Purification method of creatine kinase isoenzyme CKMB
By adding His tag and Strep tag to the co-expression vector, we ensure that the creatine kinase M subunit dimer and B subtype gene are co-expressed in the vector and the tag is exposed in the protein complex, thereby achieving efficient purification of CKMB, which solves the problem of insufficient purification efficiency and purity of CKMB in the prior art.
Patent Information
- Application Number
- CN202510227231.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-27
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art is difficult to efficiently purify high-purity CKMB under the premise of ensuring the activity of the protein complex in the form of the creatine kinase isozyme CKMB.
Through the co-expression vector construction, the genes of the creatine kinase M subunit dimer (CKM) and creatine kinase B subtype (CKB) were placed into the co-expression vector, and the His tag and Strep tag were added to the N-terminal or C-terminal of different target genes respectively to ensure that both tags were completely exposed to the surface of the complex when the dimer was formed, and thus efficient purification was carried out.
On the premise of ensuring the activity of the protein complex in the form of creatine kinase isozyme (CKMB), efficient purification is achieved, with high yield and purity.
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Figure CN119979500A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of protein processing, and in particular to a method for purifying creatine kinase isoenzyme CKMB. Background Art
[0002] Creatine kinase (CK) is a dimeric enzyme with four isoenzyme forms, namely muscle type (CK-MM), brain type (CK-BB), hybrid type (CK-MB) and mitochondrial type (CK-Mt). MM type is mainly present in various muscle cells, BB type is mainly present in brain cells, MB type is mainly present in cardiomyocytes, and Mt type is mainly present in cardiac and skeletal muscle mitochondria.
[0003] Protein dimers are macromolecular complexes or polymers formed by two protein monomers, and are a form of protein quaternary structure. There are two types of protein dimers: homodimers: composed of two identical protein molecules, a process called homodimerization. For example, VE-cadherin, a component of cell adhesion junctions, forms homodimers between cells and then binds to another dimer on the surface of a cell. Heterodimers: formed by two different protein molecules, called heterodimerization. For example, integrin is a typical heterodimeric protein, composed of two different subunits, alpha and beta.
[0004] The reasons for its formation are: 1) Chemical reaction: A chemical reaction occurs between two molecules to form a covalent bond, thereby combining into a dimer. This reaction can be spontaneous or induced under specific conditions; 2) Physical interaction: Physical interactions such as electrostatic interactions, hydrogen bonds, and van der Waals forces between molecules can combine two protein molecules to form a dimer; 3) Biological process: In many biological processes, protein dimers are key regulatory factors, and their formation is crucial for the normal function of proteins. For example, in the process of cell signal transduction, many signal proteins will form dimers to transmit signals; 4) Environmental factors: Environmental factors such as temperature, pressure, and pH value will affect the chemical properties and physical state of molecules, thereby affecting the formation of dimers.
[0005] Common examples and functions of protein dimers include: 1) Receptor tyrosine kinases: They usually function as dimers and play an important role in cell signal transduction. They can receive extracellular signals and activate intracellular signal pathways, thereby regulating cell growth, differentiation, proliferation and other processes; 2) Transcription factors: Some transcription factors with leucine zipper motifs will form dimers. They enhance their binding ability to DNA through dimerization, thereby more effectively regulating gene expression; 3) Nuclear receptors: After binding with ligands, nuclear receptors often form dimers and then enter the cell nucleus to regulate gene transcription, participating in the regulation of cell physiological functions and developmental processes; 4) G protein-coupled receptors: Some G protein-coupled receptors can form dimers. This dimerization may be closely related to receptor activation, regulation of signal transduction, and receptor endocytosis and recycling.
[0006] Dimers are often associated with some diseases, such as Alzheimer's disease: its pathogenesis is related to the misfolding and aggregation of amyloid proteins. Amyloid proteins may form dimers and higher-level aggregates, which are toxic to neurons, leading to damage and death of nerve cells, and then triggering symptoms of Alzheimer's disease. Cancer: The dimerization state of some proteins in tumor cells may change, thereby affecting the cell's signal transduction, proliferation, apoptosis and other processes, and promoting the occurrence and development of tumors. For example, proteins encoded by certain oncogenes may activate carcinogenic signaling pathways by forming dimers, while proteins encoded by some tumor suppressor genes may lose their tumor suppressor function due to abnormal dimerization.
[0007] Some existing schemes for obtaining dimers have some defects to a greater or lesser extent. For example, some schemes choose to incubate dimers in vitro, and the dimers obtained in this way may not represent their natural state. Some schemes expose dimers to extreme environments during protein treatment, which may cause the dimers to dissolve. Some schemes have too complicated purification methods, and the acquisition rate and purity cannot be guaranteed.
[0008] Therefore, it is necessary to propose a purification method for creatine kinase isoenzyme CKMB, which can be efficiently purified by a specific co-expression and purification method while ensuring the activity of the protein complex in the form of creatine kinase isoenzyme (CKMB). Summary of the invention
[0009] The technical problem to be solved by the present invention is to provide a method for purifying creatine kinase isoenzyme CKMB, which can be efficiently purified by a specific co-expression and purification method while ensuring the activity of the protein complex in the form of creatine kinase isoenzyme (CKMB).
[0010] In order to solve the above technical solution, the technical solution of the present invention is: a method for purifying the creatine kinase isoenzyme CKMB, the specific steps are: S1 co-expression vector construction: the genes of creatine kinase M subunit dimer (CKM) and creatine kinase B subtype (CKB) were placed into the co-expression vector, and His tag and Strep tag were added to the N-terminus or C-terminus of different target genes respectively; S2 conversion expression: introduce the constructed co-expression vector into the host cells and culture them for expression; S3 purification: the cells are further lysed and purified, and the purified eluted sample is dialyzed and stored to obtain creatine kinase isoenzyme CKMB.
[0011] The above technical scheme is adopted, through a specific co-expression and purification method, the genes of creatine kinase M subunit dimer (CKM) and creatine kinase B subtype (CKB) are placed in a co-expression vector, and His tag and Strep tag are added to the N-terminus or C-terminus of different target genes respectively. The requirement is that both tags must be completely exposed on the surface of the complex when the dimer is formed. Under the premise of ensuring the activity of the protein complex in the form of creatine kinase isozyme (CKMB), it is efficiently purified. The acquisition rate and purity of this method are very high.
[0012] Preferably, the co-expression vector in step S1 is an Escherichia coli expression vector.
[0013] Preferably, the gene sequence of the His tag in step S1 is as shown in SEQ NO 1, and the SEQ NO 1 is: HHHHHH; the gene sequence of the Strep tag is as shown in SEQ NO 2, and the SEQ NO 2 is: WSHPQFEK.
[0014] Preferably, the specific steps of step S2 are: S21: The constructed co-expression vector is introduced into the host cells, inoculated into the liquid culture medium containing the corresponding antibiotics, and cultured at 37°C with shaking until the logarithmic growth phase; S22: Add inducer to induce the expression of target protein. After induction, collect cells by centrifugation.
[0015] Preferably, the inducing agent in step S22 is 0.05 M IPTG, and the induction is carried out at 37° C. for 3 h.
[0016] Preferably, the filler purified in step S3 is Ni ion affinity chromatography filler and Streptactin affinity chromatography filler in a ratio of 1 to 2:1.
[0017] Preferably, the specific steps of cell lysis in step S3 are: resuspending the cells with a buffer and adding a protease inhibitor, then disrupting the cells with ultrasonication, and then collecting the supernatant containing the target protein by centrifugation, which is the pretreated sample solution. That is, resuspending the cells with a buffer (the buffer includes 20mM PB, 150mM NaCl, pH 7.4) and adding a protease inhibitor, then using ultrasonication, the power is 400W, ultrasonication for 5s, intervals of 5s, a total of ultrasonication for 20min, to disrupt the cells, then centrifuging, the centrifugation rate is 10000rpm, the centrifugation time is 15min, and the supernatant containing the target protein is collected, which is the pretreated sample solution.
[0018] Preferably, the specific steps of purification in step S3 are: S31 first equilibration: After the filler is placed in the affinity chromatography column and installed, the affinity chromatography column is equilibrated with an equilibration buffer at a flow rate of 2 ml / min and 2 column volumes; S32 Sample loading: The pretreated sample solution is passed through the affinity chromatography column that has been equilibrated for the first time at a flow rate of 0.2-0.5 mL / min; S33 second equilibration: use equilibration buffer to perform second equilibration, collect the effluent, and mark it as the second equilibration effluent, flow rate 2 ml / min, 2 column volumes; S34 elution 1: use elution buffer 1 to flow through the affinity chromatography column and collect eluate 1; S35: The third equilibration: the buffer solution is re-equilibrated for the third time, and the effluent is collected and marked as the third equilibration effluent, with a flow rate of 2 ml / min and 2 column volumes; S36 elution 2: passing elution buffer 2 through the affinity chromatography column; collecting eluate 2; S37 fourth equilibrium: repeat step S35, collect the effluent, mark it as the fourth equilibrium solution, flow rate 2 ml / min, 2 column volumes; S38 Elution Three: Use elution buffer three to flow through the affinity chromatography column, and collect eluate three, which is the target protein, to obtain a purified elution sample.
[0019] Preferably, the equilibration buffer in step S3 is a pH-neutral buffer having a formula of 20 mM PB, 150 mM NaCl, and a pH of 7.4.
[0020] Preferably, the eluent 1 in step S3 is an equilibration buffer containing 1-5 mM D-biotin, the eluent 2 is an equilibration buffer containing 150-300 mM imidazole, and the eluent 3 is an equilibration buffer containing 1-5 mM D-biotin and 150-300 mM imidazole.
[0021] Preferably, the filler purified in step S3 is Ni ion affinity chromatography filler and Streptactin affinity chromatography filler in a ratio of 1:1.
[0022] Compared with the prior art, the present invention has the following beneficial effects: through a specific co-expression and purification method, the protein is introduced when the vector is constructed, so that the activity of the protein complex in the form of creatine kinase isoenzyme (CKMB) is ensured and it is efficiently purified. This method has a high yield and purity. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The present invention is a flow chart of the method for purifying creatine kinase isoenzyme CKMB. DETAILED DESCRIPTION
[0024] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.
[0025] Example: Figure 1 As shown, the purification method of the creatine kinase isoenzyme CKMB comprises the following specific steps: S1 co-expression vector construction: the genes of creatine kinase M subunit dimer (CKM) and creatine kinase B subtype (CKB) are placed in a co-expression vector, and His tags and Strep tags are added to the N-terminus or C-terminus of different target genes respectively; the co-expression vector in step S1 described in this embodiment is an Escherichia coli expression vector; The gene sequence of the His tag in step S1 is shown in SEQ NO 1, and the SEQ NO 1 is: HHHHHH; the gene sequence of the Strep tag is shown in SEQ NO 2, and the SEQ NO 2 is: WSHPQFEK; the His tag and the Strep tag need to be placed at the N-terminus or C-terminus of different target genes, respectively, and the requirement is that both tags need to be completely exposed on the surface of the complex when the dimer is formed; S2 conversion expression: introduce the constructed co-expression vector into the host cells and culture them for expression; The specific steps of step S2 are: S21: The constructed co-expression vector is introduced into the host cells, inoculated into the liquid culture medium containing the corresponding antibiotics, and cultured at 37°C with shaking until the logarithmic growth phase; S22: adding an inducer to induce the expression of the target protein. After the induction is completed, the cells are collected by centrifugation; the inducer in step S22 is 0.05 M IPTG, and the induction is carried out at 37° C. for 3 h; S3 purification: the cells are further lysed and purified, and the purified eluted sample is dialyzed and stored to obtain creatine kinase isoenzyme CKMB.
[0026] The filler purified in step S3 is a Ni ion affinity chromatography filler and a Streptactin affinity chromatography filler in a ratio of 1 to 2:1; the specific steps of cell lysis in step S3 are: resuspending the cells with a buffer and adding a protease inhibitor, then disrupting the cells with ultrasonication, and then collecting the supernatant containing the target protein by centrifugation, which is the pretreated sample solution; that is, resuspending the cells with a buffer (the buffer includes 20mM PB, 150mM NaCl, and the pH is 7.4) and adding a protease inhibitor, then disrupting with ultrasonication at a power of 400W, 5s of ultrasonication, 5s of interval, and a total of 20min of ultrasonication to disrupt the cells, then centrifuging at a centrifugal speed of 10000rpm and a centrifugal time of 15min, and collecting the supernatant containing the target protein, which is the pretreated sample solution; The specific steps of purification in step S3 are: S31 first equilibration: After the filler is placed in the affinity chromatography column and installed, the affinity chromatography column is equilibrated with an equilibration buffer at a flow rate of 2 ml / min and 2 column volumes; S32 Sample loading: The pretreated sample solution is passed through the affinity chromatography column that has been equilibrated for the first time at a flow rate of 0.2-0.5 mL / min; S33 second equilibration: use equilibration buffer to perform second equilibration, collect the effluent, and mark it as the second equilibration effluent, flow rate 2 ml / min, 2 column volumes; S34 elution 1: use elution buffer 1 to flow through the affinity chromatography column and collect eluate 1; S35: The third equilibration: the buffer solution is re-equilibrated for the third time, and the effluent is collected and marked as the third equilibration effluent, with a flow rate of 2 ml / min and 2 column volumes; S36 elution 2: passing elution buffer 2 through the affinity chromatography column; collecting eluate 2; S37 fourth equilibrium: repeat step S35, collect the effluent, mark it as the fourth equilibrium solution, flow rate 2 ml / min, 2 column volumes; S38 Elution 3: Use elution buffer 3 to flow through the affinity chromatography column, collect the eluate 3 which is the target protein, and thus obtain the purified elution sample; The equilibration buffer in step S3 is a pH-neutral buffer having a formula of 20 mM PB, 150 mM NaCl, and a pH of 7.4; In step S3, the first eluent is an equilibration buffer containing 1-5 mM D-biotin, the second eluent is an equilibration buffer containing 150-300 mM imidazole, and the third eluent is an equilibration buffer containing 1-5 mM D-biotin and 150-300 mM imidazole; In this embodiment, the filler purified in step S3 is Ni ion affinity chromatography filler and Streptactin affinity chromatography filler in a ratio of 1:1.
[0027] The purified eluted samples obtained by the purification method of creatine kinase isoenzyme CKMB were respectively subjected to recombinant CKMM detection, recombinant CKBB detection and CKMB detection, and the detection results are shown in Table 1.
[0028] Table 1 Results of recombinant CKMM, recombinant CKBB and CKMB assays
[0029] As can be seen from Table 1, the CKMB detection reagent was used to detect the three elution products. When detecting recombinant CKMB, the concentration and the reaction value were well positively correlated, and the reaction value was very strong; while when detecting recombinant CKMM and CKBB, the reaction value was very weak, and the high concentration antigen detection value was not significantly different from the background. This means that the target protein CKMB was separated from the recombinant protein mixture by this method.
[0030] For ordinary technicians in this field, the specific embodiments are only illustrative descriptions of the present invention. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned methods. As long as various non-substantial improvements are made using the method concepts and technical solutions of the present invention, or the concepts and technical solutions of the present invention are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.
[0031] Gene sequence list SEQ NO 1: HHHHHH.
[0032] SEQ NO 2: WSHPQFEK.
Claims
1. A method for purifying creatine kinase isoenzyme CKMB, characterized in that: The specific steps are: S1 co-expression vector construction: the genes of creatine kinase M subunit dimer and creatine kinase B isoform were placed into the co-expression vector, and His tag and Strep tag were added to the N-terminus or C-terminus of different target genes respectively; S2 conversion expression: introduce the constructed co-expression vector into the host cells and culture them for expression; S3 purification: the cells are further lysed and purified, and the purified eluted sample is dialyzed and stored to obtain creatine kinase isoenzyme CKMB.
2. The method for purifying creatine kinase isoenzyme CKMB according to claim 1, characterized in that: The co-expression vector in step S1 is an Escherichia coli expression vector.
3. The method for purifying creatine kinase isoenzyme CKMB according to claim 1, characterized in that: The gene sequence of the His tag in the step S1 is shown as SEQ NO 1, and the SEQ NO 1 is: HHHHHH; the gene sequence of the Strep tag is shown as SEQ NO 2, and the SEQ NO 2 is: WSHPQFEK.
4. The method for purifying creatine kinase isoenzyme CKMB according to claim 2, characterized in that: The specific steps of step S2 are: S21: The constructed co-expression vector is introduced into the host cells, inoculated into the liquid culture medium containing the corresponding antibiotics, and cultured at 37°C with shaking until the logarithmic growth phase; S22: Add inducer to induce the expression of target protein. After induction, collect cells by centrifugation.
5. The method for purifying creatine kinase isoenzyme CKMB according to claim 4, characterized in that: The filler purified in step S3 is Ni ion affinity chromatography filler and Streptactin affinity chromatography filler in a ratio of 1 to 2:
1.
6. The method for purifying creatine kinase isoenzyme CKMB according to claim 5, characterized in that: The induction agent in step S22 is 0.05 M IPTG, and the induction is carried out at 37° C. for 3 h.
7. The method for purifying creatine kinase isoenzyme CKMB according to claim 5, characterized in that: The specific steps of cell lysis in step S3 are: resuspending the cells with a buffer and adding a protease inhibitor, then disrupting the cells with ultrasonication, and then centrifuging to collect the supernatant containing the target protein, which is the pretreated sample solution.
8. The method for purifying creatine kinase isoenzyme CKMB according to claim 7, characterized in that: The specific steps of purification in step S3 are: S31 first equilibration: After the filler is placed in the affinity chromatography column and installed, the affinity chromatography column is equilibrated with an equilibration buffer at a flow rate of 2 ml / min and 2 column volumes; S32 Sample loading: The pretreated sample solution is passed through the affinity chromatography column that has been equilibrated for the first time at a flow rate of 0.2-0.5 mL / min; S33 second equilibration: use equilibration buffer to perform second equilibration, collect the effluent, and mark it as the second equilibration effluent, flow rate 2 ml / min, 2 column volumes; S34 elution 1: use elution buffer 1 to flow through the affinity chromatography column and collect eluate 1; S35: The third equilibration: the buffer solution is re-equilibrated for the third time, and the effluent is collected and marked as the third equilibration effluent, with a flow rate of 2 ml / min and 2 column volumes; S36 elution 2: passing elution buffer 2 through the affinity chromatography column; collecting eluate 2; S37 fourth equilibrium: repeat step S35, collect the effluent, mark it as the fourth equilibrium solution, flow rate 2 ml / min, 2 column volumes; S38 Elution Three: Use elution buffer three to flow through the affinity chromatography column, and collect eluate three, which is the target protein, to obtain a purified elution sample.
9. The method for purifying creatine kinase isoenzyme CKMB according to claim 7, characterized in that: The equilibration buffer in step S3 is a pH-neutral buffer having a formula of 20 mM PB, 150 mM NaCl, and a pH of 7.4; the first eluent is an equilibration buffer containing 1 to 5 mM D-biotin, the second eluent is an equilibration buffer containing 150 to 300 mM imidazole, and the third eluent is an equilibration buffer containing 1 to 5 mM D-biotin and 150 to 300 mM imidazole.
10. The method for purifying creatine kinase isoenzyme CKMB according to claim 5, characterized in that: The filler purified in step S3 is a Ni ion affinity chromatography filler and a Streptactin affinity chromatography filler in a ratio of 1:1.