CN117024606A - Purification method of human mitochondrial respiratory chain super-complex CICIII2CIV - Google Patents

Purification method of human mitochondrial respiratory chain super-complex CICIII2CIV Download PDF

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CN117024606A
CN117024606A CN202311132534.5A CN202311132534A CN117024606A CN 117024606 A CN117024606 A CN 117024606A CN 202311132534 A CN202311132534 A CN 202311132534A CN 117024606 A CN117024606 A CN 117024606A
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贡红日
杜占强
饶子和
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Nankai University
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Abstract

The invention provides a human mitochondrial respiratory chain super-compound CICIII 2 A purification method of CIV belongs to the technical field of protein purification. The invention provides a human mitochondrial total protein solution, which comprises a human mitochondrial respiratory chain super complex CICICIII assembled by human mitochondrial respiratory chain complexes I, III and IV 2 CIV, the C-terminal of NDUFS3 subunit of the humanized mitochondrial respiratory chain complex I is fused with a 3 xFlag labelSigning; then sequentially performing Flag tag affinity column chromatography purification and gel exclusion column chromatography purification on the human mitochondrial total protein solution to obtain a human mitochondrial respiratory chain super-complex CICIII 2 CIV pure product. The purification method provided by the invention can obtain the human mitochondrial respiratory chain super-compound CICIII with high purity, high yield and good physiological activity 2 CIV。

Description

Purification method of human mitochondrial respiratory chain super-complex CICIII2CIV
Technical Field
The invention belongs to the technical field of protein purification, and in particular relates to a method for purifying proteinHuman mitochondrial respiratory chain super complex CICIII 2 Method for purifying CIV.
Background
The mitochondrial oxidative phosphorylation (Oxidative phosphorylation, OXPHOS) system consists of mitochondrial respiratory chain complex I, II, III, IV (CI, CII, CIII, CIV) and ATP synthase, providing the energy substance ATP for various activities of the living body. The mitochondrial respiratory chain complexes I, II, III and IV can not only function independently, but also can be assembled into mitochondrial respiratory chain super complexes to cooperatively complete the electron transfer process. The mitochondrial respiratory chain super-complex is found to be involved in a number of physiological processes, including regulation of ROS production and acceleration of oxidative phosphorylation processes in the body. Mutation of the constituent subunits of the human mitochondrial respiratory chain results in impaired activity and thus many clinically relevant diseases, including metabolic-related diseases, tumors, etc. The research on the structural function of the human mitochondrial respiratory chain super complex is of great significance for understanding the assembly mechanism, the catalytic mechanism, the mutation pathogenic mechanism, the drug discovery and the like of the mitochondrial respiratory chain super complex.
At present, the density gradient centrifugation and gel exclusion chromatography are mainly used for purifying the human mitochondrial respiratory chain super complex, and the method belongs to endogenous extraction, but has the defects of higher requirements on equipment, more complex purification process and low yield of the obtained protein. Therefore, a simple and efficient purification method of the human mitochondrial respiratory chain super complex is developed, which has important significance for subsequent research of the structural function and drug discovery.
Disclosure of Invention
The invention aims to provide a purification method of a human mitochondrial respiratory chain super-compound, which can obtain the human mitochondrial respiratory chain super-compound CICIII with high purity, high yield and good physiological activity 2 CIV。
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a human mitochondrial respiratory chain super-compound CICIII 2 A method of purifying CIV comprising the steps of:
providing a human mitochondrial total protein solution comprising a super complex CICIII 2 CIV, the humanized mitochondrial respiratory chain super complex CICIII 2 CIV is assembled from a humanized mitochondrial respiratory chain complex I, III and IV, wherein the NDUFS3 subunit C-terminus of the humanized mitochondrial respiratory chain complex I is fused with a 3 xflag tag;
performing Flag tag affinity column chromatography purification on the humanized mitochondrial total protein solution to obtain a humanized mitochondrial respiratory chain super-complex CICIII 2 Crude CIV extract; the chromatographic column used for the Flag tag affinity column chromatographic purification is a chromatographic column filled with Anti-Flag filler;
the human mitochondrial respiratory chain super complex CICIII 2 Purifying the CIV crude extract by gel exclusion column chromatography to obtain human mitochondrial respiratory chain super complex CICIII 2 CIV pure product.
Preferably, the chromatographic column used for the chromatographic purification of the gel exclusion column is a Superose6in 10/300 gel exclusion chromatographic column.
Preferably, the Flag-tag affinity column chromatography purification preferably comprises a first elution and a second elution performed sequentially; the first eluent used for the first elution comprises 3-morpholinopropane sulfonic acid, naCl and a first detergent; the second eluent used for the second elution comprises 3-morpholinopropane sulfonic acid, naCl, a second detergent and 1×Flagpeptides.
Preferably, in the first eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20-30 mmol/L, the molar concentration of the NaCl is 50-100 mmol/L, and the mass percent of the first detergent is 0.004% or 0.1%; the first detergent is lauryl maltose neopentyl glycol and/or digitalis saponin.
Preferably, in the second eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20-30 mmol/L, the molar concentration of the NaCl is 50-100 mmol/L, the mass percentage of the second detergent is 0.004% or 0.1%, and the mass concentration of the 1 xFlag peptide is 200-300 mug/mL; the second detergent is lauryl maltose neopentyl glycol and/or digitalis saponin.
Preferably, the third eluent used for the gel exclusion column chromatographic purification comprises 3-morpholinopropane sulfonic acid, naCl and a third detergent; in the third eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20-30 mmol/L, the molar concentration of the NaCl is 50-100 mmol/L, and the mass percentage of the third detergent is 0.004% or 0.1%; the third eluent is lauryl maltose neopentyl glycol and/or digitalis saponin.
Preferably, the preparation method of the human mitochondrial total protein solution comprises the following steps:
a pQCIP plasmid is adopted to establish a humanized cell-NDUFS 3-3 xFlag over-expression stable transfer cell line;
mixing the humanized cell-NDUFS 3-3 xFlag over-expression stable transfer cell line with a first buffer solution, grinding, and carrying out solid-liquid separation to obtain a humanized mitochondrial crude extract;
and mixing the crude human mitochondrial extract with a second buffer solution, and performing mitochondrial membrane dissolution to obtain a human mitochondrial total protein solution.
Preferably, the first buffer solution comprises sucrose, 3-morpholinopropane sulfonic acid and ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid; in the first buffer solution, the molar concentration of sucrose is 250mmol/L, the molar concentration of 3-morpholinopropane sulfonic acid is 20-30 mmol/L, and the molar concentration of ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid is 1mmol/L.
Preferably, the second buffer solution comprises 3-morpholinopropane sulfonic acid, naCl, and a fourth detergent; in the second buffer solution, the molar concentration of the 3-morpholinopropane sulfonic acid is 20-30 mmol/L, the molar concentration of NaCl is 50-100 mmol/L, and the mass percentage of the fourth detergent is 1%; the fourth detergent comprises lauryl maltose neopentyl glycol and/or digitalis saponin.
Preferably, the method for establishing the humanized cell-NDUFS 3-3 xFlag over-expression stable transgenic cell line comprises the following steps:
fusing 3 xFlag tags at the C end of NDUFS3 subunit of the humanized mitochondrial respiratory chain complex I by adopting a molecular cloning method to construct pQCIP-NDUFS 3-3 xFlag plasmid;
chemically transfecting the humanized cells by using the pQCTIP-NDUFS 3-3 xFlag plasmid through a transfection reagent to obtain a humanized cell fluid transfected with the pQCTIP-NDUFS 3-3 xFlag plasmid;
and mixing the human cell sap transfected with pQCIP-NDUFS 3-3 xFlag plasmid with puromycin for cell screening to obtain a human cell-NDUFS 3-3 xFlag over-expression stable transfer cell line.
The invention provides a human mitochondrial respiratory chain super-compound CICIII 2 A method of purifying CIV comprising the steps of: providing a human mitochondrial total protein solution comprising human mitochondrial respiratory chain super complex CICIII 2 CIV, the humanized mitochondrial respiratory chain super complex CICIII 2 CIV is assembled from a humanized mitochondrial respiratory chain complex I, III and IV, wherein the NDUFS3 subunit C-terminus of the humanized mitochondrial respiratory chain complex I is fused with a 3 xflag tag; performing Flag tag affinity column chromatography purification on the humanized mitochondrial total protein solution to obtain a humanized mitochondrial respiratory chain super-complex CICIII 2 Crude CIV extract; the human mitochondrial respiratory chain super complex CICIII 2 Purifying the CIV crude extract by gel exclusion column chromatography to obtain human mitochondrial respiratory chain super complex CICIII 2 CIV pure product. The purification method provided by the invention uses the 3 xFlag tag at the C end of the NDUFS3 subunit of the human mitochondrial respiratory chain complex I, adopts a chromatographic column filled with Anti-Flag filler to carry out affinity chromatography purification, and uses the Anti-Flag filler to specifically identify the 3 xFlag tag, thereby effectively obtaining the super-complex CICICIII rich in human mitochondrial respiratory chain 2 The crude extract of CIV is then subjected to gel exclusion chromatography to obtain purified human mitochondrial respiratory chain super complex CICIII 2 CIV. The purification method provided by the invention has the advantages of simple operation flow and low experimental cost, and can obtain the humanized mitochondrial respiratory chain super-compound CICIII with high purity, high yield and good physiological activity 2 CIV is convenient for subsequent structural function and drug discovery research.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a graph of the elution results of a gel exclusion chromatographic column, the abscissa in FIG. 1 being the elution volume of the chromatographic column in mL; the left ordinate is 280nm ultraviolet absorption, and the unit is mAU;
FIG. 2 is a diagram showing the result of SDS-PAGE electrophoresis using gel exclusion chromatography column; the left side of the figure is Marker, and the right side of the figure is sample component identification;
FIG. 3 is a graph of the Western Blotting identification result of gel exclusion chromatography column sampling; in FIG. 3, the left is Marker, and the right is identification of CI subunit NDUFA8, CII subunit SDHA, CIII subunit UQCRC1, and CIV subunit COXIV in the sample components;
FIG. 4 shows gel exclusion chromatography column sampling BN-PAGE and NBT (Nitrotetrazolium blue chloride) staining signatures; in FIG. 4, the left is Marker, and the right is NBT staining identification of CI, CII and CIV in sample BN-PAGE identification and sample fractions.
Detailed Description
The invention provides a human mitochondrial respiratory chain super-compound CICIII 2 A method of purifying CIV comprising the steps of:
providing a human mitochondrial total protein solution comprising human mitochondrial respiratory chain super complex CICIII 2 CIV, the humanized mitochondrial respiratory chain super complex CICIII 2 CIV is assembled from a humanized mitochondrial respiratory chain complex I, III and IV, wherein the NDUFS3 subunit C-terminus of the humanized mitochondrial respiratory chain complex I is fused with a 3 xflag tag;
performing Flag tag affinity column chromatography purification on the humanized mitochondrial total protein solution to obtain a humanized mitochondrial respiratory chain super-complex CICIII 2 Crude CIV extract; the chromatographic column used for the Flag tag affinity column chromatographic purification is a chromatographic column filled with Anti-Flag filler;
the human mitochondrial respiratory chain super complex CICIII 2 Purifying the CIV crude extract by gel exclusion column chromatography to obtain human mitochondrial respiratory chain super complex CICIII 2 CIV pure product.
In the present invention, the raw materials used are commercially available products well known to those skilled in the art unless otherwise specified.
The invention provides a human mitochondrial total protein solution, which comprises human mitochondrial respiratory chain super-complex CICIII 2 CIV, the humanized mitochondrial respiratory chain super complex CICIII 2 CIV is assembled from a humanized mitochondrial respiratory chain complex I, III and IV, fused at the C-terminus to the NDUFS3 subunit of the humanized mitochondrial respiratory chain complex I with a 3 xflag tag. In the present invention, the preparation method of the human mitochondrial total protein solution preferably comprises the following steps: a pQCIP plasmid is adopted to establish a humanized cell-NDUFS 3-3 xFlag over-expression stable transfer cell line; mixing the humanized cell-NDUFS 3-3 xFlag over-expression stable transfer cell line with a first buffer solution, grinding, and carrying out solid-liquid separation to obtain a humanized mitochondrial crude extract; and mixing the crude human mitochondrial extract with a second buffer solution, and performing mitochondrial membrane dissolution to obtain a human mitochondrial total protein solution.
In the invention, the method for establishing the humanized cell-NDUFS 3-3 xFlag over-expression stable transgenic cell line comprises the following steps: fusing 3 xFlag tags at the C end of NDUFS3 subunit of the humanized mitochondrial respiratory chain complex I by adopting a molecular cloning method to construct pQCIP-NDUFS 3-3 xFlag plasmid; chemically transfecting the humanized cells by using the pQCTIP-NDUFS 3-3 xFlag plasmid through a transfection reagent to obtain a humanized cell fluid transfected with the pQCTIP-NDUFS 3-3 xFlag plasmid; and mixing the human cell sap transfected with pQCIP-NDUFS 3-3 xFlag plasmid with puromycin for cell screening to obtain a human cell-NDUFS 3-3 xFlag over-expression stable transfer cell line.
The invention adopts a molecular cloning method to fuse 3 xFlag label at the C end of NDUFS3 subunit of the humanized mitochondrial respiratory chain complex I to construct pQCIP-NDUFS 3-3 xFlag plasmid. The present invention has no special requirement on the molecular cloning method, and the molecular cloning method well known to the person skilled in the art can be adopted.
After the pQCTIP-NDUFS 3-3 xFlag plasmid is obtained, the invention adopts the pQCTIP-NDUFS 3-3 xFlag plasmid to chemically transfect human cells through a transfection reagentHuman cell sap transfected with pQCXIP-NDUFS3-3 xFlag plasmid was obtained. In the present invention, the transfection reagent is preferably PEI chemical reagent. In the present invention, the human-derived cell is particularly preferably a HEK293F cell. In the present invention, the mass ratio of the PEI chemical reagent to the pQCIP-NDUFS 3-3 xFlag plasmid at the time of the chemical transfection is preferably 4:1. in the present invention, the cell density of the human cells at the time of the chemical transfection is preferably 0.5X10 6 ～2.0×10 6 And more preferably 0.5X10 g/mL 6 And each mL. In the present invention, the time for the chemical transfection is preferably 12 to 48 hours, more preferably 48 hours. The specific implementation process of the chemical transfection is not particularly required, and the chemical transfection method well known to the person skilled in the art can be adopted.
After obtaining the human cell sap transfected with pQCTIS-NDUFS 3-3 xFlag plasmid, the invention mixes the human cell sap transfected with pQCTIS-NDUFS 3-3 xFlag plasmid with puromycin for cell screening to obtain the human cell-NDUFS 3-3 xFlag over-expression stable transfer cell line. In the present invention, the mass concentration of puromycin in the mixed solution obtained by mixing the puromycin with the human cell sap transfected with pQCIP-NDUFS 3-3 XFlag plasmid is preferably 1 to 10. Mu.g/mL, more preferably 5. Mu.g/mL. In the present invention, the time for cell selection is preferably 2 to 4 weeks, more preferably 4 weeks.
After obtaining a human cell-NDUFS 3-3 xFlag over-expression stable transfer cell line, the invention mixes the human cell-NDUFS 3-3 xFlag over-expression stable transfer cell line with a first buffer solution, grinds, and performs solid-liquid separation to obtain a crude extract of human mitochondria.
The present invention preferably uses Phosphate Buffer (PBS) to wash the human cells-NDUFS 3-3 xFlag over-expressed stably transformed cells before the milling, and then collects the cell pellet.
In the present invention, the cell density of the human cell-NDUFS 3-3 xFlag overexpressing stably transformed cells is preferably 4.0X10 6 ～6.0×10 6 And more preferably 6.0X10 g/mL 6 individual/mL; the volume of the humanized cell-NDUFS 3-3 xFlag over-expression stable transfer cell is preferably 0.5-1L, more preferably 1L.In the present invention, the first buffer solution preferably includes sucrose, 3-morpholinopropane sulfonic acid and ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid; the molar concentration of the sucrose in the first buffer solution is preferably 250mmol/L; the molar concentration of the 3-morpholinopropane sulfonic acid is preferably 20-30 mmol/L, more preferably 20mmol/L; the molar concentration of the ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid is preferably 1mmol/L. The pH of the first buffer solution according to the invention is preferably 7.4. In the invention, the first buffer solution provides proper osmotic pressure, and the cells can be lysed after the humanized cell-NDUFS 3-3 xFlag over-expressed stable transfer cells are mixed and ground with the first buffer solution.
In the present invention, the grinding preferably includes the steps of: mixing and grinding the humanized cell-NDUFS 3-3 xFlag over-expression stable transfer cells and part of the first buffer solution, and then performing first solid-liquid separation to obtain a first supernatant and a first precipitate; performing second solid-liquid separation on the first supernatant to obtain a second supernatant and a second precipitate; the second precipitate is crude extract of human mitochondria. In the present invention, the grinding is preferably performed in a glass tissue homogenizer, and the number of times of each grinding is preferably 15 to 20. In the grinding process, the volume of the first buffer solution is preferably 4-6 times of the volume of the human cell-NDUFS 3-3 xFlag over-expressed stable transfer cell, and more preferably 6 times of the volume of the human cell-NDUFS 3-3 xFlag over-expressed stable transfer cell. In the present invention, the first solid-liquid separation is preferably centrifugation; the rotational speed of the centrifugation is preferably 1000g; the time is preferably 10 to 15 minutes, more preferably 10 minutes, and the temperature is preferably 4 ℃. In the present invention, the second solid-liquid separation means is preferably centrifugation; the rotational speed of the centrifugation is preferably 10000 to 12000g, more preferably 10000g; the time is preferably 10 to 15 minutes, more preferably 15 minutes; the temperature is preferably 4 ℃. The invention can obtain the coarse mitochondrial extract precipitate through twice solid-liquid separation.
After obtaining the crude extract of human mitochondria, the invention mixes the crude extract of human mitochondria with a second buffer solution to carry out mitochondrial membrane dissolution, thus obtaining the total protein solution of human mitochondria.
In the present invention, the second buffer solution preferably includes 3-morpholinopropane sulfonic acid, naCl and a fourth detergent; the molar concentration of the 3-morpholinopropane sulfonic acid in the second buffer solution is preferably 20-30 mmol/L, more preferably 20mmol/L; the molar concentration of NaCl is preferably 50-100 mmol/L, more preferably 50mmol/L; the mass percentage of the fourth detergent is preferably 1%; the fourth detergent preferably comprises lauryl maltoneopentyl glycol and/or digitalis saponin, more preferably lauryl maltoneopentyl glycol or digitalis saponin. The pH of the second buffer solution according to the invention is preferably 7.4. In an embodiment of the present invention, specifically, the second buffer solution is used to precipitate crude human mitochondrial extract to 30mL. In the present invention, the time for dissolving the mitochondrial membrane is preferably 40 to 60 minutes. After the mitochondria are dissolved in the membrane, the invention preferably carries out third solid-liquid separation to obtain the human-source mitochondrial total protein solution. In the present invention, the third solid-liquid separation means is preferably centrifugation; the rotational speed of the centrifugation is preferably 40000g; the time is preferably 40 to 60 minutes, more preferably 40 minutes; the temperature is preferably 4 ℃. In the invention, the second buffer solution is used for dissolving the mitochondria membrane to obtain a human mitochondrial total protein solution.
After obtaining a human mitochondrial total protein solution, the invention carries out Flag tag affinity column chromatography purification on the human mitochondrial total protein solution to obtain a human mitochondrial respiratory chain super-complex CICIII 2 CIV crude extract. In the present invention, the column used for the Flag-tag affinity column chromatography purification is preferably a column packed with Anti-Flag packing. Before performing Flag-tag affinity column chromatography purification, the Anti-Flag packing is preferably loaded into a gravity column, and balanced by adopting a first eluent, so as to obtain the chromatographic column filled with the Anti-Flag packing. In the present invention, at said equilibrium, the volume of the first eluent is preferably 2 times the volume of the chromatographic column packed with Anti-Flag packing. The Flag-tag affinity column chromatography purification according to the present invention preferably includes a first elution and a second elution performed sequentially. The pH value of the first eluent used for the first elution is preferably 7.4; the first eluent preferably comprises 3-morpholinopropane sulfonic acid, naCl and a first eluentA detergent; the molar concentration of the 3-morpholinopropane sulfonic acid is 20-30 mmol/L, more preferably 20mmol/L; the molar concentration of NaCl is preferably 50-100 mmol/L, more preferably 50mmol/L; the mass percentage of the first detergent is preferably 0.004% or 0.1%; the first detergent is preferably lauryl maltoneopentyl glycol and/or digitalis saponin, more preferably lauryl maltoneopentyl glycol or digitalis saponin. In the present invention, the first elution serves to remove impurities. In the present invention, when the first detergent is lauryl maltose neopentyl glycol, the mass percentage of the first detergent is 0.004%; when the first detergent is digitonin, the mass percentage of the first detergent is 0.1%.
In the present invention, the second elution is preferably a competitive elution; the pH of the second eluent used for the second elution is preferably 7.4; the second eluent preferably comprises 3-morpholinopropane sulfonic acid, naCl, a second detergent and 1×flag peptide; the molar concentration of the 3-morpholinopropane sulfonic acid is preferably 20-30 mmol/L, more preferably 20mmol/L; the molar concentration of NaCl is preferably 50-100 mmol/L, more preferably 50mmol/L; the mass percentage of the second detergent is preferably 0.004% or 0.1%; the second detergent is lauryl maltoneopentyl glycol and/or digitalis saponin, more preferably lauryl maltoneopentyl glycol or digitalis saponin. In the present invention, when the second detergent is lauryl maltose neopentyl glycol, the mass percentage of the second detergent is 0.004%; when the second detergent is digitonin, the mass percentage of the second detergent is 0.1%. In the present invention, the mass concentration of the 1 XFlag peptide is preferably 200 to 300. Mu.g/mL, more preferably 200. Mu.g/mL. The amino acid sequence of the 1 xFlag peptide is shown as SEQ ID NO. 1; the SEQ ID NO.1 is: dykdddk. In the invention, the second elution is used for competitive elution to obtain the human mitochondrial respiratory chain super complex CICIII 2 CIV crude extract.
In the invention, the Flag-tag affinity column chromatography is further used for carrying out centrifugal concentration on the eluent obtained by the second elution by adopting a concentration tube with the molecular weight cut-off of 100 Kda. The invention has no special requirements on the specific operation of the centrifugal concentration, and the operation modes which are well known to the person skilled in the art can be adopted. In the examples of the present invention, the eluate obtained by the second elution was concentrated to 500. Mu.L or less using a 100kDa cut-off concentration tube.
Obtaining the human mitochondrial respiratory chain super complex CICIII 2 After the CIV crude extract, the invention leads the humanized mitochondrial respiratory chain super complex CICIII 2 Purifying the CIV crude extract by gel exclusion column chromatography to obtain human mitochondrial respiratory chain super complex CICIII 2 CIV pure product. In the present invention, the column used for the gel exclusion column chromatography purification is preferably a Superose6in 10/300 gel exclusion column. The present invention preferably balances the gel exclusion chromatography column with a third eluent prior to gel exclusion chromatography purification. In the present invention, the volume of the third eluent at the equilibrium is preferably 1 time the volume of the Superose6in 10/300 gel exclusion chromatography column. The gel exclusion column chromatographic purification according to the invention preferably comprises a third elution. In the present invention, the pH of the third eluent used in the third elution is preferably 7.4; the third eluent used for the third elution preferably comprises 3-morpholinopropane sulfonic acid, naCl and a third detergent; the molar concentration of the 3-morpholinopropane sulfonic acid is preferably 20-30 mmol/L, more preferably 20mmol/L; the molar concentration of NaCl is preferably 50-100 mmol/L, more preferably 50mmol/L; the mass percentage of the third detergent is preferably 0.004% or 0.1%; the third eluent is preferably lauryl maltose neopentyl glycol and/or digitalis saponin, more preferably lauryl maltose neopentyl glycol or digitalis saponin. In the present invention, when the third detergent is lauryl maltose neopentyl glycol, the mass percentage of the third detergent is 0.004%; when the third detergent is digitonin, the mass percentage of the third detergent is 0.1%. In the present invention, the third elution is used for obtaining the human mitochondrial respiratory chain super complex CICIII 2 CIV pure product.
The invention uses HEK293F-NDUFS33 xFlag over-expression stable transformation cell line as experimental material, adopts the mode of combining affinity chromatography purification and gel exclusion chromatography by adopting Anti-Flag filler, has the advantages of simple experimental steps, short time consumption, easy standardization operation and the like, and has low requirements on experimental equipment and greatly reduces time cost and capital cost compared with the current common density gradient centrifugation method and anion-cation exchange column purification.
The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings.
In the embodiment of the invention, a chromatographic column used for the Flag tag Affinity column chromatography purification is a commercial commodity, and commodity information is Anti-DYKDDDDK G1 Affinity Resin (Kirschner Biotech Co., ltd.); the column used for the gel exclusion chromatography purification is commercially available and the commercial information is Superose6in 10/300 (GE Healthcare).
Example 1
With ddH 2 And O is a solvent to prepare a first eluent, a second eluent, a third eluent, a first buffer solution and a second buffer solution:
first eluent (ph=7.4): 20 mmol/L3-morpholinopropane sulfonic acid, 50mmol/LNaCl, digitalis saponin with mass percent of 0.1%;
a second eluent (ph=7.4); 20 mmol/L3-morpholinopropane sulfonic acid, 50mmol/LNaCl, digitonin with mass percent of 0.02%, 200 mug/mL 1 XFlag peptide;
a third eluent (ph=7.4); 20 mmol/L3-morpholinopropane sulfonic acid, 50mmol/LNaCl, digitalis saponin with mass percent of 0.1%;
first buffer solution (ph=7.4); 25mmol/L sucrose, 20 mmol/L3-morpholinopropane sulfonic acid, 1mmol/L ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid;
second buffer solution (ph=7.4): 20 mmol/L3-morpholinopropane sulfonic acid, 50mmol/LNaCl, digitonin with a mass percentage of 1%.
Fusing 3 xFlag tags at the C end of NDUFS3 subunit of the humanized mitochondrial respiratory chain complex I by adopting a molecular cloning method to construct pQCIP-NDUFS 3-3 xFlag plasmid; passing the constructed pQCIP-NDUFS 3-3 xFlag plasmid through PEI chemical reagentHEK293F cells were transfected with PEI chemistry and pQCIRP-NDUFS 3-3 XFlag plasmid in a mass ratio of 4:1, HEK293F cell density at transfection was 0.5X10 6 And (3) adding puromycin with the concentration of 5 mug/mL into the obtained human cell sap transfected with pQCIP-NDUFS 3-3 xFlag plasmid for 4 weeks after 48h of transfection, and obtaining a human HEK293F-NDUFS33 xFlag over-expression stable transgenic cell line.
Culturing 1L of humanized HEK293F-NDUFS33 xFlag over-expressed stable cell until the cell density is 6.0x10 6 The harvested cell volume was about 30mL per mL. Adding 100mL of phosphate buffer to resuspend the cells, and centrifugally collecting cell sediment; adding a first buffer solution into the cell sediment to resuspend cells, grinding the cells for 20 times up and down by using a glass tissue homogenizer, adding the first buffer solution until the total volume is 200mL, and centrifuging 1000g for 10min at 4 ℃ to obtain a supernatant; centrifuging 10000g of the obtained supernatant at 4deg.C for 15min, and collecting precipitate to obtain crude extract of human mitochondria;
the crude human mitochondrial extract was resuspended to 30mL with a second buffer solution and mitochondrial membrane dissolution was performed for 60min. After the mitochondrial membrane is dissolved, 40000g is centrifugated for 40min at the temperature of 4 ℃, and supernatant fluid is collected to obtain a humanized mitochondrial total protein solution;
loading 5mL of Anti-Flag filler into a gravity column, balancing 2 column volumes by adopting a first eluent, loading the human mitochondrial total protein solution into a chromatographic column filled with the Anti-Flag filler, and allowing the chromatographic column to flow through the hanging column for 5 times; the first eluent is adopted to carry out the first elution, the second eluent is adopted to carry out the second elution, the eluent obtained by the second elution is collected, and the eluent obtained by the second elution is concentrated to below 500 mu L by a concentration tube with the molecular weight cut off of 100kDa, thus obtaining the human mitochondrial respiratory chain super compound CICIII 2 Crude CIV extract;
the Superose6increase10/300 gel exclusion chromatography column was equilibrated with a third eluent of 1 column volume (25 mL), after which the human mitochondrial respiratory chain super complex CICIII obtained in the previous step was purified 2 The CIV crude extract is loaded and eluted by a third eluent, the result is shown in figure 1, and the abscissa in the figure is the elution volume of the chromatographic column, and the unit is mL; ultraviolet absorption in 280nm on the ordinatemAU. As can be seen from FIG. 1, the humanized mitochondrial respiratory chain super complex CICIII 2 CIV shows a characteristic peak at about 11.49 mL.
Collecting a sample at the characteristic peak position to obtain a human mitochondrial respiratory chain super complex CICIII 2 CIV pure; the obtained human mitochondrial respiratory chain super complex CICIII 2 CIV pure products are subjected to SDS-PAGE electrophoresis, detection is shown in figure 2, and a characteristic peak position sample comprises mitochondrial respiratory chain complex I, III and IV components through mass spectrum identification.
WesternBlotting identification was performed on the characteristic peak position samples, and the results are shown in FIG. 3, which further indicate that the characteristic peak position sample components contain human mitochondrial respiratory chain complex I, III and IV components, and no complex II component exists.
According to reference "Structural basis ofmitochondrial membrane bending by the I-II-III 2 –IV 2 Supercomplex "(Muhleip A, flygaard R K, baradaran R, et al Nature,2023,615 (7954):934-938) the samples at the characteristic peak positions of example 1 were subjected to mitochondrial respiratory chain complex activity testing, i.e.NBT staining, and the results are shown in FIG. 4, further indicating the presence of complex I and complex IV components and the absence of complex II components in the resulting purified samples. The results of FIG. 3 are combined to demonstrate that the humanized mitochondrial respiratory chain super complex CICIII purified according to the example of the invention 2 CIV contains human mitochondrial respiratory chain complexes I, III and IV and has good physiological activity.
Compared with a density gradient centrifugal extraction method, the purification method provided by the invention has the characteristics of simple operation, low experimental cost, long time and the like, and the obtained humanized mitochondrial respiratory chain super-compound CICIII 2 The CIV protein has high purity and good physiological activity, and can be used for subsequent structural function and drug discovery research.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, according to which one can obtain other embodiments without inventiveness, these embodiments are all within the scope of the invention.

Claims (10)

1. Human mitochondrial respiratory chain super-complex CICIII 2 A method of purifying CIV comprising the steps of:
providing a human mitochondrial total protein solution comprising human mitochondrial respiratory chain super complex CICIII 2 CIV, the humanized mitochondrial respiratory chain super complex CICIII 2 CIV is assembled from a humanized mitochondrial respiratory chain complex I, III and IV, wherein the NDUFS3 subunit C-terminus of the humanized mitochondrial respiratory chain complex I is fused with a 3 xflag tag;
performing Flag tag affinity column chromatography purification on the humanized mitochondrial total protein solution to obtain a humanized mitochondrial respiratory chain super-complex CICIII 2 Crude CIV extract; the chromatographic column used for the Flag tag affinity column chromatography purification is a chromatographic column filled with Anti-Flag filler;
the human mitochondrial respiratory chain super complex CICIII 2 Purifying the CIV crude extract by gel exclusion column chromatography to obtain human mitochondrial respiratory chain super complex CICIII 2 CIV pure product.
2. The method according to claim 1, wherein the column used for the gel exclusion column chromatography purification is a Superose6increase10/300 gel exclusion column.
3. The purification method according to claim 1, wherein the Flag-tag affinity column chromatography purification comprises sequentially performing a first elution and a second elution; the first eluent used for the first elution comprises 3-morpholinopropane sulfonic acid, naCl and a first detergent; the second eluent used for the second elution comprises 3-morpholinopropane sulfonic acid, naCl, a second detergent and 1×Flagpeptides.
4. A purification method according to claim 3, wherein in the first eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20-30 mmol/L, the molar concentration of the NaCl is 50-100 mmol/L, and the mass percentage of the first detergent is 0.004% or 0.1%; the first detergent is lauryl maltose neopentyl glycol and/or digitalis saponin.
5. A purification method according to claim 3, wherein in the second eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20-30 mmol/L, the molar concentration of the NaCl is 50-100 mmol/L, the mass percentage of the second detergent is 0.004% or 0.1%, and the mass concentration of the 1 xflagpeptide is 200-300 μg/mL; the second detergent is lauryl maltose neopentyl glycol and/or digitalis saponin.
6. The method of claim 1, wherein the third eluent used for the gel exclusion column chromatography purification comprises 3-morpholinopropane sulfonic acid, naCl, and a third detergent; in the third eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20-30 mmol/L, the molar concentration of the NaCl is 50-100 mmol/L, and the mass percentage of the third detergent is 0.004% or 0.1%; the third eluent is lauryl maltose neopentyl glycol and/or digitalis saponin.
7. The method of purification according to claim 1, wherein the method of preparation of the human mitochondrial total protein solution comprises the steps of:
a pQCIP plasmid is adopted to establish a humanized cell-NDUFS 3-3 xFlag over-expression stable transfer cell line;
mixing the humanized cell-NDUFS 3-3 xFlag over-expression stable transfer cell line with a first buffer solution, grinding, and carrying out solid-liquid separation to obtain a humanized mitochondrial crude extract;
and mixing the crude human mitochondrial extract with a second buffer solution, and performing mitochondrial membrane dissolution to obtain a human mitochondrial total protein solution.
8. The purification method of claim 7, wherein the first buffer solution comprises sucrose, 3-morpholinopropane sulfonic acid, and ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid; in the first buffer solution, the molar concentration of sucrose is 250mmol/L, the molar concentration of 3-morpholinopropane sulfonic acid is 20-30 mmol/L, and the molar concentration of ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid is 1mmol/L.
9. The purification method of claim 7, wherein the second buffer solution comprises 3-morpholinopropane sulfonic acid, naCl, and a fourth detergent; in the second buffer solution, the molar concentration of the 3-morpholinopropane sulfonic acid is 20-30 mmol/L, the molar concentration of NaCl is 50-100 mmol/L, and the mass percentage of the fourth detergent is 1%; the fourth detergent comprises lauryl maltose neopentyl glycol and/or digitalis saponin.
10. The method for purifying the human cell-NDUFS 3-3 xflag over-expression stable transgenic cell line according to claim 7, wherein the method for establishing the human cell-NDUFS 3-3 xflag over-expression stable transgenic cell line comprises the following steps:
fusing 3 xFlag tags at the C end of NDUFS3 subunit of the humanized mitochondrial respiratory chain complex I by adopting a molecular cloning method to construct pQCIP-NDUFS 3-3 xFlag plasmid;
chemically transfecting the humanized cells by using the pQCTIP-NDUFS 3-3 xFlag plasmid through a transfection reagent to obtain a humanized cell fluid transfected with the pQCTIP-NDUFS 3-3 xFlag plasmid;
and mixing the human cell sap transfected with pQCIP-NDUFS 3-3 xFlag plasmid with puromycin for cell screening to obtain a human cell-NDUFS 3-3 xFlag over-expression stable transfer cell line.
CN202311132534.5A 2023-09-05 2023-09-05 Purification method of human mitochondrial respiratory chain super-complex CICIII2CIV Pending CN117024606A (en)

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