CN114591926B - Purification method of humanized respiratory chain compound I - Google Patents
Purification method of humanized respiratory chain compound I Download PDFInfo
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- C12N9/0004—Oxidoreductases (1.)
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- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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Abstract
The invention provides a purification method of a humanized respiratory chain compound I, and relates to the technical field of protein purification. The method sequentially adopts the methods of cation exchange chromatography, anion exchange chromatography and molecular sieve chromatography to purify the humanized respiratory chain compound I, has low requirements on experimental equipment compared with the current common method by means of density gradient centrifugation, and has the advantages of simple steps, short time consumption, good safety, easiness in standardization operation and the like. The anion-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 purification method can be further advanced to large-scale separation and purification of the humanized respiratory chain compound I.
Description
Technical Field
The invention belongs to the technical field of protein purification, and particularly relates to a purification method of a humanized respiratory chain complex I.
Background
Mitochondria are the energy factories of eukaryotic cells, and energy supply 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, is also a respiratory chain inlet, transfers electrons to receptor quinone through oxidizing NADN, and generates a transmembrane proton gradient, so that ATP synthase is finally promoted to synthesize ATP and supply energy for biological activities.
About 40% of mitochondrial system diseases are found to be associated with complex I dysfunction, such as crensel syndrome and the like. In addition, more and more researches indicate that the compound I is also an important antitumor drug target. Therefore, the research of the respiratory chain compound I has important significance for understanding the organism energy metabolism mechanism and developing the medicine for curing diseases. Thus, the work of purifying the human respiratory chain complex I in vitro is beneficial to accelerating the mechanism research and the drug development aiming at the human respiratory chain complex I.
Disclosure of Invention
Therefore, the invention aims to provide a purification method of a human respiratory chain compound I, which has the advantages of simple operation method, low experimental cost, short duration, high purity, high activity of target protein and the like, and is beneficial to developing 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 proteins;
and separating and purifying the solution rich in mitochondrial proteins by cation exchange chromatography, anion exchange chromatography and molecular sieve chromatography in sequence to obtain the pure humanized respiratory chain complex I.
Preferably, the human mitochondria are extracted from human suspension cells HEK293F.
Preferably, the method for extracting human mitochondria comprises the following steps: cracking the cultured human suspension cells HEK293F, and performing differential centrifugation on the obtained cell lysate to obtain crude mitochondria;
the differential centrifugation includes: 1000g first centrifugation for 10min, collecting supernatant, and 10000g second centrifugation for 10min, collecting precipitate.
Preferably, the removing of impurities comprises re-suspending the human mitochondria by using buffer MES 50 containing a detergent, standing for 30-120 min on ice, centrifuging for 40min with 40000g, and collecting supernatant;
the buffer MES 50 is a mixed solution of 20mM MES and 50mM NaCl with the final concentration and the pH value is 6.0.
Preferably, the cation exchange chromatography comprises gradient elution of the target protein by sequentially utilizing buffer MES H containing a detergent and buffer MES L containing the detergent;
the buffer MES H is a mixed solution of 20mM MES and 1M NaCl with 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 is performed, balancing the cation exchange chromatography column by utilizing buffer MES 50 containing detergent, and keeping the flow rate at 1ml/min; the buffer MES 50 is a mixed solution of 20mM MES and 50mM NaCl with the final concentration and the pH value is 6.0.
Preferably, the anion exchange chromatography comprises gradient elution by utilizing a buffer MOPS H containing a detergent and a buffer MOPS L containing a detergent in sequence, and collecting the target protein;
the buffer MOPS H is a mixed solution of final concentration of 20mM MOPS and 1M NaCl, and the pH value is 7.4;
the buffer MOPS L was 20mM MOPS, pH7.4.
Preferably, prior to performing the anion exchange chromatography, further comprising equilibrated anion exchange chromatography columns with buffer MOPS 50 containing a detergent;
the buffer MOPS 50 is a mixed solution of final concentration of 20mM MOPS and 50mM NaCl, and the pH value is 7.4.
Preferably, the molecular sieve chromatography is gel exclusion chromatography.
Preferably, prior to performing the molecular sieve chromatography, further comprising equilibrating the molecular sieve with a buffer MOPS100 comprising a detergent;
the buffer MOPS100 is a mixed solution of final concentration of 20mM MOPS and 100mM NaCl, and pH7.4.
The beneficial effects are that: the invention provides a purification method of a human respiratory chain compound I, which sequentially adopts cation exchange chromatography, anion exchange chromatography and molecular sieve chromatography to purify the human respiratory chain compound I. The anion-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 purification method can be further advanced to large-scale separation and purification of the humanized respiratory chain compound I.
Drawings
FIG. 1 is a SP column diagram of a cation column, wherein the abscissa of the diagram is the elution volume of the column, and the unit is ml; the left ordinate represents ultraviolet absorption at 280nm in mAU; the right ordinate represents the conductivity parameter in buffer, and the unit is mS/cm;
FIG. 2 is a sample NTB staining chart of the SP column of the cationic column;
fig. 3 is a diagram of an anion column Q: the abscissa in the figure represents the elution volume of the chromatographic column in ml; the left ordinate represents ultraviolet absorption at 280nm in mAU; the right ordinate represents the conductivity parameter in buffer, and the unit is mS/cm;
FIG. 4 is a sample NTB staining chart of a cationic column Q;
fig. 5 is a gel exclusion chromatographic column diagram: the abscissa in the figure represents the elution volume of the chromatographic column in ml; the ordinate represents the UV absorption at 280nm in mAU.
Detailed Description
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 proteins;
and separating and purifying the solution rich in mitochondrial proteins by cation exchange chromatography, anion exchange chromatography and molecular sieve chromatography in sequence to obtain the pure humanized respiratory chain complex I.
The source of the human mitochondria is not particularly limited, but is preferably extracted from a cell line, and human suspension cells HEK293F are exemplified in the examples, but they are not construed as the full scope of the present invention. The invention preferably extracts mitochondria from HEK293F cells by differential centrifugation, more preferably comprising: cracking the cultured human suspension cells HEK293F, and performing differential centrifugation on the obtained cell lysate to obtain crude mitochondria; the differential centrifugation includes: 1000g first centrifugation for 10min, collecting supernatant, and 10000g second centrifugation for 10min, collecting precipitate. The source of the HEK293F cells is not particularly limited in the present invention, and is preferably isolated from HEK293 cell lines, also known as human embryonic kidney epithelial cells.
The present invention preferably further comprises culturing the HEK293F cells to about 3.5X10 before extracting crude mitochondria 6 After centrifugation at 1000g for 10 min/ml, the cells were resuspended in PBS, collected after centrifugation, resuspended in pre-chilled buffer MIB, and then groundCrushing to obtain cell lysate. The grinding according to the invention preferably comprises carrying out the grinding on a glass grinder pre-cooled on ice, preferably once up and down, for a total of 30 grinding times, followed by centrifugation at 1000g for 10min, and collecting the supernatant and precipitate. The invention preferably further comprises re-grinding after collecting the supernatant, specifically re-suspending and precipitating by utilizing precooled buffer MIB, grinding for 15 times, centrifuging for 10min at 1000g, collecting the supernatant, and combining the two supernatants to obtain the mitochondrial suspension. The invention preferably carries out 1000g centrifugation for 10min on the mitochondrial suspension, removes impurities, carries out 10000g centrifugation for 10min, and collects mitochondrial sediment. The buffer MIB of the present invention is preferably: 250mM of cross, 20mM of MOPS and 1mM of EGTA, pH7.4.
The invention preferably carries out impurity removal on the mitochondrial sediment, wherein the impurity removal preferably comprises the steps of re-suspending the human mitochondria by utilizing buffer MES 50 containing a detergent, standing for 30-120 min on ice, centrifuging for 40min by 40000g, collecting supernatant, and removing undissolved proteins and impurity proteins; the buffer MES 50 is a mixed solution of 20mM MES and 50mM NaCl with the final concentration and the pH value is 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, such as dodecyl- β -D-maltoside (DDM), glycosylase (GDN), etc. In the invention, after the membrane protein complex I is dissolved in the membrane, the buffer used for purification is added with a detergent, so that the stability of the membrane protein is maintained, the concentration of the detergent is determined according to the type of the detergent, and the final concentration is generally determined according to the type of the detergent, which is 2-5 times the critical micelle concentration of the detergent.
The invention utilizes cation exchange chromatography to carry out preliminary separation on the solution rich in mitochondrial protein, and collects eluent containing respiratory chain complex I. The cation exchange chromatography preferably comprises gradient elution of target proteins by sequentially utilizing buffer MOPS H containing a detergent and buffer MES L containing the detergent; the buffer MES H is preferably a mixed solution of 20mM MES and 1M NaCl with the final concentration, and the pH value is 6.0; the buffer MES L is preferably 20mM MES, pH 6.0. The invention preferably further comprises balancing the cation exchange chromatography column by utilizing buffer MES 50 containing detergent before the cation exchange chromatography is carried out, and keeping the flow rate to be 1ml/min; the buffer MES 50 is a mixed solution of 20mM MES and 50mM NaCl with the final concentration and the pH value is 6.0. The amount of buffer MES 50 containing detergent according to the present invention is preferably 5 times the volume of the cation exchange chromatography column. The method preferably further comprises filtering impurities from the solution rich in mitochondrial proteins by using a 0.45 μm filter membrane to prevent the column from being blocked.
The invention preferably collects and elutes the target protein, adjusts pH to 7.4, adjusts salt concentration to 50mM, then carries on anion exchange chromatography, the said anion exchange chromatography preferably includes utilizing buffer MOPS H containing detergent and buffer MOPS L gradient elution containing detergent sequentially, collect the target protein; the buffer MOPS H is preferably a mixed solution of MOPS with the final concentration of 20mM and NaCl with the final concentration of 1M, and the pH value is 7.4; the buffer MOPS L is preferably 20mM MOPS, pH7.4. The present invention preferably further comprises, prior to performing said anion exchange chromatography, a buffer MOPS 50 equilibrium anion exchange chromatography column containing a detergent; the buffer MOPS 50 is a mixed solution of final concentration of 20mM MOPS and 50mM NaCl, and the pH value is 7.4. At equilibrium according to the invention, the volume of buffer MOPS 50 containing detergent is preferably 5 times the volume of the anion exchange chromatography column. The invention is carried out after the balancing, and the method preferably further comprises the steps of filtering the collected target protein solution by using a 0.45 mu m filter membrane to remove impurities and proteins with poor properties before the loading, and then carrying out the loading.
The invention carries out molecular sieve chromatography on the target protein solution collected from the anion exchange chromatography column, and preferably comprises balancing molecular sieve by utilizing buffer MOPS100 containing detergent before carrying out the molecular sieve chromatography; the buffer MOPS100 is preferably a mixture of final concentration 20mM MOPS and 100mM NaCl, pH7.4. At equilibrium according to the invention, the volume of buffer MOPS100 containing detergent is preferably the same as the molecular sieve chromatography volume. The molecular sieve chromatography preparation comprises a 100kDa cut-off molecular weight concentration tube for concentration before sample loading, sample loading and target protein collection.
The purification method of the human respiratory chain complex I provided in the present invention is described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
The composition of each buffer solution used in the embodiment of the invention is 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
Mitochondrial preparation
Cell density was harvested to about 3.5X10 6 HEK293F cells were cultured in a suspension culture of human source/ml, centrifuged for 10min at 140g, and the cell volume was 40ml; 100ml of 1 XPBS was added to resuspend cells, 1000g centrifuged for 10min,4℃and the cell pellet was collected and this step repeated. The cells were resuspended by adding buffer MIB in a total volume of 200ml.
Pre-cooling a glass grinder on ice, grinding the glass grinder up and down for 30 times, centrifuging for 10min at 1000g, and collecting supernatant; the sediment was resuspended in buffer MIB, the total volume was 200ml, again ground on ice 15 times, centrifuged at 1000g for 10min at 4 ℃, and the supernatant was collected. The mitochondrial suspension obtained twice was homogenized, centrifuged again at 1000g for 10min at 4℃to remove impurities and the supernatant was collected. 10000g of supernatant is centrifuged for 10min at 4 ℃ and mitochondrial precipitate is collected.
(II) preparation of protein solution
The mitochondrial pellet was resuspended in 30ml buffer MES 50, mixed well, 4ml of 10% (w/v) lauryl maltoneopentyl glycol (LMNG) was added, the volume was fixed to 40ml, and placed on an ice shaker to dissolve the membrane for 30min. After the completion of the membrane dissolution, 40000g was centrifuged for 40min, the supernatant was carefully poured out to avoid pouring out the precipitate, and the protein solution collected by centrifugation was filtered with a 0.45 μm filter membrane, and the protein sample was ready.
(III) cation exchange chromatography column
5ml of strong cation exchange column SP is taken, buffer MES 50 balances 5 column volumes, and after balancing is finished, a prepared protein sample is loaded with the flow rate of 1ml/min. The following operations are all carried outpurification was performed on the pure system with buffer MES 50+0.004% (w/v) LMNG as the system equilibrium solution, and 50-250mM NaCl salt ion concentration gradient was formed by buffer MES L+0.004% (w/v) LMNG and buffer MES H+0.004% (w/v) LMNG for elution and collection. The conductance value of the appearance position of the target protein is 8mS/cm-180mS/cm, the complex I protein is collected, buffer MOPS L+0.004% (w/v) LMNG is added to dilute to the salt concentration of about 50mM NaCl, and 1MTris-HCl pH8.0 is used to adjust the pH value to 7.2-7.4, so that the preparation for the subsequent anion exchange chromatography is carried out.
Protein solution samples the protein solution collected by centrifugation was filtered using a 0.45 μm filter membrane and the protein samples were ready.
Dynamic sampling detection during cation exchange chromatographic column elution is carried out by using invitrogen Native PAGE-16% bis-Tris Gel electrophoresis. The results of the cation exchange chromatography column are shown in FIG. 1, with the presence of complex I in the black box.
Meanwhile, the activity of the compound I is detected by adopting an NTB (negative temperature coefficient) active gel staining method, and the result is shown in figure 2, wherein a blue band is the band of the compound I.
NTB gum staining method:
20ml buffer MOPS100+0.004% (w/v) LMNG, adding an appropriate amount of tetrazolium blue chloride (NTB) powder, adding 200. Mu.l 10mM NADH to start the reaction, and staining at 37℃for 20min.
(IV) anion exchange chromatography column
Taking 1ml of strong anion column, pre-loading the column Q, balancing 5 column volumes by buffer MOPS 50, loading prepared protein sample on ice by peristaltic pump after balancing, and avoiding air intake of the ion column at a flow rate of 1ml/min, wherein the following operations are carried outpurifying on a pure system, wherein the equilibrium solution of the system is buffer MOPS 50+0.004% (w/v) LMNG, 50-450mM NaCl salt ion concentration gradient is formed by buffer MOPS L+0.004% (w/v) LMNG and buffer MOPS H+0.004% (w/v) LMNG, eluting and collecting, the appearance position of target protein is the detection conductance value of 28mS/cm-35.5mS/cm, collecting the complex I protein, and the result of an anion column is shown in figure 3. Complex I activity was detected using NTB-reactive gel staining, and the purple band was the band of complex I as shown in fig. 4. Shut off using a 100kDa cut-off tube, centrifuge 1800g, concentrate to 500 μl, remove to EP tube and prepare protein samples.
(V) gel exclusion chromatographic column
Taking 24ml superose 6increase10/300GE gel exclusion chromatographic column, balancing buffer MOPS100+0.004% (w/v) LMNG for 25ml, loading the prepared protein, and collecting complex I protein to obtain pure protein solution.
The molecular sieve results are shown in figure 5.
The purity is higher according to the gel filtration chromatography, invitrogen Native PAGE and activity detection results, and the method is suitable for structural research and biochemical research.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (3)
1. A method for purifying a human respiratory chain complex I, comprising the steps of: removing impurities from human mitochondria to obtain a solution rich in mitochondrial proteins; the impurity removal comprises the steps of re-suspending the humanized mitochondria by using a detergent of 1% (w/v) LMNG+bufferMES 50, standing for 30-120 min on ice, centrifuging for 40min with 40000g, and collecting supernatant; the buffer MES 50 is a mixed solution of 20mM MES and 50mM NaCl with the final concentration and the pH value is 6.0;
separating and purifying the solution rich in mitochondrial proteins by cation exchange chromatography, anion exchange chromatography and molecular sieve chromatography in sequence to obtain a pure humanized respiratory chain complex I;
the cation exchange chromatography comprises the step of forming gradient elution target protein by using 0.004% (w/v) LMNG+buffer MES H and 0.004% (w/v) LMNG+buffer MES L in sequence; the buffer MES H is a mixed solution of 20mM MES and 1M NaCl with the final concentration and the pH value is 6.0; the bufferMES L was 20mM MES, pH 6.0; before the cation exchange chromatography, the method further comprises balancing a cation exchange chromatography column by using 0.004% (w/v) LMNG+bufferMES 50;
the anion exchange chromatography comprises the steps of sequentially utilizing 0.004% (w/v) LMNG+bufferMOPS H and 0.004% (w/v) LMNG+bufferMOPS L to form gradient elution, and collecting target proteins; the buffer MOPS H is a mixed solution of final concentration of 20mM MOPS and 1M NaCl, and the pH value is 7.4; the buffer MOPS L is 20mM MOPS, and the pH value is 7.4; prior to performing the anion exchange chromatography, further comprising equilibrated anion exchange chromatography columns with 0.004% (w/v) lmng+buffermops 50; the bufferMOPS 50 is a mixed solution of final concentration of 20mM MOPS and 50mM NaCl, and the pH value is 7.4;
the molecular sieve chromatography is gel exclusion chromatography, and before the molecular sieve chromatography is carried out, the method further comprises the step of balancing a molecular sieve by using 0.004% (w/v) LMNG+bufferMOPS 100; the bufferMOPS100 was a mixture of final concentration 20mM MOPS and 100mM NaCl, pH7.4.
2. The method of claim 1, wherein the method of extracting human mitochondria comprises: cracking the cultured human suspension cells HEK293F, and performing differential centrifugation on the obtained cell lysate to obtain crude mitochondria;
the differential centrifugation includes: 1000g first centrifugation for 10min, collecting supernatant, and 10000g second centrifugation for 10min, collecting precipitate.
3. The purification method according to claim 1 or 2, wherein the human mitochondria are extracted from human suspension cells HEK293F.
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| CN1324938A (en) * | 2000-05-24 | 2001-12-05 | 上海博德基因开发有限公司 | New polypeptide human HADH ubiquinone oxide-reductase 21.89 and polynucleotides for encoding same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1324938A (en) * | 2000-05-24 | 2001-12-05 | 上海博德基因开发有限公司 | New polypeptide human HADH ubiquinone oxide-reductase 21.89 and polynucleotides for encoding same |
Non-Patent Citations (3)
| Title |
|---|
| Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation;James A. Letts等;《Journal of Biological Chemistry》;第291卷(第47期);全文 * |
| Resolution of the Membrane Domain of Bovine Complex I into Subcomplexes: Implications for the Structural Organization of the Enzyme;Leonid A. Sazanov等;《Biochemistry》;第39卷(第24期);全文 * |
| 线粒体呼吸链复合体I;李凤杰等;《中国细胞生物学学报》;第36卷(第8期);第1155页左栏最后一段-右栏第一段 * |
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