol is 30-40 mug/mL.
In step 3) of the present invention, the fermentation medium is used for culturing, wherein each liter of the fermentation medium contains peptone 18-22g, yeast extract 8-12g, Na2HPO4·7H2O 6-7g，KH2PO4 3-4g，NH4Cl 2-3g，Na2SO4 0.5-1.0g，MgSO40.4-0.6g, 4-6mL of glycerol, 0.4-0.6g of glucose, 1.8-2.2g of lactose and FeCl3 0.001-0.005g。
Further, in step 3), the culture conditions are as follows: the inoculation amount is 0.5-1.5%, the culture temperature is 28-32 ℃, the stirring speed is 200-.
In step 3) of the present invention, the extracting SUMO protease from the culture solution comprises:
collecting thallus from the culture liquid, suspending the thallus in a lysis buffer solution, and ultrasonically lysing cells in an ice bath; wherein, the cell ultrasonic lysis conditions are as follows: the power is 25%, the ultrasonic treatment is stopped for 3-5 seconds after 2-3 seconds to be used as a cycle, and 360 cycles of 340-;
and centrifuging the cracking solution, and filtering the centrifuged supernatant by adopting a microporous filter membrane to obtain a crude enzyme solution.
Furthermore, the method for producing the SUMO protease further comprises purifying the crude enzyme solution.
Specifically, the purification comprises:
carrying out column chromatography on the crude enzyme solution by adopting a nickel column;
dialyzing the effluent of the column chromatography.
The implementation of the invention has at least the following advantages:
1. the SUMO protease expression vector pET30a-stag-Bsulpstar can generate a large amount of SUMO protease with high activity in bacillus subtilis by optimizing a promoter and a signal peptide, thereby greatly reducing the production cost of the SUMO protease.
2. The invention adopts a bacillus subtilis expression system for expression, has the advantages of easy control of culture conditions, high propagation speed, high secretion, relatively simple SUMO protease extraction process operation and the like, and is particularly suitable for large-scale production.
3. The method has low production cost and easy operation, the secreted SUMO protease has high concentration and strong biological activity, and the whole process has no generation of toxic substances, thereby having good application prospect in the aspects of cutting and removing the fusion tag to obtain the target protein and the like.
Drawings
FIG. 1 shows the colony morphology of Bacillus subtilis according to the present invention.
FIG. 2 is a gram stain of Bacillus subtilis according to the invention.
FIG. 3 shows a phylogenetic tree constructed by the Bacillus subtilis based on the 16S rDNA sequence.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings and the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental materials and reagents were as follows:
first, experimental material
Tryptone, Yeast Extract: original products of Oxoid corporation;
Ni2+column available from GE Healthcare;
the other reagents are domestic analytical pure reagents or foreign imported split charging products.
Second, preparation of main reagent
LB liquid Medium
Dissolving 10g/L peptone (Tryptone), 5g/L Yeast Extract (Yeast Extract), and 10g/L NaCl in purified water, and autoclaving.
LB solid Medium
Dissolving 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, 16g/L Agar (Agar) in purified water, and autoclaving for use.
3. Fermentation medium (1L)
20g peptone, 10g yeast extract, 6.7g Na2HPO4.7H2O，3.4g KH2PO4，2.6g NH4Cl，0.7g Na2SO4，0.5g MgSO45mL of Glycerol (Glycerol), 0.5g of Glucose (Glucose), 2g of Lactose (Lactose), 0.003g of FeCl3Dissolving in 1L purified water, and autoclaving.
4.0.01M PBS buffer
135mM NaCl，2.7mM KCl，1.5mM KH2PO4And 8mM K2HPO4，pH 7.2；
Weighing 7.9g NaCl, 0.2g KCl, 0.24g KH2PO4(or 1.44g Na)2HPO4) And 1.8g K2HPO4Dissolving in 800ml of distilled water, adjusting the pH value of the solution to 7.4 by using HCl, and finally adding distilled water to a constant volume of 1L; stored in a refrigerator at 4 ℃.
5. Lysis buffer
PBS buffer, 1mg/mL lysozyme, 1% Tween 20(Tween 20), 20mM Imidazole (Imidazole).
6.Binding buffer
20mM imidazole, 20mM Phosphate (Phosphate), 0.5M NaCl, pH 7.4-7.6.
7.Wash buffer
50mM imidazole, 20mM phosphate, 0.5M NaCl, pH 7.4-7.6.
8.Elution buffer
300mM imidazole, 20mM phosphate, 0.5M NaCl, pH 7.4-7.6.
9. Dialysis buffer
20mM Tris-HCl pH 8.0, 150mM NaCl, 10% glycerol (glycerol).
10.10 Xenzyme digestion reaction solution
200mM Tris-HCl pH 8.0, 1500mM NaCl, 20mM dithiothreitol (dithiothreitol).
11.10×Ulp protease store buffer
100mM Tris-HCl pH 8.0，750mM NaCl，50mM DTT，10mM EDTA。
Tricine-SDS-PAGE protein electrophoresis solution
(1) Positive electrode buffer (10 ×): 121.14g Tris was dissolved in 400ml redistilled water, adjusted to pH8.9 with 1mol/L HCl and made up to 500 ml.
(2) Negative electrode buffer (10 ×): 60.55g Tris, 89.58g Tricine and 5g SDS were dissolved in 400ml redistilled water and water was added to a final volume of 500 ml.
(3) Gel buffer (3 ×): 18.15g Tris,1.5g SDS were dissolved in 400ml redistilled water, titrated to pH8.45 with 1mol/L HCl and diluted to 500ml with water.
13. Preparation of acrylamide storage liquid
(1)3C acrylamide stock (49.5% T, 3% C): 48g of acrylamide and 1.5g of methylenebisacrylamide were dissolved in redistilled water to 100 ml.
(2)5C acrylamide stock (49.5% T, 5% C): 47g of acrylamide and 2.5g of methylenebisacrylamide were dissolved in distilled water to prepare a 100ml solution.
(3)6C acrylamide stock (49.5% T, 6% C): 46.5g of acrylamide and 3.0g of methylenebisacrylamide were dissolved in distilled water to prepare a 100ml solution.
(4) Loading buffer solution: consists of 4% SDS, 12% glycerol, 50mmol/L Tris, 2% mercaptoethanol and a little bromophenol blue, pH 6.8.
(5) The fixing solution comprises 250ml of methanol, 50ml of glacial acetic acid, 0.05mol of ammonium acetate and water to a constant volume of 500ml (0.7mm, 30 min).
(6) Staining solution 0.025% Coomassie brilliant blue G-250+ methanol 50% + 10% glacial acetic acid (0.7mm, 60 min).
(7) Destaining solution 10% glacial acetic acid + 30% methanol + ddwater (0.7mm, 2h or overnight).
14. Carnacillin (kan) solution
Dissolving 50mg/mL kanamycin sodium salt in sterilized water, filtering with microporous membrane for sterilization, subpackaging, and storing at-20 ℃ for later use.
15. Chloramphenicol (Cm) solution
Dissolving 34mg/mL chloramphenicol in sterilized anhydrous ethanol, packaging, and storing at-20 deg.C.
The invention uses Bacillus subtilis as host bacteria, which is purchased from China Center for Type Culture Collection (CCTCC).
Example 1
First, plasmid transformation expression host
mu.L of plasmid pET30a-stag-Bsulpstar was added to 150. mu.L of competent cells, gently rotated to mix the contents, ice-cooled for 30min, then heat-shocked in a water bath at 42 ℃ for 90s, and then quickly transferred to the ice bath for 2 min.
Adding 950 μ L LB culture medium into the above competent cells, resuscitating and culturing at 37 deg.C with shaking at 150rpm for 0.5-1h, centrifuging at 12000rpm for 2min, spreading 100 μ L of the mixture on LB solid culture medium containing kanamycin (50 μ g/mL) and chloramphenicol (34 μ g/mL), sucking the spread culture solution to dry at room temperature, and culturing at 37 deg.C for 12-18h by inversion.
Randomly picking a single clone the next day, performing colony PCR for the sake of insurance, uniformly mixing the positive bacteria liquid with 60% of glycerol (the final concentration of the glycerol is 20%), and storing the mixture at-80 ℃.
Expression of the Di, SUMO protease
1) The single colony preserved as described above was inoculated into 5mL of LB liquid medium (containing Kan 50. mu.g/mL, Cm 34. mu.g/mL) and cultured overnight at 37 ℃.
2) The following day, the cells were inoculated into 500mL of a fermentation medium at an inoculum size of 1% (v/v), and cultured with shaking at 30 ℃ and 220rpm for about 20 hours.
3) Centrifuging the culture solution at 6000rpm and 4 deg.C for 10min, and collecting thallus; the cells were resuspended in PBS and centrifuged again to collect the cells.
4) Cell lysis: the thalli sediment is suspended in a lysis buffer solution, is placed at 4 ℃ for half an hour, and then is used for cell lysis in an ice bath by using an ultrasonic instrument, wherein the lysis conditions are as follows: the power is 25%, ultrasonic 2 seconds and 4 seconds are taken as a cycle, and cracking is carried out for about 350 cycles; after lysis, the lysate was centrifuged at 12000rpm at 4 ℃ for 15min, the supernatant was collected and filtered through a microporous membrane to remove particulate matter.
The genetic engineering bacteria BS20210415 of the present invention has been preserved in China general microbiological culture Collection center (CGMCC) in 30/4/2021, with the addresses: no. 3 of Xilu No. 1 of Beijing, Chaoyang, the institute for microbiology of Chinese academy of sciences, with the collection number of CGMCC No.22263, and the suggested classification name is Bacillus subtilis.
The conventional physicochemical identification result of the genetically engineered bacteria is shown in fig. 1 and fig. 2: FIG. 1 shows the colony morphology of Bacillus subtilis according to the present invention; FIG. 2 is a gram stain of Bacillus subtilis according to the invention.
The evolutionary tree of the genetic engineering bacteria is shown in figure 3, the phylogenetic tree constructed by the Bacillus subtilis based on the 16S rDNA sequence is shown in the figure, and the marked line in the figure is the Bacillus subtilis.
Purification of SUMO protease
1) The nickel column was first equilibrated with a Binding buffer of 10 column volumes.
2) Loading (the sample can be added completely, the sample and the nickel matrix are mixed fully and placed at 4 ℃ for 30min), and then washing the column by using Binding buffer and Wash buffer with 3 times of column volume respectively.
3) The column was washed with 40mL of Elution buffer, and a small amount was taken for 12% SDS-PAGE to check purity.
4) The effluent was filled into dialysis bags (14 kDa dialysis bag for SUMO protease), and dialyzed overnight at 4 ℃ against dialysis buffer, with at least 2 buffer changes.
5) SUMO protease samples, 10 × Ulp protease store buffer and 100% glycerol were mixed as described in 5: 1: 6, mixing, and storing at-80 deg.C.
Fourth, in vitro cleavage of fusion protein
The Ulp protease fusion protein was mixed with the SUMO protease sample described above at a ratio of 1: 5 ratio (v/v) in 10 Xenzyme digestion reaction solution at 4 ℃ overnight (or 30 ℃ for 1-2 h); meanwhile, enzyme addition and inactivation (namely treatment at 100 ℃ for 30min) are set as negative controls.
Four, SDS-PAGE electrophoretic detection
Washing the gel plate with water and absolute ethyl alcohol in sequence, and then naturally drying or baking the gel plate; installing a glass plate and a lath, fixing the glass plate in an electrophoresis tank, and checking whether the glass plate leaks by using distilled water; the split gum and the concentrated gum were prepared separately according to the formulation in table 1.
TABLE 1 separation gel and concentrated gel formulations for SDS-PAGE electrophoretic detection
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.