CN116621993A - Method for cutting magnetic microsphere sumo enzyme - Google Patents
Method for cutting magnetic microsphere sumo enzyme Download PDFInfo
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- CN116621993A CN116621993A CN202310540132.2A CN202310540132A CN116621993A CN 116621993 A CN116621993 A CN 116621993A CN 202310540132 A CN202310540132 A CN 202310540132A CN 116621993 A CN116621993 A CN 116621993A
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- C12N15/09—Recombinant DNA-technology
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- C12N15/62—DNA sequences coding for fusion proteins
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a sumo enzyme cutting method for magnetic microspheres. Comprising the following steps: s1, placing a His tag protein purified magnetic microsphere material into a reaction container, and binding the magnetic microsphere with a tag fusion protein; s2, fully cleaning the microspheres by using the impurity-cleaning buffer solution with the volume 10 times that of the magnetic microspheres; s3, adding the prepared enzyme into the washed magnetic microspheres, mixing the mixture lightly, and carrying out a reaction overnight by light shaking; s4, absorbing the magnetic microspheres by using a magnetic rod sleeve for 10min, and collecting fusion protein liquid containing the enzyme and the labels removed. The method is simple to operate, is suitable for industrial mass production, reduces environmental pollution, is suitable for the biotechnology field, and is used for tag removal of tag proteins and purification of fusion proteins.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a biological method for obtaining fusion protein by combining immobilized metal ion affinity chromatography and tag removal through magnetic microsphere sumo enzyme cleavage.
Background
Proteins are widely used in many fields of life sciences such as new drug development, disease detection, proteomics research, and enzyme and substrate reactions.
The expression system used for the production of the recombinant protein may be bacteria, yeast, animal cells, plant cells, etc. The prokaryotic protein expression system is the most commonly used expression system and is also the most economical protein expression system. The yeast protein expression system is represented by pichia methanolica, and has the advantages of high expression quantity, inducibility, glycosylation mechanism approaching to higher eukaryote, and the like. The main advantage of mammalian and insect cell expression systems is that the mechanism of protein post-translational processing is closest to the native form in vivo, most readily preserving biological activity.
When the protein is applied to the human body, each drug must meet the corresponding quality control standard (SFDA, state Food and Drug Administration) in order to secure the safety of the use of the drug, and for example, host cell proteins, host cell nucleic acids, endotoxins, viruses, etc., which cause damage, must be removed. One or more purification steps are applied to the purification of the protein to meet the quality requirements of the product.
The methods widely used for protein purification at present are as follows: metal ion chelate affinity chromatography (e.g., ni affinity chromatography), affinity chromatography (e.g., protein a affinity chromatography), ion exchange chromatography (e.g., cation exchange, anion exchange) and mixed mode ion exchange, hydrophobic interaction, size exclusion chromatography.
The traditional enzyme digestion method comprises the steps of eluting recombinant protein with Ni and GST labels combined on a metal ion chelating affinity chromatographic column by using eluent containing imidazole and reduced glutathione, replacing the eluted recombinant protein sample with Ni and GST labels into a buffer solution suitable for enzyme digestion, adding sumo enzyme for enzyme digestion reaction, and finally separating the fusion protein after enzyme digestion and the label through the metal ion chelating affinity chromatographic column. Therefore, there is a need for an economical and efficient method of purification and production of fusion proteins that reduces environmental pollution.
Disclosure of Invention
The invention aims to solve the defects and provides a method for performing sumo enzyme cleavage on a magnetic microsphere, wherein a sumo enzyme is directly used for enzyme cleavage on the magnetic microsphere, fusion proteins are excised from a connected label, and then the fusion proteins are recovered.
In order to overcome the defects in the background art, the technical scheme adopted by the invention for solving the technical problems is as follows: a method of performing sumo enzymatic cleavage of a magnetic microsphere, the method of producing a fusion protein by performing sumo enzymatic cleavage of a magnetic microsphere on a magnetic microsphere from a protease cleavage site between the tag and the fusion protein, the method comprising:
s1, placing His tag protein purified magnetic microspheres and tagged fusion proteins in a reaction container to obtain the magnetic microspheres combined with the His tag fusion proteins;
s2, fully cleaning the magnetic microsphere combined with the fusion protein with the His tag obtained in the step S1 by using a washing buffer solution with the volume which is 10 times that of the His tag protein purified magnetic microsphere;
s3, preparing sumo enzyme of which each 40ng is dissolved by using 1ml of enzyme digestion buffer solution, adding the prepared sumo enzyme into the washed magnetic microsphere combined with the His tag fusion protein obtained in the step S2, lightly mixing, and lightly vibrating to react overnight;
s4, adsorbing the magnetic microsphere combined with the fusion protein with the His tag in the step S3 by using a magnetic rod for 10min, and collecting fusion protein liquid with the tag cut off by enzyme;
s5, washing the magnetic microspheres adsorbed by the magnetic rod in the step S4 through a small amount of buffer solution again to obtain more fusion protein solution.
According to another embodiment of the present invention, the binding time of the tagged fusion protein to the magnetic microsphere in step S1 is 5-15 minutes.
According to another embodiment of the present invention, further comprising the step S2, the wash buffer is selected from phosphoric acid or a salt thereof or Tris (hydroxymethyl) aminomethane (Tris) or a salt thereof.
According to another embodiment of the present invention, the reaction condition of the light shaking reaction overnight in the step S3 is that the enzyme digestion is performed at the temperature of 2-50 ℃ for 10-16 hours.
According to another embodiment of the present invention, it is further included that the sumo protease in the step S3 refers to a protease derived from Escherichia coli.
According to another embodiment of the invention, the tagged fusion protein further comprises different concentrations of inhibitor, non-inhibitor, 1mM EDTA,10 mM EDTA,50 mM EDTA,100mM EDTA, 1mM PMSF,5 mM PMSF, or 10 mM PMSF.
According to another embodiment of the present invention, the method further comprises the step of eluting the small amount of buffer in step S5 with imidazole.
According to another embodiment of the present invention, the reaction vessel in the step S1 is a deep hole plate hole or a centrifuge tube magnetic rack in the flux purifier.
According to another embodiment of the present invention, the magnetic rod in the step S4 is sleeved with a magnetic rod sleeve, and the magnetic microsphere is attracted to the lower top end of the magnetic rod sleeve by attraction of the magnetic rod. In the use process, a magnetic rod sleeve is sleeved on the magnetic rod, the magnetic rod and the magnetic rod sleeve slowly move up and down in the deep hole plate, and the magnetic beads are collected to the lower top end of the magnetic rod sleeve. After the magnetic rod and the magnetic rod sleeve are combined with the magnetic beads, the magnetic rod is lifted from the deep hole plate and then transferred to the next deep hole plate, the magnetic rod is separated from the magnetic rod sleeve, and then the magnetic beads fall.
The beneficial effects of the invention are as follows: the method for purifying the fusion protein by sumo enzyme cleavage of the magnetic microsphere can rapidly remove the tag to obtain the fusion protein; the method is simple to operate, suitable for industrial mass production, reduced in environmental pollution, suitable for the biotechnology field, and used for tag removal of tag proteins and purification of fusion proteins;
the time for purifying the fused protein can be greatly shortened, and the method comprises the following steps: sample crushing, sample loading, impurity washing, eluting, dialyzing, enzyme cutting and tag removing purification, wherein the eluting, dialyzing and tag removing purification are omitted, and the total time can be saved by at least 8 h. And the elution and the dialysis in the omitted steps need reagents, so that the reagent preparation of the part saves time and cost.
Drawings
FIG. 1 is a SDS-PAGE analysis of RN73D enzyme before and after cleavage in the present invention;
FIG. 2 is a diagram showing SDS-PAGE analysis before and after enzyme digestion of the Twin Strep Tag II protein at different temperatures in the examples of the present invention;
FIG. 3 is a diagram showing SDS-PAGE analysis of the Twain Strep Tag II protein before and after cleavage at different concentrations of inhibitors in the examples of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. Embodiments of the invention are described herein in terms of various specific embodiments, including those that are apparent to those of ordinary skill in the art and all that come within the scope of the invention.
A method of magnetically microsphere sumo enzymatic cleavage, the method comprising:
s1, placing His tag protein purified magnetic microspheres and tagged fusion proteins in a reaction container to obtain magnetic microspheres combined with the tagged fusion proteins;
s2, fully cleaning the magnetic microsphere combined with the fusion protein with the His tag obtained in the step S1 by using a washing buffer solution with the volume which is 10 times that of the His tag protein purified magnetic microsphere;
s3, preparing sumo enzyme of which each 40ng is dissolved by using 1ml of enzyme digestion buffer solution, adding the prepared sumo enzyme into the washed magnetic microsphere combined with the His tag fusion protein obtained in the step S2, lightly mixing, and lightly vibrating to react overnight;
s4, adsorbing the magnetic microsphere combined with the fusion protein with the His tag in the step S3 by using a magnetic rod for 10min, and collecting fusion protein liquid containing sumo enzyme, from which the tag is cut off;
s5, washing the magnetic microspheres adsorbed by the magnetic rod sleeve in the step S4 through a small amount of buffer solution again to obtain more fusion protein solution. As the Sumo protease is provided with the His tag, the Sumo protease can be combined on the magnetic microsphere, and the Sumo protease can be further removed to achieve the purification effect. The residual part on the magnetic microsphere can be eluted by using eluent with imidazole, and the digestion efficiency is calculated according to the content detection of the eluted sample.
Wherein the binding time of the fusion protein with the label and the magnetic microsphere in the step S1 is 5-15 minutes.
Wherein the wash buffer in step S2 is selected from phosphoric acid or a salt thereof or Tris (hydroxymethyl) aminomethane (Tris) or a salt thereof.
Wherein, the reaction condition of the light shaking reaction overnight in the step S3 is enzyme digestion for 10-16 hours at the temperature of 2-50 ℃.
Wherein sumo protease in step S3 refers to a protease derived from E.coli.
Wherein the tagged fusion protein contains inhibitor with different concentrations of non-inhibitor, 1mM EDTA,10 mM EDTA,50 mM EDTA,100mM EDTA, 1mM PMSF,5 mM PMSF or 10 mM PMSF.
The reaction container in the step S1 is a deep hole plate hole or a centrifuge tube magnetic rack in the flux purifier. When the magnetic microsphere is applied to a small-amount high-flux protein expression system, the magnetic microsphere can be carried out by a flux purifier combining a magnetic bead medium with P8, P16, P32 and P24, and is placed in a deep hole plate hole; when magnetic microperification requires a large volume of sample, it can be placed in a 50ml centrifuge tube magnetic rack for manual purification operation.
Wherein, the magnetic rod in step S4 is sleeved with a magnetic rod sleeve, and the magnetic microsphere is adsorbed on the lower top end of the magnetic rod sleeve by the attraction of the magnetic rod. In the use process, a magnetic rod sleeve is sleeved on the magnetic rod, the magnetic rod and the magnetic rod sleeve slowly move up and down in the deep hole plate, and the magnetic beads are collected to the lower top end of the magnetic rod sleeve. After the magnetic rod and the magnetic rod sleeve are combined with the magnetic beads, the magnetic rod is lifted from the deep hole plate and then transferred to the next deep hole plate, the magnetic rod is separated from the magnetic rod sleeve, and then the magnetic beads fall.
Example one, magnetic microsphere cleavage of His-tag tagged fusion protein and elution of fusion protein:
s1, placing 10 ml Ni Smart Magarose Beads magnetic microspheres in a 50ml centrifuge tube, and combining His-tagged proteins with 10 ml Ni Smart Magarose Beads magnetic microspheres for 5 min;
s2, washing the magnetic microsphere combined with the fusion protein with the label in the step S1 by using 0.1mM Tris,pH8.0 buffer solution of 100 ml;
s3, using sumo enzyme of 400 ng, performing enzyme digestion with enzyme digestion buffer of 10 ml, then adding the prepared sumo enzyme to the washed magnetic microsphere combined with the fusion protein with the label, gently mixing, gently shaking, and reacting at 4 ℃ for at least 12 hours to allow the sumo enzyme to cleave the combined protein, i.e. cleave His-tag and thereby release the fusion protein from the column;
s4, adsorbing the magnetic microsphere combined with the fusion protein with the His tag in the step S3 by using a magnetic rod for 10min, and collecting fusion protein liquid containing sumo enzyme, from which the tag is cut off;
s5, after enzyme cutting liquid is collected, the magnetic microspheres adsorbed by the magnetic rod sleeve are washed by 0.1mM Tris,pH8.0 buffer solution, and polypeptides cut off from the His-tag are eluted, so that protein concentration detection is carried out;
10ng each was diluted with water to a final volume of 20. Mu.l before and after cleavage, and 5. Mu.l of 5 x loading Buffer was added for SDS-PAGE analysis.
The results before and after cleavage are shown in FIG. 1, wherein:
m: protein molecular weight markers;
1: before protease cleavage;
2: after protease cleavage;
the yield of the excised and recovered fusion protein was 60% to 75%.
| Step (a) | Amount bound to microsphere (mg) | Amount of enzyme-digested-liquid recovered (mg) | Recovery (%) |
| Loading | 530 | N/A | 100 |
| Elution | N/A | 372 | 70.18 |
In the table, N/a=inapplicable.
Example two, magnetic microsphere cleavage of Twain Strep Tag II fusion protein and elution of fusion protein:
s1, placing 10 ml Ni Smart Magarose Beads magnetic microspheres in a 50ml centrifuge tube, and combining His-tagged proteins with 10 ml Ni Smart Magarose Beads magnetic microspheres for 5 min;
s2, washing the magnetic microsphere combined with the fusion protein with the label in the step S1 by using 0.1mM Tris,pH8.0 buffer solution of 100 ml;
s3, using 400 ng sumo enzyme, performing enzyme digestion with 10 ml enzyme digestion buffer solution, adding the prepared sumo enzyme into the washed magnetic microspheres, gently mixing, and gently shaking to react at 4 ℃, 30 ℃, 37 ℃ and 50 ℃ for 1 hour respectively to allow the sumo enzyme to cleave the bound protein, i.e. cleave His-tag and thereby release fusion protein from the column;
s4, adsorbing the magnetic microsphere combined with the fusion protein with the His tag in the step S3 by using a magnetic rod for 10min, and collecting fusion protein liquid containing sumo enzyme, from which the tag is cut off;
s5, after enzyme cutting liquid is collected, the magnetic microspheres adsorbed by the magnetic rod sleeve are washed by 0.1mM Tris,pH8.0 buffer solution, and polypeptides cut off from the His-tag are eluted, so that protein concentration detection is carried out;
10ng of each was diluted with water to a final volume of 20. Mu.l before and after cleavage, and 5. Mu.l of 5℃loading Buffer was added for SDS-PAGE analysis.
The results before and after cleavage are shown in FIG. 2, wherein:
m: protein molecular weight markers;
1: before protease cleavage;
2: protease cutting at 4 ℃;
3: protease cutting at 30 ℃;
4: protease cutting at 37 ℃;
5: protease cutting at 50 ℃;
the yield of the excised and recovered fusion protein was 60% to 75%.
| Step (a) | Amount bound to column (mg) | Amount recovered from column (mg) | Recovery (%) |
| Loading | 500 | N/A | 100 |
| Elution (4 ℃ C.) | N/A | 345 | 69 |
| Elution (30 ℃ C.) | N/A | 335 | 67 |
| Elution (37 ℃ C.) | N/A | 325 | 65 |
| Elution (50 ℃ C.) | N/A | 305 | 61 |
In the table, N/a=inapplicable.
Example three, on-column cleavage of Twin Strep Tag II fusion protein and elution of fusion protein:
s1, 10 ml Ni Smart Magarose Beads magnetic microspheres are placed in a 50ml centrifuge tube, his-tagged proteins are combined with the 10 ml Ni Smart Magarose Beads magnetic microspheres, and inhibitors with different concentrations are added into each group of proteins: no inhibitor, 1mM EDTA,10 mM EDTA,50 mM EDTA,100mM EDTA,1 mM PMSF,5 mM PMSF,10 mM PMSF, binding time 5 min;
s2, washing the magnetic microsphere combined with the fusion protein with the label in the step S1 by using 0.1mM Tris, pH8.0 buffer solution of 100 ml;
s3, using sumo protease of 400 ng, performing enzyme digestion with enzyme digestion buffer of 10 ml, then adding the prepared enzyme into the washed magnetic microsphere, gently mixing, and gently shaking at 4 ℃ for reaction for at least 12 hours to allow sumo enzyme to cleave the bound protein, i.e. cleave His-tag and thereby release fusion protein from the column;
s4, adsorbing the magnetic microsphere combined with the fusion protein with the His tag in the step S3 by using a magnetic rod for 10min, and collecting fusion protein liquid containing sumo enzyme, from which the tag is cut off;
s5, after enzyme cutting liquid is collected, the magnetic microspheres adsorbed by the magnetic rod sleeve are washed by 0.1mM Tris,pH8.0 buffer solution, and polypeptides cut off from the His-tag are eluted, so that protein concentration detection is carried out;
10ng of each was diluted with water to a final volume of 20. Mu.l before and after protease cleavage, and 5. Mu.l of 5 x loading Buffer was added for SDS-PAGE analysis.
The results before and after cleavage are shown in FIG. 3, wherein:
m: protein molecular weight markers;
1: before protease cleavage;
2: protease cutting without inhibitor;
3: 1mM EDTA protease;
4:10 After cleavage with mM EDTA protease;
5:50 After cleavage with mM EDTA protease;
6:100 After cleavage with mM EDTA protease;
7: 1mM PMSF protease cleavage;
8: after cleavage with 5 mM PMSF protease;
9:10 After cleavage with mM PMSF protease;
the yield of the excised and recovered fusion protein was 60% to 75%.
| Step (a) | Amount bound to column (mg) | Amount recovered from column (mg) | Recovery (%) |
| Loading | 500 | N/A | 100 |
| Elution 1 (without inhibitor) | N/A | 357.4 | 71.48 |
| Elution 2 (1 mM EDTA) | N/A | 351.7 | 70.34 |
| Elution 3 (10 mM EDTA) | N/A | 35.82 | 71.64 |
| Elution 4 (50 mM EDTA) | N/A | 351.25 | 70.25 |
| Elution 5 (100 mM EDTA) | N/A | 349.2 | 69.84 |
| Elution 6 (1 mM PMSF) | N/A | 342.9 | 68.58 |
| Elution 7 (5 mM PMSF) | N/A | 346.7 | 69.34 |
| Elution 8 (10 mM PMSF) | N/A | 356.25 | 71.25 |
In the table, N/a=inapplicable.
Because of the magnetism, the magnetic microsphere can directionally move to a specific position under the action of a magnetic field or can be quickly separated from surrounding media. The performances lead the preparation to have extremely wide application prospect, thus having wide application prospect in the fields of cell separation, protein separation and purification, immobilized enzyme, immunoassay determination, targeted drugs, DNA separation, nucleic acid hybridization and the like.
The purification of proteins using magnetic microspheres as solid phase medium is an emerging protein separation technique. The traditional protein separation method, such as salting out, organic solvent, membrane separation technology, ion exchange technology, chromatography technology and the like, achieves the purpose of separation by changing factors such as pH value, temperature, ionic strength, dielectric constant and the like, has complicated separation process and large loss of target protein. The magnetic separation of the protein is carried out by modifying the surface of the magnetic microsphere, covalently binding the ligand which can be recognized and reversibly bound by the target protein, and then separating the target protein. In the magnetic separation process, the magnetic microspheres are directly put into a mixed solution containing target proteins, the target proteins are tightly combined with the magnetic microspheres, and then the separation is carried out by using an external magnetic field. The whole separation process does not need to adjust the pH value, the temperature, the ionic strength and the dielectric constant of the mixed solution, thereby avoiding the loss of protein in the traditional separation process. Compared with the traditional separation method, the magnetic separation technology of the protein has the advantages of rapidness, high purity, high yield and the like.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (9)
1. A method for sumo enzymatic cleavage of magnetic microspheres, comprising:
s1, placing His tag protein purified magnetic microspheres and tagged fusion proteins in a reaction container to obtain the magnetic microspheres combined with the His tag fusion proteins;
s2, fully cleaning the magnetic microsphere combined with the fusion protein with the His tag obtained in the step S1 by using a washing buffer solution with the volume which is 10 times that of the His tag protein purified magnetic microsphere;
s3, preparing sumo enzyme of which each 40ng is dissolved by using 1ml of enzyme digestion buffer solution, adding the prepared sumo enzyme into the washed magnetic microsphere combined with the His tag fusion protein obtained in the step S2, lightly mixing, and lightly vibrating to react overnight;
s4, adsorbing the magnetic microsphere combined with the fusion protein with the His tag in the step S3 by using a magnetic rod for 10min, and collecting fusion protein liquid with the tag cut off by enzyme;
s5, washing the magnetic microspheres adsorbed by the magnetic rod in the step S4 through a small amount of buffer solution again to obtain more fusion protein solution.
2. The method of sumo enzymatic cleavage of magnetic microspheres according to claim 1, wherein: the binding time of the tagged fusion protein and the magnetic microsphere in the step S1 is 5-15 minutes.
3. The method of sumo enzymatic cleavage of magnetic microspheres according to claim 1, wherein: the washing buffer in the step S2 is selected from phosphoric acid or a salt thereof or Tris (hydroxymethyl) aminomethane (Tris) or a salt thereof.
4. The method of sumo enzymatic cleavage of magnetic microspheres according to claim 1, wherein: the reaction condition of the light shaking reaction overnight in the step S3 is that the enzyme digestion is carried out for 10 to 16 hours at the temperature of 2 to 50 ℃.
5. The method of sumo enzymatic cleavage of magnetic microspheres according to claim 1, wherein: the sumo enzyme in step S3 refers to a protease derived from Escherichia coli.
6. The method of sumo enzymatic cleavage of magnetic microspheres according to claim 1, wherein: the tagged fusion protein contains different concentrations of inhibitor, uninhibited, 1mM EDTA,10 mM EDTA,50 mM EDTA,100mM EDTA, 1mM PMSF,5 mM PMSF, or 10 mM PMSF.
7. The method of sumo enzymatic cleavage of magnetic microspheres according to claim 1, wherein: the small amount of buffer solution in the step S5 is eluent with imidazole.
8. The method of sumo enzymatic cleavage of magnetic microspheres according to claim 1, wherein: the reaction vessel in the step S1 is a deep hole plate hole or a centrifuge tube magnetic rack in the flux purifier.
9. The method of sumo enzymatic cleavage of magnetic microspheres according to claim 1, wherein: and the magnetic rod in the step S4 is sleeved with a magnetic rod sleeve, and the magnetic microspheres are adsorbed on the lower top end of the magnetic rod sleeve through the magnetic rod.
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