CN114591926A

CN114591926A - Purification method of human respiratory chain compound I

Info

Publication number: CN114591926A
Application number: CN202210351749.5A
Authority: CN
Inventors: 贡红日; 徐金旭; 周珊
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2022-06-07
Anticipated expiration: 2042-04-02
Also published as: CN114591926B

Abstract

The invention provides a purification method of a human respiratory chain compound I, and relates to the technical field of protein purification. The invention adopts the methods of cation exchange chromatography, anion exchange chromatography and molecular sieve chromatography in sequence to purify the human respiratory chain compound I, has low requirements on experimental equipment compared with the prior common density gradient centrifugation method, and has the advantages of simple steps, short time consumption, good safety, easy standardized operation and the like. The anion and cation filler and the molecular sieve filler used in the purification method are simple and easy to obtain, the purification process is easy to amplify, and the method can be further promoted to large-scale separation and purification of the human respiratory chain compound I.

Description

Purification method of human respiratory chain compound I

Technical Field

The invention belongs to the technical field of protein purification, and particularly relates to a purification method of a human respiratory chain compound I.

Background

Mitochondria are energy factories of eukaryotic cells, and providing energy is one of the most important functions of mitochondria. Eukaryotic mitochondrial respiratory chain complexes include complex I (NADH-ubiquinone oxidoreductase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (ATP synthase). The complex I is used as a protein complex with the largest respiratory chain and is also used as a respiratory chain inlet, electrons are transferred to acceptor quinone through oxidation of NADN, a transmembrane proton gradient is generated, and finally ATP synthetase is promoted to synthesize ATP to supply energy for biological activities.

About 40% of mitochondrial diseases were found to be associated with complex I dysfunction, such as crensell syndrome and the like. In addition, more and more studies show that the compound I is also an important antitumor drug target. Therefore, the research on the respiratory chain compound I has important significance for understanding the energy metabolism mechanism of organisms and developing medicines for curing diseases. Therefore, the work of purifying the human respiratory chain compound I in vitro is beneficial to accelerating the mechanism research and the drug research and development of the human respiratory chain compound I.

Disclosure of Invention

In view of the above, the present invention aims to provide a purification method of a human respiratory chain complex I, which has the advantages of simple operation method, low experiment cost, short duration, high purity, high target protein activity, and the like, and is beneficial to the development of subsequent structural function research and drug research and development.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a purification method of a human respiratory chain compound I, which comprises the following steps: removing impurities from human mitochondria to obtain a solution rich in mitochondrial protein;

and sequentially separating and purifying the solution rich in the mitochondrial protein by using cation exchange chromatography, anion exchange chromatography and molecular sieve chromatography to obtain a pure human respiratory chain compound I.

Preferably, the human mitochondria are extracted from human suspension cells HEK 293F.

Preferably, the method for extracting human mitochondria comprises: cracking cultured human-derived suspension cells HEK293F, and performing differential centrifugation on the obtained cell lysate to obtain coarse mitochondria;

the differential centrifugation comprises: centrifuging 1000g for 10min, collecting supernatant, centrifuging 10000g for 10min, and collecting precipitate.

Preferably, the impurity removal comprises the steps of resuspending the human mitochondria by using buffer MES 50 containing a detergent, standing on ice for 30-120 min, centrifuging at 40000g for 40min, and collecting supernatant;

the buffer MES 50 was a mixture of 20mM MES and 50mM NaCl at a final concentration, pH 6.0.

Preferably, the cation exchange chromatography comprises eluting the target protein by sequentially using a buffer MES H containing a detergent and a buffer MES L containing a detergent in a gradient manner;

the buffer MES H is a mixed solution of 20mM MES and 1M NaCl at the final concentration, and the pH value is 6.0;

the buffer MES L was 20mM MES, pH 6.0.

Preferably, before the cation exchange chromatography, the method further comprises balancing the cation exchange chromatography column by using buffer MES 50 containing detergent, and keeping the flow rate at 1 ml/min; the buffer MES 50 was a mixture of 20mM MES and 50mM NaCl at a final concentration, pH 6.0.

Preferably, the anion exchange chromatography comprises gradient elution by sequentially using buffer MOPS H containing a detergent and buffer MOPS L containing a detergent, and collecting the target protein;

the buffer MOPS H is a mixed solution of 20mM MOPS and 1M NaCl at the final concentration, and the pH value is 7.4;

the buffer MOPS L was 20mM MOPS, pH 7.4.

Preferably, before the anion exchange chromatography, the method further comprises the step of balancing the anion exchange chromatography column by using buffer MOPS 50 containing a detergent;

the buffer MOPS 50 is a mixture of 20mM MOPS and 50mM NaCl at a final concentration, pH 7.4.

Preferably, the molecular sieve chromatography is gel exclusion chromatography.

Preferably, before the molecular sieve chromatography, the method further comprises the step of balancing the molecular sieve by using a buffer MOPS100 containing a detergent;

the buffer MOPS100 is a mixture of 20mM MOPS and 100mM NaCl at a final concentration, pH 7.4.

Has the advantages that: the invention provides a purification method of a human respiratory chain compound I, which adopts cation exchange chromatography, anion exchange chromatography and molecular sieve chromatography in sequence to purify the human respiratory chain compound I. The anion and cation filler and the molecular sieve used in the purification method are simple and easy to obtain, the purification process is easy to amplify, and the method can be further promoted to large-scale separation and purification of the human respiratory chain compound I.

Drawings

FIG. 1 is a diagram of a cationic column SP column with the abscissa representing the elution volume of the column in ml; the left ordinate represents the ultraviolet absorption at 280nm, in mAU; the right ordinate represents the conductance parameter in the buffer, and the unit is mS/cm;

FIG. 2 is a NTB staining pattern of cation column SP column sampling;

fig. 3 is a Q-column diagram of an anion column: the abscissa of the figure is the elution volume of the column in ml; the left ordinate represents the ultraviolet absorption at 280nm, in mAU; the right ordinate represents the conductance parameter in the buffer, and the unit is mS/cm;

FIG. 4 is a NTB staining pattern of a cation column Q column sample;

FIG. 5 is a diagram of a gel exclusion chromatography column: the abscissa of the graph is the elution volume of the column in ml; the ordinate represents the UV absorption at 280nm in mAU.

Detailed Description

The source of the human mitochondria is not particularly limited in the present invention, and is preferably extracted from a cell line, and in the examples, human suspension cell HEK293F is exemplified, but it is not to be construed as the full scope of the present invention. The present invention preferably extracts mitochondria from HEK293F cells by differential centrifugation, more preferably comprising: cracking cultured human-derived suspension cells HEK293F, and performing differential centrifugation on the obtained cell lysate to obtain coarse mitochondria; the differential centrifugation comprises: centrifuging 1000g for 10min, collecting supernatant, centrifuging 10000g for 10min, and collecting precipitate. The source of the HEK293F cells is not particularly limited in the present invention, and the HEK293 cells are preferably isolated from the HEK293 cell line, also known as human embryonic kidney epithelial cells.

Before the HEK293F cell is used for extracting the pachytene, the invention preferably also comprises the step of culturing the cell to about 3.5X 10⁶And (2) per ml, centrifuging 1000g for 10min, then resuspending the cells by using PBS, centrifuging, then collecting the cells, then resuspending the cells by using precooled buffer MIB, and then grinding and crushing to obtain cell lysate. The grinding of the invention preferably comprises grinding on a glass grinder precooled on ice, wherein the grinding is preferably carried out once for 30 times, then 1000g centrifugation is carried out for 10min, and supernatant and precipitate are collected. The method preferably comprises the step of grinding again after collecting the supernatant, specifically, the pre-cooled buffer MIB is used for re-suspending and precipitating, grinding is carried out for 15 times, then 1000g of the precipitate is centrifuged for 10min, the supernatant is collected, and the two supernatants are combined to obtain the mitochondrial suspension. The method preferably centrifuges the mitochondrial suspension for 10min at 1000g, removes impurities, centrifuges the mitochondrial suspension for 10min at 10000g and collects mitochondrial precipitates at 4 ℃. The buffer MIB is preferably as follows: 250mM sucrose, 20mM MOPS and 1mM EGTA, pH 7.4.

Preferably, the mitochondrial precipitation is subjected to impurity removal, and the impurity removal preferably comprises the steps of resuspending the human mitochondria by using a buffer MES 50 containing a detergent, standing on ice for 30-120 min, centrifuging at 40000g for 40min, collecting supernatant, and removing undissolved protein and impurity protein; the buffer MES 50 was a mixture of 20mM MES and 50mM NaCl at a final concentration, pH 6.0. The concentration of the detergent of the present invention is preferably 1% (w/v), and the kind of the detergent may be adjusted according to the actual situation, for example, dodecyl- β -D-maltoside (DDM), glycolytic hormone (GDN), etc. In the invention, after the membrane protein compound I is subjected to membrane lysis, a detergent is added into a buffer used for purification, the stability of the membrane protein is kept, the concentration of the detergent is determined according to the type of the detergent, and the final concentration is determined according to the type of the detergent, wherein the concentration of the detergent is 2-5 times of the critical micelle concentration of the detergent.

The invention uses cation exchange chromatography to carry out primary separation on the solution rich in mitochondrial protein, and collects the eluent containing the respiratory chain compound I. The cation exchange chromatography of the invention preferably comprises the following steps of sequentially utilizing buffer MOPS H containing a detergent and buffer MESL containing a detergent to carry out gradient elution on the target protein; the buffer MES H is preferably a mixture of 20mM MES and 1M NaCl at a final concentration of pH 6.0; the buffer MES L is preferably 20mM MES, pH 6.0. Before the cation exchange chromatography is carried out, the method preferably further comprises balancing the cation exchange chromatography column by using a buffer MES 50 containing a detergent, and keeping the flow rate at 1 ml/min; the buffer MES 50 was a mixture of 20mM MES and 50mM NaCl at a final concentration, pH 6.0. The detergent containing buffer MES 50 of the present invention is preferably used in an amount of 5 times the volume of the cation exchange chromatography column. Before loading, the present invention preferably further comprises filtering impurities from the mitochondrial protein-rich solution with a 0.45 μm filter to prevent clogging of the column.

Preferably, the target protein is collected and eluted, the pH is adjusted to 7.4, the salt concentration is adjusted to 50mM, then anion exchange chromatography is carried out, the anion exchange chromatography preferably comprises gradient elution by sequentially utilizing buffer MOPS H containing a detergent and buffer MOPS L containing a detergent, and the target protein is collected; the buffer MOPS H is preferably a mixed solution of 20mM MOPS and 1M NaCl at the final concentration, and the pH value is 7.4; the buffer MOPS L is preferably 20mM MOPS, pH 7.4. Before the anion exchange chromatography, the invention preferably further comprises a buffer MOPS 50 equilibrium anion exchange chromatography column containing a detergent; the buffer MOPS 50 is a mixture of 20mM MOPS and 50mM NaCl at a final concentration, pH 7.4. In the equilibrium of the present invention, the volume of buffer MOPS 50 containing detergent is preferably 5 times the volume of the anion exchange chromatography column. The present invention performs loading after the equilibration, and preferably further comprises, before the loading, filtering the collected target protein solution with a 0.45 μm filter to remove impurities and poor proteins, and then performing loading.

The invention carries out molecular sieve chromatography on the target protein solution collected from an anion exchange chromatography column, and preferably also comprises a buffer MOPS100 equilibrium molecular sieve containing a detergent before the molecular sieve chromatography is carried out; the buffer MOPS100 is preferably a mixture of 20mM MOPS and 100mM NaCl at a final concentration, pH 7.4. In the equilibrium of the present invention, the volume of buffer MOPS100 containing detergent is preferably the same as the chromatography volume of the molecular sieve. The preparation before the molecular sieve chromatography of the invention comprises the steps of concentrating a 100KDa molecular weight cut-off concentration pipe, loading and collecting target protein.

The following examples are provided to illustrate the purification method of human respiratory chain complex I provided by the present invention in detail, but they should not be construed as limiting the scope of the present invention.

The compositions of the buffer solutions used in the examples of the present invention were as follows:

buffer MIB：250mM sucrose，20mM MOPS，1mM EGTA，pH7.4；

Buffer MES 50：20mM MES，50mM NaCl，pH 6.0；

buffer MES H：20mM MES，1M NaCl，pH 6.0；

buffer MES L：20mM MES，pH 6.0；

buffer MOPS 50：20mM MOPS，50mM NaCl，pH 7.4；

buffer MOPS H：20mM MOPS，1M NaCl，pH 7.4；

buffer MOPS L：20mM MOPS，pH 7.4；

buffer MOPS 100：20mM MOPS，100mM NaCl，pH7.4。

example 1

(I) mitochondrial preparation

Collecting cells at a density of about 3.5X 10⁶3L/ml human source suspension culture HEK293F cells, centrifuging for 10min at 140g, and obtaining the cell volume of 40 ml; 100ml of 1 XPBS was added to resuspend the cells, centrifuged at 1000g for 10min, the cell pellet was collected at 4 ℃ and the procedure was repeated. Buffer MIB was added to resuspend the cells in a total volume of 200 ml.

Pre-cooling a glass grinder on ice, grinding the glass grinder up and down for 30 times, centrifuging the ground glass for 10min at 1000g, and collecting supernatant at 4 ℃; adding buffer MIB to resuspend the precipitate to a total volume of 200ml, grinding again on ice for 15 times, centrifuging at 1000g for 10min, and collecting the supernatant at 4 ℃. And mixing the mitochondria suspensions obtained in the two steps, centrifuging the mixture for 10min at 1000g again, removing impurities at 4 ℃, and collecting the supernatant. Centrifuging the supernatant at 10000g for 10min, and collecting the mitochondrial precipitate at 4 ℃.

(II) preparation of protein solution

Adding 30ml buffer MES 50 into the mitochondria precipitate, re-suspending, mixing, adding 4ml 10% (w/v) Lauryl Maltose Neopentyl Glycol (LMNG), fixing the volume to 40ml, placing on a shaking table on ice, and dissolving the membrane for 30 min. After the membrane dissolution is finished, 40000g is centrifuged for 40min, the supernatant is carefully poured out to avoid pouring out the precipitate, and the protein solution collected by centrifugation is filtered by using a 0.45 mu m filter membrane, so that the preparation of the protein sample is finished.

(III) cation exchange chromatography column

Taking 5ml strong cation exchange column SP column, buffer MES 50 balancing 5 column volumes, after balancing, loading the prepared protein sample at flow rate of 1 ml/min. The following operations are all carried out

purification was performed on the pure system, the equilibrium solution of the system was buffer MES 50+ 0.004% (w/v) LMNG, and elution was performed using buffer MES L + 0.004% (w/v) LMNG and buffer MES H + 0.004% (w/v) LMNG with a salt ion concentration gradient of 50-250mM NaCl and collected. The conductance value of the target protein appearance position is 8mS/cm-180mS/cm, the compound I protein is collected, buffer MOPS L + 0.004% (w/v) LMNG is added for dilution until the salt concentration is about 50mM NaCl, and the pH is adjusted to 7.2-7.4 by using 1M Tris-HCl pH8.0, so as to prepare for subsequent anion exchange chromatography.

Protein solution samples the protein solution collected by centrifugation was filtered using a 0.45 μm filter and the protein samples were prepared.

Dynamic sampling detection during elution of the cation exchange chromatography column was performed by invitrogen Native PAGE 4-16% Bis-Tris Gel electrophoresis. The results of the cation exchange chromatography column are shown in FIG. 1, with the black boxes showing the appearance of complex I.

Meanwhile, the NTB active gel staining method is adopted to detect the activity of the compound I, and the result is shown in figure 2, and a blue strip is a strip of the compound I.

NTB gel staining method:

20ml buffer MOPS100+ 0.004% (w/v) LMNG, appropriate amount of tetrazolium chloride blue (NTB) powder was added, reaction was initiated by adding 200. mu.l of 10mM NADH, and staining was carried out at 37 ℃ for 20 min.

(IV) anion exchange chromatography column

Taking 1ml strong anion column prepackage column Q column, balancing 5 column volumes by buffer MOPS 50, after balancing, loading the prepared protein sample on ice by a peristaltic pump with the flow rate of 1ml/min to avoid air intake of the ion column, wherein the following operations are all performed

purifying on a pure system, wherein a balance solution of the system is buffer MOPS 50+ 0.004% (w/v) LMNG, a 50-450mM NaCl salt ion concentration gradient is formed by using buffer MOPS L + 0.004% (w/v) LMNG and buffer MOPS H + 0.004% (w/v) LMNG for elution and collection, the appearance position of the target protein is a detection conductance value of 28-35.5 mS/cm, the compound I protein is collected, and the result of an anion column is shown in figure 3. The activity of the complex I was detected by NTB staining with active gel, and the result is shown in FIG. 4, in which the purple band is the band of the complex I. The protein sample was prepared by blocking with a 100kDa stop tube, centrifuging at 1800g, concentrating to 500. mu.l, and removing to an EP tube.

(V) gel exclusion chromatography column

Taking 24ml superose 6 incrasse 10/300GE gel exclusion chromatographic column, balancing 25ml buffer MOPS100+ 0.004% (w/v) LMNG, loading the prepared protein, collecting the compound I protein, and preparing the pure protein solution.

The molecular sieve results are shown in FIG. 5.

According to the results of gel filtration chromatography, invitrogen Native PAGE and activity detection, the purity is high, and the method is suitable for structural research and biochemical research.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for purifying a human respiratory chain complex I, which is characterized by comprising the following steps: removing impurities from human mitochondria to obtain a solution rich in mitochondrial protein;

2. The purification method according to claim 1, wherein the human mitochondria are extracted from human suspension cells HEK 293F.

3. The purification method according to claim 1 or 2, wherein the method for extracting human mitochondria comprises: cracking cultured human-derived suspension cells HEK293F, and performing differential centrifugation on the obtained cell lysate to obtain coarse mitochondria;

4. The purification method according to claim 1, wherein the removing comprises resuspending the human mitochondria using buffer MES 50 containing detergent, standing on ice for 30-120 min, centrifuging at 40000g for 40min, and collecting the supernatant;

the buffermES 50 is a mixture of 20mM MES and 50mM NaCl at a final concentration, pH 6.0.

5. The purification method according to claim 1, wherein the cation exchange chromatography comprises sequentially forming a gradient elution of the target protein using buffer MES H containing a detergent and buffer MES L containing a detergent;

the buffermES H is a mixed solution of 20mM MES and 1M NaCl at the final concentration, and the pH value is 6.0;

the buffermES L was 20mM MES, pH 6.0.

6. The purification method according to claim 1 or 5, further comprising, before the cation exchange chromatography, equilibrating the cation exchange chromatography column with a buffer MES 50 containing detergent at a flow rate of 1 ml/min; the buffer MES 50 was a mixture of 20mM MES and 50mM NaCl at a final concentration, pH 6.0.

7. The purification method according to claim 1, wherein the anion exchange chromatography comprises sequentially performing gradient elution by using buffer MOPS H containing a detergent and buffer MOPS L containing a detergent, and collecting the target protein;

the buffermOPS L was 20mM MOPS, pH 7.4.

8. The purification method according to claim 1 or 7, further comprising, before the anion exchange chromatography, equilibrating the anion exchange chromatography column with bufferMOPS 50 containing a detergent;

the buffermOPS 50 is a mixture of 20mM MOPS and 50mM NaCl at a final concentration, pH 7.4.

9. The purification method of claim 1, wherein the molecular sieve chromatography is gel exclusion chromatography.

10. The purification method according to claim 1 or 9, further comprising, before the molecular sieve chromatography, equilibrating the molecular sieve with bufferMOPS 100 containing a detergent;

the buffermOPS 100 is a mixture of MOPS and NaCl at a final concentration of 20mM and 100mM, and has a pH of 7.4.