CN116103251A - Purification method of humanized mitochondrial respiratory chain complex II - Google Patents

Purification method of humanized mitochondrial respiratory chain complex II Download PDF

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CN116103251A
CN116103251A CN202211600937.3A CN202211600937A CN116103251A CN 116103251 A CN116103251 A CN 116103251A CN 202211600937 A CN202211600937 A CN 202211600937A CN 116103251 A CN116103251 A CN 116103251A
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respiratory chain
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贡红日
饶子和
杜占强
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Nankai University
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Abstract

The invention belongs to the technical field of protein purification, and particularly relates to a purification method of a human mitochondrial respiratory chain complex II. The purification method provided by the invention comprises the following steps: loading the total protein solution of the human mitochondrial complex into a chromatographic column filled with Anti-Flag filler for affinity column chromatography purification, loading the obtained crude extract of the human mitochondrial respiratory chain complex II into a gel exclusion chromatographic column for gel exclusion column chromatography purification, and collecting a second eluent after second elution by using a second eluent to obtain a pure product of the human mitochondrial respiratory chain complex II; the SDHD subunit C-terminal of the human mitochondrial respiratory chain complex II in the human mitochondrial complex total protein solution is fused with a 3 xFlag tag. The purification method provided by the invention has the advantages of simple operation flow, low experimental cost, high purity of the obtained humanized mitochondrial respiratory chain complex II and good physiological activity, and can be used for subsequent structural function and drug discovery research.

Description

Purification method of humanized mitochondrial respiratory chain complex II
Technical Field
The invention belongs to the technical field of protein purification, and particularly relates to a purification method of a human mitochondrial respiratory chain complex II.
Background
Respiratory chain complex ii, also known as Succinate Dehydrogenase (SDH), can participate in two important vital active processes, tricarboxylic acid cycle (TCA) and oxidative phosphorylation (Oxidative phosphorylation, OXPHOS), simultaneously, and couple these two energy generation processes together in a ligament fashion. Since respiratory chain complex II plays an important role in the normal life activities of organisms, mutation of subunits of human mitochondrial respiratory chain complex II (hC II) causes impaired activity and causes a series of clinically relevant mitochondrial diseases such as metabolic abnormality-related diseases, tumors, etc.
The structure and the function of the human mitochondrial respiratory chain complex II are researched, and the method has significance for deep understanding of the catalysis mechanism, the mutation pathogenesis mechanism, the drug discovery and the like of the human mitochondrial respiratory chain complex II. In recent years, great progress has been made in the structural and functional studies of the mitochondrial respiratory chain complex II of mammals such as swine heart, but no report of purification and structural analysis of the mitochondrial respiratory chain complex II of human origin has been made so far.
The main means for researching the humanized mitochondrial respiratory chain protein compound is density gradient centrifugation combined with anion-cation exchange column purification, but the method needs an ultracentrifuge, has higher requirements on equipment and complex process, and the purity of the obtained protein is not high.
Disclosure of Invention
Therefore, the invention aims to provide a purification method of a human mitochondrial respiratory chain complex II, which can obtain the high-purity human mitochondrial respiratory chain complex II and has low production cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a purification method of a human mitochondrial respiratory chain complex II, which comprises the following steps:
loading the total protein solution of the human mitochondrial complex into a first chromatographic column for affinity column chromatography purification, and collecting a first eluent after first elution by adopting the first eluent to obtain a crude extract of the human mitochondrial respiratory chain complex II; the first chromatographic column is a chromatographic column filled with Anti-Flag filler; the SDHD subunit C end of the human mitochondrial respiratory chain complex II in the human mitochondrial complex total protein solution is fused with a 3 xFlag tag;
loading the crude extract of the human mitochondrial respiratory chain complex II into a second chromatographic column for gel exclusion column chromatography purification, and collecting a second eluent after second elution by adopting a second eluent to obtain a pure product of the human mitochondrial respiratory chain complex II; the second chromatographic column is a gel exclusion chromatographic column.
Preferably, the first eluent comprises 3-morpholinopropane sulfonic acid, naCl, a first detergent and 1×flag peptide; in the first eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, the mass percentage of the first detergent is 0.004%, 0.02% or 0.03%, and the mass concentration of the 1×flagpeptide is 200 mug/mL.
Preferably, before the first eluting, pre-eluting with impurity removing agent to remove impurities; the impurity removing agent comprises 3-morpholinopropane sulfonic acid, naCl and a second detergent; in the impurity removing agent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the second impurity removing agent is 0.004%, 0.02% or 0.03%.
Preferably, the second eluent comprises 3-morpholinopropane sulfonic acid, naCl and a third detergent; in the second eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the third detergent is 0.004%, 0.02% or 0.03%.
Preferably, in the second elution, the flow rate of the second eluent is 0.5mL/min; the pressure of the second elution was 1.5MPa.
Preferably, the first detergent, the second detergent and the third detergent are each lauryl maltoneopentyl glycol (LMNG) 、 Sugar ferment (GND) or dodecyl-beta-D-maltoside (DDM).
Preferably, after the first eluent is obtained, the method further comprises: loading the first eluent into a concentration tube, and centrifugally concentrating to obtain the crude extract of the humanized mitochondrial respiratory chain complex II; the molecular weight cut-off of the concentration tube is 100kDa.
Preferably, the preparation method of the human mitochondrial complex total protein solution comprises the following steps:
the pQCIP plasmid is adopted to establish a human cell-SDHD-3 xFlag over-expression stable transfer cell line;
mixing and grinding the humanized cell-SDHD-3 xFlag over-expression stable transformation cell line and a first buffer solution, and carrying out solid-liquid separation to obtain a humanized mitochondrial crude extract; the first buffer solution comprises sucrose (sucrose), 3-morpholinopropane sulfonic acid (MOPS) and ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid (EGTA); in the first buffer solution, the molar concentration of the cross is 250mmol/L, the molar concentration of the MOPS is 20mmol/L, and the molar concentration of the EGTA is 1mmol/L;
mixing the crude human mitochondrial extract with a second buffer solution to perform mitochondrial membrane dissolution, and performing solid-liquid separation to obtain a total protein solution of the human mitochondrial complex; the second buffer solution comprises 3-morpholinopropane sulfonic acid, naCl and a fourth detergent; in the second mixed solution, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the fourth detergent is 1%.
Preferably, the fourth detergent comprises lauryl maltoneopentyl glycol (LMNG) and/or dodecyl- β -D-maltoside (DDM); the time for dissolving the mitochondrial membrane is 30-60 min.
Preferably, the method for establishing the humanized cell-SDHD-3 xFlag over-expression stable transgenic cell line comprises the following steps:
a molecular cloning method is adopted to fuse a 3 xFlag label at the C end of an SDHD subunit of a human mitochondrial respiratory chain complex II, so as to construct pQCIP-SDHD-3 xFlag plasmid;
chemically transfecting the human cells by using the pQCIP-SDHD-3 xFlag plasmid through a transfection reagent to obtain a human cell sap transfected with the pQCIP-SDHD-3 xFlag plasmid;
and mixing the human cell sap transfected with pQCIP-SDHD-3 xFlag plasmid with puromycin for cell screening to obtain a human cell-SDHD-3 xFlag over-expression stable transfer cell line.
The invention provides a purification method of a human mitochondrial respiratory chain complex II, which comprises the following steps: loading the total protein solution of the human mitochondrial complex into a first chromatographic column for affinity column chromatography purification, and collecting a first eluent after first elution by adopting the first eluent to obtain a crude extract of the human mitochondrial respiratory chain complex II; the first chromatographic column is a chromatographic column filled with Anti-Flag filler; the SDHD subunit C end of the human mitochondrial respiratory chain complex II in the human mitochondrial complex total protein solution is fused with a 3 xFlag tag; loading the crude extract of the human mitochondrial respiratory chain complex II into a second chromatographic column for gel exclusion column chromatography purification, and collecting a second eluent after second elution by adopting a second eluent to obtain a pure product of the human mitochondrial respiratory chain complex II; the second chromatographic column is a gel exclusion chromatographic column. According to the purification method provided by the invention, the 3 xFlag tag at the C end of the SDHD subunit of the human mitochondrial respiratory chain complex II is utilized, the chromatographic column filled with the Anti-Flag filler is adopted for affinity chromatography purification, the 3 xFlag tag is specifically identified by the Anti-Flag filler, the crude extract rich in the human mitochondrial respiratory chain complex II can be effectively obtained, and then the purified human mitochondrial respiratory chain complex II is obtained by gel exclusion chromatography. The purification method provided by the invention has the advantages of simple operation flow, low experimental cost, high purity of the obtained humanized mitochondrial respiratory chain complex II and good physiological activity, and can be used 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 shows the results of gel exclusion chromatography, the abscissa in FIG. 1 shows the column elution volume in mL; the left ordinate is 280nm ultraviolet absorption, and the unit is mAU;
FIG. 2 shows the result of SDS-PAGE electrophoresis of gel exclusion chromatography column samples; in fig. 2, the left is Marker, and the right is sample component identification by adhesive tape mass spectrum;
FIG. 3 is a human mitochondrial respiratory chain complex II activity assay including succinic dehydrogenase activity (SDH) and ubiquinone Succinate reductase activity (SQR).
Detailed Description
The invention provides a purification method of a human mitochondrial respiratory chain complex II, which comprises the following steps:
loading the total protein solution of the human mitochondrial complex into a first chromatographic column for affinity column chromatography purification, and collecting a first eluent after first elution by adopting the first eluent to obtain a crude extract of the human mitochondrial respiratory chain complex II; the first chromatographic column is a chromatographic column filled with Anti-Flag filler; the SDHD subunit C end of the human mitochondrial respiratory chain complex II in the human mitochondrial complex total protein solution is fused with a 3 xFlag tag;
loading the crude extract of the human mitochondrial respiratory chain complex II into a second chromatographic column for gel exclusion column chromatography purification, and collecting a second eluent after second elution by adopting a second eluent to obtain a pure product of the human mitochondrial respiratory chain complex II; the second chromatographic column is a gel exclusion chromatographic column.
In the present invention, the raw materials used are commercially available products well known to those skilled in the art unless otherwise specified.
Loading a total protein solution of a human mitochondrial complex into a first chromatographic column for affinity column chromatography purification, and collecting a first eluent after first elution by adopting a first eluent to obtain a crude extract of a human mitochondrial respiratory chain complex II; the first chromatographic column is a chromatographic column filled with Anti-Flag filler; the SDHD subunit C-terminal of the human mitochondrial respiratory chain complex II in the human mitochondrial complex total protein solution is fused with a 3 xFlag tag.
In the present invention, the preparation method of the human mitochondrial complex total protein solution preferably comprises the following steps:
the pQCIP plasmid is adopted to establish a human cell-SDHD-3 xFlag over-expression stable transfer cell line;
mixing and grinding human cells-SDHD-3 xFlag over-expressed stable transformed cells and a first buffer solution, and carrying out solid-liquid separation to obtain a crude extract of human mitochondria; the first buffer solution comprises sucroses, 3-morpholinopropane sulfonic acid and ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid; in the first buffer solution, the molar concentration of the cross is 250mmol/L, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, and the molar concentration of the ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid is 1mmol/L;
mixing the crude human mitochondrial extract with a second buffer solution to perform mitochondrial membrane dissolution, and performing solid-liquid separation to obtain a total protein solution of the human mitochondrial complex; the second buffer solution comprises 3-morpholinopropane sulfonic acid, naCl and a fourth detergent; in the second mixed solution, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the fourth detergent is 1%.
The invention adopts pQCIP plasmid to build human cell-SDHD-3 xFlag over-expression stable transfer cell line.
In the present invention, the method for establishing the humanized cell-SDHD-3 xFlag over-expression stable transgenic cell line preferably comprises the following steps:
a molecular cloning method is adopted to fuse a 3 xFlag label at the C end of an SDHD subunit of a human mitochondrial respiratory chain complex II, so as to construct pQCIP-SDHD-3 xFlag plasmid;
chemically transfecting the human cells by using the pQCIP-SDHD-3 xFlag plasmid through a transfection reagent to obtain a human cell sap transfected with the pQCIP-SDHD-3 xFlag plasmid;
and mixing the human cell sap transfected with pQCIP-SDHD-3 xFlag plasmid with puromycin for cell screening to obtain a human cell-SDHD-3 xFlag over-expression stable transfer cell line.
The invention adopts a molecular cloning method to fuse 3 xFlag tags at the C end of SDHD subunit of a humanized mitochondrial respiratory chain complex II to construct pQCIP-SDHD-3 xFlag plasmid. The present invention has no special requirement on the molecular cloning method, and molecular cloning well known to those skilled in the art can be adopted.
After the pQCIP-SDHD-3 xFlag plasmid is obtained, the pQCIP-SDHD-3 xFlag plasmid is adopted to chemically transfect the human cells through a transfection reagent, so that the human cell sap for transfecting the pQCIP-SDHD-3 xFlag plasmid is obtained.
In the present invention, the transfection reagent is particularly 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 pQCIRP-SDHD-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 And each mL.
In the present invention, the time of the chemical transfection was 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 pQCIP-SDHD-3 xFlag plasmid, the invention mixes the human cell sap transfected with pQCIP-SDHD-3 xFlag plasmid with puromycin for cell screening to obtain the human cell-SDHD-3 xFlag over-expression stable transfection cell line.
In the present invention, the mass concentration of puromycin in the mixture of the puromycin and the human cell sap transfected with pQCIP-SDHD-3 XFlag plasmid is preferably 5. Mu.g/mL.
In the present invention, the time for cell selection is preferably 2 weeks.
In the invention, the human cell-SDHD-3 xFlag over-expression stable transfer cell line is particularly preferably a human HEK293F-SDHD-3 xFlag over-expression stable transfer cell line.
After obtaining a humanized cell-SDHD-3 xFlag over-expression stable transfer cell line, mixing and grinding the humanized cell-SDHD-3 xFlag over-expression stable transfer cell line and a first buffer solution, and carrying out solid-liquid separation to obtain a humanized mitochondrial crude extract; the first buffer solution comprises sucroses, 3-morpholinopropane sulfonic acid and ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid; in the first buffer solution, the molar concentration of the cross is 250mmol/L, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, and the molar concentration of the ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid is 1mmol/L.
In the present inventionThe cell density of the humanized cell-SDHD-3 xFlag overexpressing stably transformed cells is preferably 4.0x10 6 And each mL. The volume of the humanized cell-SDHD-3 xFlag over-expressed stably transformed cells is preferably 2L.
The invention preferably washes the human cells-SDHD-3 xFlag over-expressed stably transformed cells prior to the mixed milling. In the present invention, the washing preferably includes the steps of: adding the humanized cell-SDHD-3 xFlag over-expressed stable transformed cells into Phosphate Buffer (PBS) to resuspend the cells, centrifugally separating, and collecting cell sediment, wherein the volume of the PBS is 100mL; the rotational speed of the centrifugation is preferably 1000g, and the time of the centrifugation is preferably 10min. Adding the cell sediment into PBS to resuspend the cells, centrifugally separating, and collecting the cell sediment; the volume of the PBS is 100mL; the rotational speed of the centrifugation is preferably 1000g, and the time of the centrifugation is preferably 10min.
In the present invention, the pH of the first buffer solution is preferably 7.4.
In the invention, the first buffer solution provides proper osmotic pressure, and the human cell-SDHD-3 xFlag over-expressed stable transfer cell can be cracked after being mixed and ground with the first buffer solution.
In the present invention, the number of times of the mixed grinding is preferably two.
In the present invention, the mixed grinding is preferably performed in a glass tissue homogenizer, and the number of grinding times in the first mixed grinding is preferably 20; in the second mixed grinding, the number of grinding is preferably 10.
In the present invention, the mixed grinding preferably includes the steps of:
mixing and grinding the humanized cell-SDHD-3 xFlag over-expression stable transfer cell and part of the first buffer solution for the first time, and carrying out the first solid-liquid separation to obtain a first supernatant and a first precipitate;
mixing and grinding the first precipitate and the rest of the first buffer solution for the second time, and carrying out solid-liquid separation for the second time to obtain a second supernatant;
and mixing the first supernatant and the second supernatant, performing third solid-liquid separation, and taking a third precipitate to obtain the crude extract of the human mitochondria.
The invention mixes and grinds the human cell-SDHD-3 xFlag over-expression stable transfer cell and part of the first buffer solution for the first time, and separates the first solid from the liquid for the first time, thus obtaining a first supernatant and a first precipitate. In the first mixed grinding, the volume of part of the first buffer solution is preferably 5 times that of the human cell-SDHD-3 xFlag over-expressed stable transfer cell (cell sediment). The first solid-liquid separation is preferably centrifugation, the rotation speed of the centrifugation is preferably 1000g, and the time of the centrifugation is preferably 10min.
After the first precipitate is obtained, the first precipitate and the rest of the first buffer solution are mixed and ground for the second time, and the second supernatant is obtained through the second solid-liquid separation. The present invention preferably allows the first precipitation to be resuspended with the remaining first buffer solution and subjected to a second milling. In the second mixed grinding, the volume of the residual first buffer solution is preferably 5 times the volume of the first precipitate. In the present invention, the second solid-liquid separation is preferably performed by centrifugation, the rotational speed of the centrifugation is preferably 1000g, and the time of the centrifugation is preferably 10min.
After the first supernatant and the second supernatant are obtained, the first supernatant and the second supernatant are preferably mixed to obtain total supernatant, the total supernatant is subjected to third solid-liquid separation, and third precipitate is taken to obtain the crude human mitochondrial extract. In the present invention, the total supernatant is preferably subjected to a third solid-liquid separation, preferably centrifugation, at a rotational speed of 10000g for a time of preferably 10min. The centrifugation according to the invention enables mitochondrial precipitation.
The invention directly uses the humanized HEK293F-SDHD3 xFlag over-expression stable transformation cell line as an experimental material, and directly obtains the mitochondria crude extract from the cell respectively, and has large experimental sample, low cost and simple operation.
After the crude extract of the human mitochondria is obtained, the crude extract of the human mitochondria and a second buffer solution are mixed for mitochondrial membrane dissolution, and the solid-liquid separation is carried out to obtain the total protein solution of the human mitochondrial complex; the second buffer solution comprises 3-morpholinopropane sulfonic acid, naCl and a fourth detergent; in the second mixed solution, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the fourth detergent is 1%.
In the present invention, the fourth detergent preferably comprises LMNG and/or DDM, more preferably DDM or LMNG.
In the present invention, the pH of the second buffer solution is preferably 7.4.
In the present invention, the time for dissolving the mitochondrial membrane is preferably 30 to 60 minutes, more preferably 40 to 60 minutes.
In the present invention, the mitochondrial membrane is preferably performed on ice.
In the invention, the volume ratio of the crude human mitochondrial extract to the fourth detergent in the second buffer solution is preferably 2-4 times of the critical micelle concentration; the critical micelle concentration refers to the minimum concentration of micelles formed by the fourth detergent, and is determined by the nature of the fourth detergent itself. In the practice of the invention, the fourth detergent is exemplified by detergent LMNG, but is not to be construed as merely limiting the scope of the invention.
In the present invention, after the mitochondrial membrane is dissolved, the present invention preferably performs a fourth solid-liquid separation to obtain the total protein solution of the humanized mitochondrial complex. In the present invention, the fourth solid-liquid separation is preferably centrifugation, so as to obtain the total protein solution of the mitochondrial complex. The rotational speed of the centrifuge according to the invention is preferably 40000g; the time of the centrifugation is preferably 40min and the temperature of the centrifugation is preferably 4 ℃. In the present invention, the fourth detergent is capable of removing insoluble substances.
In the present invention, the Anti-Flag filler is preferably purchased from zenith and biotechnology limited.
Preferably, the Anti-Flag filler is filled into a gravity column to obtain a first chromatographic column before the first elution is carried out; and balancing the first chromatographic column by adopting a impurity removing agent. At this equilibrium, the volume of the impurity removing agent is preferably 2 times the volume of the first chromatographic column.
In the present invention, the first eluent preferably comprises 3-morpholinopropane sulfonic acid, naCl, a first detergent and 1×Flagpeptides; in the first eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, the mass percentage of the first detergent is 0.004%, 0.02% or 0.03%, and the mass concentration of the 1×flagpeptide is 200 mug/mL.
In the invention, the amino acid sequence of the 1×flagpeptide is shown in SEQ ID No. 1.
The SEQ ID NO.1 is: dykdddk.
As one or more specific embodiments of the present invention, the first detergent is preferably LMNG, and the mass percentage of the first detergent is 0.004%.
As one or more embodiments of the present invention, the first detergent is preferably DDM, and the mass percentage of the first detergent is preferably 0.03%.
As one or more specific embodiments of the present invention, the first detergent is preferably GDN, and the mass percentage of the first detergent is preferably 0.02%.
In the present invention, before the first elution, the present invention preferably further includes pre-eluting with a impurity removing agent to remove impurities; the impurity removing agent preferably comprises 3-morpholinopropane sulfonic acid, naCl and a second detergent; in the impurity removing agent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the second impurity removing agent is 0.004%, 0.02% or 0.03%.
As one or more specific embodiments of the present invention, the first detergent is preferably LMNG, and the mass percentage of the first detergent is 0.004%.
As one or more embodiments of the present invention, the first detergent is preferably DDM, and the mass percentage of the first detergent is preferably 0.03%.
As one or more specific embodiments of the present invention, the first detergent is preferably GDN, and the mass percentage of the first detergent is preferably 0.02%.
In the present invention, the first elution is preferably a competitive elution.
The invention preferably loads the human mitochondrial complex total protein solution to an affinity chromatography column loaded with Anti-Flag filler, and runs through the column for 5 times.
In the present invention, after the first eluent is obtained, the present invention preferably further comprises: loading the first eluent into a concentration tube, and centrifugally concentrating to obtain the crude extract of the humanized mitochondrial respiratory chain complex II; the molecular weight cut-off of the concentrator tube is preferably 100kDa.
After obtaining the crude extract of the human mitochondrial respiratory chain complex II, loading the crude extract of the human mitochondrial respiratory chain complex II into a second chromatographic column for gel exclusion column chromatography purification, and collecting a second eluent after second elution by using a second eluent to obtain a pure product of the human mitochondrial respiratory chain complex II; the second chromatographic column is a gel exclusion chromatographic column.
In the present invention, the gel exclusion chromatography column is particularly preferably a Superose6in create 10/300 (GEHealthcare) gel exclusion chromatography column.
In the present invention, the volume of the gel exclusion chromatography column is preferably 25mL.
In the present invention, the gel exclusion chromatography column is preferably equilibrated with a second eluent before the second elution is performed. In the present invention, the volume of the second eluent at the time of the equilibration is preferably 1 time the volume of the gel exclusion chromatography column, i.e., when the volume of the gel exclusion chromatography column is 25mL, 25mL of the second eluent is used for equilibration.
In the present invention, the second eluent preferably comprises 3-morpholinopropane sulfonic acid, naCl and a third detergent; in the second eluent, the molar concentration of the 3-morpholinopropane sulfonic acid (MOPS) is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the third detergent is 0.004%, 0.02% or 0.03%.
As one or more specific embodiments of the present invention, the first detergent is preferably LMNG, and the mass percentage of the first detergent is 0.004%.
As one or more embodiments of the present invention, the first detergent is preferably DDM, and the mass percentage of the first detergent is preferably 0.03%.
As one or more specific embodiments of the present invention, the first detergent is preferably GDN, and the mass percentage of the first detergent is preferably 0.02%.
In the present invention, the pH of the second eluent is preferably 7.4.
In the present invention, the first detergent, the second detergent, and the third detergent are different in kind from the fourth detergent.
In the present invention, the flow rate of the second eluent is preferably 0.5mL/min at the time of the second elution; the pressure of the second elution is preferably 1.5MPa.
The invention uses HEK293F-SDHD3 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 steps, short time consumption, good safety, 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 scheme of the invention will be clearly and completely described below by combining the embodiment of the invention and the attached drawings.
Example 1
Buffer used in this implementation:
buffer a (first buffer solution): 250mM cross, 20mM3- (N-morpholino) propanesulfonic acid (MOPS, 3-morpholinopropanesulfonic acid), 1mMethyleneglycoltetraacetic acid (EGTA, ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid), pH7.4;
buffer B (impurity removal agent and second eluent): 20mM MPS, 100mM NaCl,0.02% W/VGDN, pH7.4;
buffer C (second buffer solution): 20mM MPS, 100mM NaCl,1% W/VLMNG (lauryl maltose neopentyl glycol), pH7.4;
first eluent: 20mM MPS, 100mM NaCl,0.02% W/VGDN, 200. Mu.g/mL 1 XFlagpeptide, pH7.4;
a molecular cloning method is adopted to fuse a 3 xFlag label at the C end of an SDHD subunit of a human mitochondrial respiratory chain complex II, so as to construct pQCIP-SDHD-3 xFlag plasmid; transfecting HEK293F cells with the constructed pQCIP-SDHD-3 xFlag plasmid by using a PEI chemical reagent, wherein the mass ratio of the PEI chemical reagent to the pQCIP-SDHD-3 xFlag plasmid is 4:1, and the cell density is 0.5x10 when the cells are transfected 6 Adding puromycin (with the concentration of 5 mug/mL) into the obtained human cell fluid transfected with pQCIP-SDHD-3 xFlag plasmid after 48h transfection, and screening for 2 weeks to obtain a human HEK293F-SDHD3 xFlag over-expression stable transfer cell line;
culturing 2L of humanized HEK293F-SDHD3 xFlag over-expressed stable suspension cells to a cell density of 4.0x10 6 The harvested cell volume was about 40mL per mL. Adding 100mL of Phosphate Buffer (PBS) to re-suspend the cells, centrifuging 1000g for 10min, and collecting cell sediment; 100mL of Phosphate Buffer (PBS) was added again to resuspend the cells, and 1000g was centrifuged for 10min to collect the cell pellet. Adding buffer solution A into the cell sediment to re-suspend the cells, wherein the total volume is 200mL; grinding with a glass tissue homogenizer for 20 times (one time for one time), centrifuging for 10min at 1000g, and collecting supernatant and precipitate; the pellet was resuspended in buffer A, the total volume was 200mL, and the pellet was again ground 10 times and centrifuged at 1000g for 10min and the supernatant collected. Combining the supernatants obtained in the two steps, centrifuging 10000g for 10min, and collecting mitochondrial sediment and crude extract of human mitochondria;
the crude mitochondrial extract was resuspended to 40mL using buffer C, placed on ice and the membrane slowly shaken for 40min. After the membrane dissolution is finished, 40000g is centrifugated for 40min at 4 ℃, and supernatant fluid is collected to obtain a total protein solution of the humanized mitochondrial complex;
loading 5mL of Anti-Flag filler (Changzhou heaven and earth and biotechnology limited company) into a gravity column, balancing 2 column volumes by adopting a buffer solution B (the buffer solution B is 2 times of the volume of a chromatographic column), loading the total protein solution of the humanized mitochondrial complex into an affinity chromatographic column loaded with the Anti-Flag filler, and allowing the total protein solution to flow through the hanging column for 5 times;
eluting with buffer B containing 1×Flagpeptidide (DYKDDDK) at concentration of 200 μg/mL; the first eluting mode is competitive eluting, eluting with a first eluent after impurity removal by using a impurity-removing agent, and collecting a component containing a human mitochondrial respiratory chain complex II to obtain a first eluent; concentrating the collected first eluent to below 500 mu L by using a concentration tube with a molecular weight cut-off of 100kDa to obtain crude extract of the humanized mitochondrial respiratory chain complex II;
the Superose6 inch 10/300 (GEHealthcare) gel exclusion column was equilibrated with buffer B, 1 column volume, i.e., when the volume of the gel exclusion column was 25mL, 25mL buffer B was used; the crude extract of human mitochondrial respiratory chain complex II was loaded and eluted with buffer B, and the results are shown in figure 1, with the abscissa representing the column elution volume in mL. The ordinate is 280nm ultraviolet absorption in mAU. As can be seen from FIG. 1, the mitochondrial respiratory chain complex II exhibited a characteristic peak at a position around 15.2 mL;
collecting a sample at the characteristic peak position to obtain a humanized mitochondrial respiratory chain complex II solution; and carrying out SDS-PAGE (SDS-PAGE) electrophoresis on the collected solution of the human mitochondrial respiratory chain complex II, wherein the detection is shown in figure 2, and according to the figure 2, the elution sample of the gel exclusion chromatographic column has no obvious impurity band, and the composition of the mitochondrial respiratory chain complex II is identified to be complete by mass spectrum.
Example 2
The mitochondrial respiratory chain complex II activity test was performed on samples at characteristic peak positions of example 1 according to reference "Architecture of the mycobacterial Succinate dehydrogenase with a membrane-embedded Rieske FeS cluster [ J ]" (Zhou X, gao Y, wang W, et al proceedings of the National Academy of Sciences,2021,118 (15)), and as a result, as shown in FIG. 3, the human mitochondrial respiratory chain complex II activity test included succinic dehydrogenase activity (Succinate dehydrogenase, SDH) and succinic ubiquinone reductase activity (Succinate: quinone reductase, SQR). According to fig. 3, it can be seen that after the intermediate electron carrier UQ1 is added into the enzyme activity reaction system, the reaction rate of the purified sample obtained in the invention can be obviously improved compared with the reaction system without UQ1, which indicates that the humanized mitochondrial respiratory chain complex ii purified in the embodiment of the invention has higher physiological activity.
The purification method provided by the invention has the characteristics of simple operation method, low experimental cost and short time, and the obtained humanized mitochondrial respiratory chain complex II 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. A method for purifying a human mitochondrial respiratory chain complex ii, comprising the steps of:
loading the total protein solution of the human mitochondrial complex into a first chromatographic column for affinity column chromatography purification, and collecting a first eluent after first elution by adopting the first eluent to obtain a crude extract of the human mitochondrial respiratory chain complex II; the first chromatographic column is a chromatographic column filled with Anti-Flag filler; the SDHD subunit C end of the human mitochondrial respiratory chain complex II in the human mitochondrial complex total protein solution is fused with a 3 xFlag tag;
loading the crude extract of the human mitochondrial respiratory chain complex II into a second chromatographic column for gel exclusion column chromatography purification, and collecting a second eluent after second elution by adopting a second eluent to obtain a pure product of the human mitochondrial respiratory chain complex II; the second chromatographic column is a gel exclusion chromatographic column.
2. The purification method according to claim 1, wherein the first eluent comprises 3-morpholinopropane sulfonic acid, naCl, a first detergent, and 1 x Flagpeptide; in the first eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, the mass percentage of the first detergent is 0.004%, 0.02% or 0.03%, and the mass concentration of the 1×flagpeptide is 200 mug/mL.
3. The purification method of claim 1 or 2, further comprising pre-eluting with a contaminant removal agent prior to the first eluting; the impurity removing agent comprises 3-morpholinopropane sulfonic acid, naCl and a second detergent; in the impurity removing agent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the second impurity removing agent is 0.004%, 0.02% or 0.03%.
4. The purification method of claim 1, wherein the second eluent comprises 3-morpholinopropane sulfonic acid, naCl, and a third detergent; in the second eluent, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the third detergent is 0.004%, 0.02% or 0.03%.
5. The purification method according to claim 1 or 4, wherein the flow rate of the second eluent at the time of the second elution is 0.3 to 0.5mL/min; the pressure of the second elution is 1.5-1.8 MPa.
6. The method of purifying according to claim 3, wherein the first detergent, the second detergent and the third detergent are lauryl maltose neopentyl glycol 、 Sugar ferment or dodecyl-beta-D-maltoside.
7. The method of purifying according to claim 1, further comprising, after obtaining the first eluent: loading the first eluent into a concentration tube, and centrifugally concentrating to obtain the crude extract of the humanized mitochondrial respiratory chain complex II; the molecular weight cut-off of the concentration tube is 100kDa.
8. The method of purification according to claim 1, wherein the method of preparing the human mitochondrial complex total protein solution comprises the steps of:
the pQCIP plasmid is adopted to establish a human cell-SDHD-3 xFlag over-expression stable transfer cell line;
mixing and grinding the humanized cell-SDHD-3 xFlag over-expression stable transformation cell line and a first buffer solution, and carrying out solid-liquid separation to obtain a humanized mitochondrial crude extract; 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 20mmol/L, and the molar concentration of ethylene glycol-bis- (2-aminoethyl ether) tetraacetic acid is 1mmol/L;
mixing the crude human mitochondrial extract with a second buffer solution to perform mitochondrial membrane dissolution, and performing solid-liquid separation to obtain a total protein solution of the human mitochondrial complex; the second buffer solution comprises 3-morpholinopropane sulfonic acid, naCl and a fourth detergent; in the second mixed solution, the molar concentration of the 3-morpholinopropane sulfonic acid is 20mmol/L, the molar concentration of the NaCl is 100mmol/L, and the mass percentage of the fourth detergent is 1%.
9. The purification method of claim 8, wherein the fourth detergent comprises lauryl maltoneopentyl glycol and/or dodecyl- β -D-maltoside; the time for dissolving the mitochondrial membrane is 30-60 min.
10. The method for purifying the cell line according to claim 8, wherein the method for establishing the human cell-SDHD-3 xFlag over-expression stable transgenic cell line comprises the following steps:
a molecular cloning method is adopted to fuse a 3 xFlag label at the C end of an SDHD subunit of a human mitochondrial respiratory chain complex II, so as to construct pQCIP-SDHD-3 xFlag plasmid;
chemically transfecting the human cells by using the pQCIP-SDHD-3 xFlag plasmid through a transfection reagent to obtain a human cell sap transfected with the pQCIP-SDHD-3 xFlag plasmid;
and mixing the human cell sap transfected with pQCIP-SDHD-3 xFlag plasmid with puromycin for cell screening to obtain a human cell-SDHD-3 xFlag over-expression stable transfer cell line.
CN202211600937.3A 2022-12-13 2022-12-13 Purification method of humanized mitochondrial respiratory chain complex II Pending CN116103251A (en)

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