CN115197889A - Method for rapidly extracting active mitochondria by using nano antibody - Google Patents
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Abstract
The invention provides a method for rapidly extracting active mitochondria by using a nano antibody, belonging to the technical field of biotechnology and biomedical treatment, wherein nematodes marked with protein tags on mitochondrial outer membrane proteins are placed in a mitochondrial separating liquid and homogenized to obtain homogenate; centrifuging the homogenate at a low speed, and taking a supernatant; adding a solid phase carrier into the supernatant, incubating and cleaning to obtain mitochondria adsorbed on the solid phase carrier; the solid phase carrier is coupled with a protein label nano antibody; the protein tag nano antibody can be specifically combined with a protein tag for marking the mitochondria outer membrane protein. The method utilizes the antigen-antibody combination principle and utilizes the solid phase carrier coupled with the protein label nano antibody to adsorb mitochondria, thereby realizing selective affinity purification of the mitochondria with the corresponding protein label.
Description
Technical Field
The invention belongs to the technical field of biotechnology and biomedical treatment, and particularly relates to a method for quickly extracting active mitochondria by using a nano antibody.
Background
Mitochondria (mitochondrion) is a bilayer membrane organelle in eukaryotic cells, about 0.5 to 1 micron in diameter, that mediates aerobic oxidation of glucose, anaerobic oxidation, and the synthesis of ATP by oxidation of free fatty acids to provide energy for cell life activities is called an energy factory. Mitochondria are widely studied, and as an important organelle in cells, the functional diversity of mitochondria enables the mitochondria to participate in a large number of intracellular life activities, and is very important for maintaining the normal physiological functions of cells and responding to external stimuli of cells. Based on the diversity and complexity of mitochondrial function, it plays an important role in the development of many clinical diseases. Such as autism, muscular dystrophy and diabetes caused by mitochondrial gene mutations; there are also cardiovascular and cerebrovascular diseases, cancer and aging, etc. mediated and involved by mitochondrial dysfunction. Mitochondrial function studies have long been one of the important research hotspots in the field of cell biology.
In the study of mitochondrial function, especially mitochondrial proteins, it is often necessary to isolate mitochondria from cells and tissues. In recent decades, the mitochondria separation methods applied in scientific research are mainly sucrose density gradient centrifugation, percoll density gradient centrifugation and commercialized mitochondria separation kits. Sucrose density gradient centrifugation typically requires multiple ultracentrifugation steps, is cumbersome, requires a long time, and is wasteful of the sample. Compared with sucrose density gradient centrifugation, the Percoll density gradient centrifugation and commercialized mitochondrial separation kit reduces the cost on separation time, but the sample consumption is still not small, the mitochondrial purity is greatly reduced, and other organelles are easily mixed. Has not met the requirements of the current precise medical development.
Disclosure of Invention
In view of the above, the present invention provides a method for rapidly extracting active mitochondria by using nanobody, which can reduce the separation time of nematode mitochondria and improve the separation purity.
The invention provides a method for rapidly extracting active mitochondria by using a nano antibody, which comprises the following steps:
placing the nematodes in the mitochondrial separation liquid, and homogenizing to obtain a homogenate; centrifuging the homogenate and taking a supernatant; the centrifugal force of the centrifugation is 600-1000 g; the mitochondrial outer membrane protein of the nematode is marked with a protein tag; the centrifugation time is 8-10 min;
adding a solid phase carrier into the supernatant, incubating, and cleaning the incubated solid phase carrier by adopting a mitochondria separating medium to obtain mitochondria adsorbed on the solid phase carrier; the incubation time is 30-60 min;
the solid phase carrier is coupled with a protein label nano antibody; the protein tag nano antibody can be specifically combined with a protein tag for marking the mitochondria outer membrane protein.
Preferably, the protein tag comprises GFP, FLAG, RFP or HIS.
Preferably, the solid phase carrier comprises agarose beads or magnetic beads.
Preferably, the mitochondrial outer membrane protein comprises TOMM-20, TOMM-40 or TOMM-70.
Preferably, the incubation temperature is 0-4 ℃; the incubation time is 30-60 min.
Preferably, the temperature of the centrifugation is 0-4 ℃; the centrifugation time is 8-10 min.
Preferably, the mitochondrial separating fluid takes water as a solvent and comprises the following components in concentration: 200mM mannitol, 50mM sucrose, 10mM KCL, 10mM HEPES-KOH, 1mM EDTA and BSA with the mass volume percentage of 0.1%; the pH value of the HEPES-KOH is 7.4; the mitochondrial separation fluid further comprises a protease inhibitor; the protease inhibitor is added before the mitochondrial separation fluid is used.
Preferably, the ratio of the number of nematodes to the volume of the mitochondrial separation fluid is (5000 to 20000) one: 2mL.
Preferably, after the incubated solid phase carrier is washed by a mitochondrial separation fluid, the method further comprises washing the solid phase carrier again by KPBS; the KPBS takes water as a solvent and comprises the following components in concentration: 136mM KCl and 10mM KH 2 PO 4 (ii) a The KPBS has a pH of 7.25.
The invention provides a method for rapidly extracting active mitochondria by using a nano antibody, which comprises the steps of placing nematodes marked with protein tags by mitochondrial outer membrane proteins into mitochondrial separating liquid, and homogenizing to obtain homogenate; centrifuging the homogenate at low speed under the condition of 600-1000 g of centrifugal force, and taking supernatant; adding a solid phase carrier into the supernatant, incubating, and cleaning the incubated solid phase carrier by adopting a mitochondria separating medium to obtain mitochondria adsorbed on the solid phase carrier; the solid phase carrier is coupled with a protein label nano antibody; the protein tag nano antibody can be specifically combined with a protein tag for marking the mitochondria outer membrane protein.
The invention utilizes the principle of combining antigen and antibody and utilizes the solid phase carrier coupled with protein label nano antibody to adsorb mitochondria, thereby realizing the selective affinity purification of mitochondria with corresponding protein labels. For example, the outer mitochondrial membrane protein TOMM-20 is labeled with GFP and then can be combined with a GFP nanobody, so that the mitochondria with TOMM-20:: GFP label can be separated by the GFP nanobody.
In the invention, the preparation of materials (nematodes) is easy, the preparation of endogenous stable transgenic nematodes is more convenient than that of cells and mice, and transgenic strains marked by different mitochondrial proteins can be prepared in a short time according to experimental requirements.
The invention adopts the nano antibody as the tool for affinity purification for the first time. The nano antibody has a special antibody which is only composed of two heavy chains in camels and cartilaginous fishes, has the molecular weight of only 1/10 (about 15 KD) of that of the traditional antibody, and still has complete antigen recognition capability. Compared with the traditional antibody, the nano antibody has small molecular weight and good hydrophilicity, can be combined with sites which can not be combined by the traditional antibody, has stronger target binding specificity, is easier to modify and optimize, can be subjected to in vitro recombinant expression by escherichia coli, and is suitable for industrial large-scale production. The method is simple and convenient, and the mitochondria can be obtained after the nanobody is added into the homogenate of the mitochondria for incubation, thereby saving the loss of the mitochondria caused by multi-step centrifugation, improving the yield of the mitochondria, simultaneously ensuring the integrity of the mitochondria and effectively solving the problem of difficult separation of the mitochondria of the nematodes.
Drawings
FIG. 1 is a schematic diagram of the labeling of nematode mitochondrial outer membrane protein TOMM-20 with endogenous GFP using CRISPR technology, wherein A is the labeled TOMM-20: 10 fold of GFP; b is a green fluorescence picture shot under 100 times of laser confocal condition;
FIG. 2 shows that GFPbeads can effectively adsorb mitochondria with GFP labels in an affinity manner and verify the integrity of the mitochondria, wherein A is the change of green fluorescence in crude mitochondrial extracts before and after the GFPbeads carry out the affinity adsorption on crude mitochondrial extracts; b is the change of green fluorescence on beads after affinity adsorption of GFPbeads and IgGbaseds without GFP nano antibody; detecting wild type and TOMM-20 by immunoblotting, wherein the GFP nematode strain is separated from mitochondria by the method and then expresses GFP, a mitochondria specific protein VDAC-1, a cell nucleus specific protein HISTONE H3 and a cytoskeleton specific protein Actin; the expression of mitochondrial DNA and cell nucleus DNA of the GFP nematode strain after the mitochondria are separated by the method is shown in the specification D as RT-PCR detection of wild type and TOMM-20; e is the TOMM-20 observed by a transmission electron microscope, the GFP nematode strain utilizes the method to separate the ultrastructure of mitochondria on GFPbeads after the mitochondria;
FIG. 3 is a graph comparing the difference in mitochondrial content obtained by incubation of GFPbeads in mitochondrial homogenate (600 g later supernatant) and in crude mitochondrial extract (600 g later supernatant, 12000g later pellet); wherein A is a schematic diagram of the difference between the two in the mitochondrial separation step; FIGS. B to F are graphs comparing the difference in the amount of mitochondrial acquisition by the two methods, by counting the green fluorescence values on GFP beads, measuring the protein concentration, detecting the GFP expression level and analyzing the silver staining of the protein.
Detailed Description
The invention provides a method for rapidly extracting active mitochondria by using a nano antibody, which comprises the following steps: placing the nematodes in the mitochondrial separation liquid, and homogenizing to obtain a homogenate; centrifuging the homogenate and taking a supernatant; the centrifugal force of the centrifugation is 600-1000 g; the mitochondrial outer membrane protein of the nematode is marked with a protein tag; the centrifugation time is 8-10 min; adding a solid phase carrier into the supernatant, incubating, and cleaning the incubated solid phase carrier by adopting a mitochondria separating medium to obtain mitochondria adsorbed on the solid phase carrier; the incubation time is 30-60 min; the solid phase carrier is coupled with a protein label nano antibody; the protein tag nano antibody can be specifically combined with a protein tag for marking the mitochondria outer membrane protein.
Firstly, placing nematodes in a mitochondria separating medium, and homogenizing to obtain homogenate; centrifuging the homogenate and taking a supernatant; the centrifugal force of the centrifugation is 600-1000 g; the mitochondrial outer membrane protein of the nematode is marked with a protein tag; the centrifugation time is 8-10 min.
In the present invention, the mitochondrial separation fluid uses water as a solvent, and preferably comprises the following components in the following concentrations: 200mM mannitol, 50mM sucrose, 10mM KCL, 10mM HEPES-KOH, 1mM EDTA and BSA with mass volume percentage of 0.1%; the pH value of the HEPES-KOH is 7.4; the mitochondrial separation fluid further comprises a protease inhibitor; the protease inhibitor is added before the mitochondrial separating fluid is used.
In the present invention, the protease inhibitor is used in an amount that is conventional in the art.
In the present invention, the nematode is preferably caenorhabditis elegans; the ratio of the number of nematodes to the volume of the mitochondrial separation fluid is preferably (5000 to 20000): 1mL, more preferably 10000 strips: 1mL.
In the invention, before the nematode is placed in the mitochondrial separation liquid, the nematode is preferably washed, wherein the washing preferably comprises washing the nematode by sequentially using M9 and deionized water, the number of times of washing by using M9 is preferably 2, and the number of times of washing by using deionized water is preferably 1.
In the invention, the M9 takes water as a solvent and comprises the following components in concentration: KH (natural Kill) 2 PO 4 3g/L、Na 2 HPO 4 5.8g/L, naCl 0.5g/L and NH 4 Cl 1g/L; the water is preferably deionized water.
In the present invention, the temperature of the centrifugation is preferably 0 to 4 ℃.
After obtaining the supernatant, adding a solid phase carrier into the supernatant, incubating, and cleaning the incubated solid phase carrier by adopting a mitochondria separating medium to obtain mitochondria adsorbed on the solid phase carrier; the solid phase carrier is coupled with a protein label nano antibody; the protein tag nano antibody can be specifically combined with a protein tag for marking the mitochondrial outer membrane protein; the incubation time is 30-60 min.
In the present invention, before adding the solid phase carrier into the supernatant, the solid phase carrier coupled with the protein-labeled nanobody is preferably washed, and the reagent used for washing is preferably a mitochondrial separation fluid; the number of washing is preferably 2.
In the present invention, the solid phase carrier preferably includes agarose beads or magnetic beads.
In the present invention, the protein tag preferably includes GFP, FLAG, RFP or HIS. In the invention, the fluorescent protein has various choices, can provide various affinity purification schemes for different organelles and protein characteristics, and provides a brand-new purification tool for research on multi-organelle functions and proteins.
In one embodiment of the present invention, the solid phase carrier is agarose beads, and the solid phase carrier coupled with the GFP nanobody is commercially available from dda apack bio corporation.
In the present invention, the mitochondrial outer membrane protein preferably includes TOMM-20, TOMM-40 or TOMM-70. According to the invention, mitochondrial outer membrane protein is marked by an endogenous protein tag, and nematode mitochondria are subjected to affinity purification by using a protein tag nano antibody.
In the present invention, the incubation temperature is preferably 0 to 4 ℃; the incubation time is preferably 40 to 50min.
In the present invention, the number of times of washing the solid phase carrier after incubation with the mitochondrial separation fluid is preferably 3 to 5 times to remove unnecessary impurities and non-specific adsorption.
After the incubated solid phase carrier is cleaned by adopting a mitochondria separating medium, the invention preferably further comprises the step of cleaning the solid phase carrier again by adopting KPBS (Kernel Perkin-Biochemical) to clean more impurities and non-specific adsorption; the KPBS takes water as a solvent and comprises the following components in concentration: 136mM KCl and 10mM KH 2 PO 4 (ii) a The KPBS has a pH of 7.25. In the invention, KPBS is adopted to wash the solid phase carrier againThe number of times (c) is preferably 2 to 3.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
The TOMM-20 comprises the following steps of rapidly separating GFP Knock In (KI) nematode strain mitochondria:
s1, collecting 10000-20000 nematodes, washing in M9 for 2 times, and washing with deionized water for 1 time;
s2, adding 2ml of mitochondria separating medium, and placing on ice for homogenate;
s3, centrifuging the obtained homogenate for 10min at the temperature of 4 ℃ at 600g, and keeping a supernatant;
s4, taking 50 mu l of GFPbeads, washing with a mitochondria separating medium for 2 times, adding the supernatant, and incubating for 30min at 4 ℃ in a shaking table;
s5, washing the incubated GFPbeads for 5 times by using a mitochondria separating medium to remove redundant impurities and non-specific adsorption; the beads are combined to form purified mitochondria.
Example 2
The TOMM-20 comprises the following steps of rapidly separating GFP Knock In (KI) nematode strain mitochondria:
s1, collecting 10000-20000 nematodes, washing in M9 for 2 times, and washing with deionized water for 1 time;
s2, adding 2ml of mitochondria separating medium, and placing on ice for homogenate;
s3, centrifuging the obtained homogenate for 10min at the temperature of 4 ℃ at 600g, and keeping a supernatant;
s4, taking 50 mu l of GFPbeads, washing with a mitochondria separating medium for 2 times, adding the supernatant, and incubating for 60min at 4 ℃ in a shaking table;
s5, washing the incubated GFPbeads for 5 times by using a mitochondria separating medium to remove redundant impurities and non-specific adsorption; the beads are combined to form purified mitochondria.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (9)
1. A method for rapidly extracting active mitochondria by using a nanobody comprises the following steps:
placing the nematodes in the mitochondrial separation liquid, and homogenizing to obtain a homogenate; centrifuging the homogenate and taking a supernatant; the centrifugal force of the centrifugation is 600-1000 g; the mitochondrial outer membrane protein of the nematode is marked with a protein tag; the centrifugation time is 8-10 min;
adding a solid phase carrier into the supernatant, incubating, and cleaning the incubated solid phase carrier by adopting a mitochondria separating medium to obtain mitochondria adsorbed on the solid phase carrier; the incubation time is 30-60 min;
the solid phase carrier is coupled with a protein label nano antibody; the protein tag nano antibody can be specifically combined with a protein tag for marking the mitochondria outer membrane protein.
2. The method of claim 1, wherein the protein tag comprises GFP, FLAG, RFP or HIS.
3. The method of claim 1, wherein the solid support comprises agarose beads or magnetic beads.
4. The method of claim 1, wherein the mitochondrial outer membrane protein comprises TOMM-20, TOMM-40, or TOMM-70.
5. The method according to claim 1, wherein the incubation temperature is 0 to 4 ℃; .
6. The method of claim 1, wherein the temperature of the centrifugation is 0 to 4 ℃.
7. The method of claim 1, wherein the mitochondrial separation fluid is water as a solvent and comprises the following concentrations of components: 200mM mannitol, 50mM sucrose, 10mM KCL, 10mM HEPES-KOH, 1mM EDTA and BSA with mass volume percentage of 0.1%; the pH value of the HEPES-KOH is 7.4; the mitochondrial separation fluid further comprises a protease inhibitor; the protease inhibitor is added before the mitochondrial separation fluid is used.
8. The method of claim 1, wherein the ratio of the number of nematodes to the volume of mitochondrial separation fluid is (5000-20000): 2mL.
9. The method of claim 1, wherein after the step of washing the incubated solid phase carrier with a mitochondrial separation fluid, further comprising washing the solid phase carrier again with KPBS; the KPBS takes water as a solvent and comprises the following components in concentration: 136mM KCl and 10mM KH 2 PO 4 (ii) a The KPBS has a pH of 7.25.
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| CN102827276A (en) * | 2011-06-14 | 2012-12-19 | 北京华大蛋白质研发中心有限公司 | Antibody of cytochrome b5 type B and application thereof in purifying mitochondria |
| CN109265566A (en) * | 2018-10-16 | 2019-01-25 | 生工生物工程（上海）股份有限公司 | Purification process, kit and its application of 6 × HIS fusion protein |
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