Mitochondrial membrane protein peptide and mitochondrial separation method

Abstract

The invention discloses a linear mitochondrial separation method, which comprises the following steps: step one, expressing mitochondrial protein peptide with a modified amino acid sequence of NH2-Gly-Gly-Gly-Phe-(D) Arg-Phe-Lys-Phe-(D) Arg-Phe-Lys (Ac)-CON-H2 on a microsphere, so as to obtain a polypeptide modified microsphere with closed GLy, and modifying the mitochondrial protein peptide with the modified amino acid sequence of NH2-Gly-Gly-Gly-Phe-(D) Arg-Phe-Lys-Phe-(D) Arg-Phe-Lys (Ac)-CON-H2; and 2, carrying out mitochondrial extraction on a target tissue by using the GLy-closed polypeptide modified microspheres. The purity of the obtained mitochondria is high, pollution of subcellular organelles and cell debris except the mitochondria is avoided to a certain extent, and the phenomenon that the detection result deviates due to the purity of the mitochondria when the mitochondria with the high purity is detected by a high-sensitivity detection instrument is avoided. And the cost of the mitochondria obtained by the method is controllable, and stable and reliable results can be brought to experiments.

Description

Technical Field

[0001] The invention relates to the technical field of mitochondrial extraction, in particular to a mitochondrial membrane protein peptide and a mitochondrial separation method. Background Art

[0002] Mitochondria isolation kits are laboratory tools used to extract purified mitochondria from cells. Such kits usually contain all the necessary buffers, centrifugation steps, and protocols, allowing researchers to efficiently isolate mitochondria from different types of tissue or cell samples. Mitochondria are the energy factories of cells and are essential for studying biological processes such as energy metabolism, oxidative stress, and apoptosis.

[0003] As the main organelle for cellular material and energy metabolism, mitochondria play a key role in maintaining cellular physiological homeostasis, and are closely related to the occurrence and development of many diseases. Specific isolation and analysis of mitochondria from highly complex cellular components is of great significance for the study of their functional molecular mechanisms and chemical intervention, but there are still difficulties and challenges.

[0004] Currently, existing mitochondrial isolation kits, ultracentrifugation density gradient centrifugation and other technologies are widely used in mitochondrial separation, but there are still problems such as limited product purity and susceptibility to contamination and interference by impurities such as cell debris. In addition, the existing detection instruments have high precision and sensitivity, which can easily lead to deviations in test results when the samples are impure. For this reason, we propose a mitochondrial membrane protein peptide and a mitochondrial separation method. Summary of the invention

[0005] The purpose of the present invention is to provide a mitochondrial membrane protein peptide and a mitochondrial separation method to solve the problems that need to be solved in the background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a mitochondrial membrane protein peptide, the amino acid sequence of the mitochondrial membrane protein peptide is: NH2-Gly-Gly-Gly-Phe-(D)Arg-Phe-Lys-Phe-(D)Arg-Phe-Lys(Ac)-CON-H2.

[0007] As a further description of the above technical solution: A method for isolating mitochondria, comprising the following steps: Step 1: Modify the mitochondrial protein peptide on the surface of the microspheres to obtain GLy-blocked peptide-modified microspheres, and store the peptide-modified microspheres in ultrapure water; Step 2: Use GLy-blocked polypeptide-modified microspheres to extract mitochondria from the target tissue to obtain separated mitochondria.

[0008] Currently, existing mitochondrial separation kits, ultracentrifugation density gradient centrifugation and other technologies are widely used in mitochondrial separation, but there are still problems such as limited product purity and susceptibility to contamination and interference by impurities such as cell debris, and the existing detection instruments have high precision and sensitivity, which can easily lead to deviations in the detection results when the sample is impure. The present invention is simple to operate and takes a short time. The mitochondria obtained by this method have high purity, which to a certain extent avoids the contamination of subcellular organelles and cell fragments outside the mitochondria, and the mitochondria with high purity will not cause deviations in the detection results due to mitochondrial purity when detected by high-sensitivity detection instruments. The mitochondria obtained by this method have controllable costs, can bring stable and reliable results to the experiment, and are convenient for various downstream experimental operations for mitochondrial research. In addition, the technology of the present invention adopts the principle of mitochondrial membrane protein-specific affinity adsorption, without centrifugation, and directly enriches and separates by adsorption, with higher enrichment efficiency and better purity, providing a better foundation for the next step of mitochondrial function and molecular mechanism research and protein metabolism research.

[0009] As a further description of the above technical solution: The microsphere surface modification polypeptide in step 1 comprises the following steps: Step A1: Take 5±0.3 mg of microspheres containing sulfate groups and aldehyde groups, and add 500 uL of 0.4 mg / ml mitochondrial protein peptide aqueous solution; Step A2: After mixing evenly, add 5 ml of reducing agent and react for 12-13 hours. The N-terminal amino group of the mitochondrial protein peptide reacts with the aldehyde group on the microsphere to undergo aminomethylation reaction, thereby bonding the peptide to the surface of the microsphere; Step A3: Add 500uL of 1mol / L glycine aqueous solution, react for 2±0.5h, and wash with ultrapure water 3-5 times to obtain GLy-blocked polypeptide-modified microspheres, and store the obtained GLy-blocked polypeptide-modified microspheres in ultrapure water.

[0010] As a further description of the above technical solution: The extraction of mitochondria from tissue in step 2 comprises the following steps: Step S1: Weigh 50-100 ml of target tissue and add PBS to wash at least once; Step S2: Cut the target tissue for extraction, add 500±3uL of homogenate, mix well, and use a glass homogenizer to break the cells to obtain tissue homogenate; Step S3: Weigh 600±5 g of tissue homogenate and centrifuge at 4±0.5°C for 5-10 min; Step S4: Take 200uL of the supernatant from the tissue homogenate into a centrifuge tube; Step S5: Add 1 mg of peptide-modified microspheres to the centrifuge tube and incubate at 4±0.5°C for 2-3 hours; Step S6: centrifuge the tissue homogenate and polypeptide-modified beads in the centrifuge tube, discard the supernatant after centrifugation, retain the precipitate, and then add 200±5uL of buffer to the centrifuge tube for washing, so that mitochondria can be obtained.

[0011] As a further description of the above technical solution: In the step A2, the reducing agent is sodium cyanoborohydride.

[0012] As a further description of the above technical solution: In the step A2, after adding the reducing agent, the reaction is carried out at 20-25° C. for 12-13 hours. In the step A3, after adding the glycine aqueous solution, the reaction is carried out at 20-25° C. for 2±0.5 hours.

[0013] As a further description of the above technical solution: The 500uL homogenate added in step S2 contains 10mmol / L potassium dihydrogen phosphate and 136mmol / L potassium chloride, and the pH value of the homogenate is 7.25.

[0014] As a further description of the above technical solution: The 200uL buffer added in step S6 contains 10mmol / L potassium dihydrogen phosphate and 136mmol / L potassium chloride, and the pH value of the buffer is 7.25.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention is simple to operate and takes a short time. The mitochondria obtained by the method have a high purity, which avoids the contamination of subcellular organelles and cell fragments outside the mitochondria to a certain extent. The mitochondria with high purity will not cause deviations in the test results due to the purity of the mitochondria when tested by a high-sensitivity detection instrument. The mitochondria obtained by the method have a controllable cost, can bring stable and reliable results to the experiment, and are convenient for various downstream experimental operations for mitochondrial research; 2. The technology of the present invention adopts the principle of specific affinity adsorption of mitochondrial membrane proteins. It does not require centrifugation and directly enriches and separates by adsorption. It has higher enrichment efficiency and better purity, providing a better foundation for the next step of mitochondrial function and molecular mechanism research and protein metabolism research. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the front view structure of the present invention; Figure 2It is a schematic diagram comparing the specific capture ability of mitochondria of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Embodiment 1:

[0018] See also Figure 1 and Figure 2 The present invention provides a technical solution: a mitochondrial membrane protein peptide, the amino acid sequence of the mitochondrial membrane protein peptide is: NH2-Gly-Gly-Gly-Phe-(D)Arg-Phe-Lys-Phe-(D)Arg-Phe-Lys(Ac)-CON-H2.

[0019] The preparation of mitochondrial membrane protein peptides includes the following steps: Step B1: Dissolve Fmoc-amino acid, HBTU and HOBt in a DMF solution containing 0.4 mol / L N-methylmorpholine in an amount of 4 times the theoretical amount of amino acid loaded on the resin to activate the amino acid; Step B2: The activated amino acid solution is added to the resin for coupling, and the deprotection and coupling steps are repeated to complete the coupling of all amino acids; Step B3: Using a mixed solution of hydroxylamine hydrochloride (1.8 mmol) and imidazole (1.35 mmol) (the solvent is 5 mL of N-methylpyrrolidone and 1 ml of dichloromethane) as a removal agent for the Dde protecting group of the C-terminal lysine side chain, adding a mixed solution of acetic anhydride / pyridine / DMF (1:1:1:8, V / V / V) to acetylate the amino group of the C-terminal lysine side chain; Step B4: preparing a pre-cooled lysis solution, wherein the lysis solution includes TFA, water and TIS, and the volume ratio of TFA, water and TIS is 95:2.5:2.5, cleaving the polypeptide from the solid phase resin, and collecting the lysis solution; Step B5: Most of the TFA was removed by rotary evaporator, and pre-cooled ether was added to precipitate the polypeptide, and the mitochondrial membrane protein peptide was obtained after centrifugation and vacuum drying.

[0020] The mitochondrial membrane protein peptide obtained by the above manufacturing method uses the mitochondrial penetrating peptide that specifically interacts with the mitochondrial membrane as the recognition element. The introduction of Gly-Gly-Gly (glycine triad) in the manufactured mitochondrial membrane protein peptide can be regarded as a space arm molecule, because glycine has the smallest side chain, which enables it to form a flexible connection area in the peptide chain, which helps to increase the spatial stretchability of the peptide, and the (D)Arg-Phe-Lys-Phe-(D)Arg-Phe-Lys(Ac) part is designed as a ligand with specific affinity. This part contains some positively charged arginine residues (Arg) and hydrophobic phenylalanine (Phe), as well as an acetylated lysine (Lys(Ac)). These characteristics combined together make the peptide segment have a higher affinity for certain specific receptors or proteins, and then the polymer matrix microspheres rich in aldehyde groups on the surface are used as affinity matrix to capture mitochondria. Embodiment 2:

[0021] A method for isolating mitochondria, comprising the following steps: Step 1: Modify the mitochondrial protein peptide on the surface of the microspheres to obtain GLy-blocked peptide-modified microspheres, and store the peptide-modified microspheres in ultrapure water; Step 2: Use GLy-blocked polypeptide-modified microspheres to extract mitochondria from the target tissue to obtain separated mitochondria.

[0022] Among them, when separating mitochondria, a mitochondrial protein peptide with an amino acid sequence of NH2-Gly-Gly-Gly-Phe-(D)Arg-Phe-Lys-Phe-(D)Arg-Phe-Lys(Ac)-CON-H2 is used as a manufacturing raw material, and the principle of mitochondrial membrane protein-specific affinity adsorption is adopted. No centrifugation is required, and the enrichment and separation are directly carried out by adsorption. The enrichment efficiency is higher and the purity is better. In the process of mitochondrial extraction, GLy-blocked polypeptide-modified microspheres can be first obtained, and the obtained GLy-blocked polypeptide-modified microspheres can be used to extract mitochondria from the target tissue. The mitochondria extracted by the GLy-blocked polypeptide-modified microspheres are enriched and separated by direct adsorption, which has a higher enrichment efficiency and better purity, and no centrifugation step is required during the extraction process. Embodiment three:

[0023] The microsphere surface modification polypeptide in step 1 comprises the following steps: Step A1: Take 5 mg of microspheres containing sulfate groups and aldehyde groups, and add 500 uL of 0.4 mg / ml mitochondrial protein peptide aqueous solution; Step A2: After mixing evenly, add 5 ml of reducing agent and react for 12.5 hours. The N-terminal amino group of the mitochondrial protein peptide reacts with the aldehyde group on the microsphere to undergo aminomethylation reaction, thereby bonding the peptide to the surface of the microsphere; Step A3: Add 500uL of 1mol / L glycine aqueous solution, react for 2h, and wash 4 times with ultrapure water to obtain GLy-blocked polypeptide-modified microspheres, and store the obtained GLy-blocked polypeptide-modified microspheres in ultrapure water.

[0024] The extraction of mitochondria from tissue in step 2 comprises the following steps: Step S1: Weigh 75 ml of target tissue and add PBS to wash at least once; Step S2: Cut the target tissue for extraction, add 500uL of homogenate, mix well, and use a glass homogenizer to break the cells to obtain tissue homogenate; Step S3: Weigh 600 g of tissue homogenate and centrifuge at 4°C for 8 min; The centrifugation step in this step is aimed at removing cell nuclei, tissue debris and unlysed cells; Step S4: Take 200uL of the supernatant from the tissue homogenate into a centrifuge tube; Step S5: Add 1 mg of peptide-modified microspheres to the centrifuge tube and incubate at 4°C for 2.5 h; Step S6: centrifuge the tissue homogenate and polypeptide-modified beads in the centrifuge tube, discard the supernatant after centrifugation, retain the precipitate, and then add 200uL of buffer to the centrifuge tube for washing, so that mitochondria can be obtained.

[0025] In the step A2, the reducing agent is sodium cyanoborohydride.

[0026] Among them, the reducing agent is sodium cyanoborocyanide, which is used to provide the reducing agent required for the methylation reaction. Sodium cyanoborohydride reducing agent is a commonly used mild reducing agent. Under specific conditions, it can effectively reduce imines, ketones or aldehyde compounds to corresponding amines or alcohols without significantly affecting other functional groups, thereby preventing damage to mitochondrial membranes or other sensitive cell components.

[0027] In the step A2, the reducing agent is added and the reaction is carried out at 23° C. for 12.5 hours. In the step A3, the glycine aqueous solution is added and the reaction is carried out at 23° C. for 2 hours.

[0028] The 500uL homogenate added in step S2 contains 10mmol / L potassium dihydrogen phosphate and 136mmol / L potassium chloride, and the pH value of the homogenate is 7.25.

[0029] The 200uL buffer added in step S6 contains 10mmol / L potassium dihydrogen phosphate and 136mmol / L potassium chloride, and the pH value of the buffer is 7.25.

[0030] Among them, the buffer with specific composition added in the above steps S2 and S6 is very important because it helps to maintain the structural and functional integrity of the organelles, ensures the pressure difference between the inside and outside of the mitochondrial membrane, and prevents mitochondrial rupture. Embodiment 4:

[0031] The microsphere surface modification polypeptide in step 1 comprises the following steps: Step A1: Take 4.7 mg of microspheres containing sulfate groups and aldehyde groups, and add 500 uL of 0.4 mg / ml mitochondrial protein peptide aqueous solution; Step A2: After mixing evenly, add 5 ml of reducing agent and react for 12 hours. The N-terminal amino group of the mitochondrial protein peptide reacts with the aldehyde group on the microsphere to undergo aminomethylation reaction, thereby bonding the peptide to the surface of the microsphere; Step A3: Add 500uL of 1mol / L glycine aqueous solution, react for 1.5h, and wash with ultrapure water 3 times to obtain GLy-blocked polypeptide-modified microspheres, and store the obtained GLy-blocked polypeptide-modified microspheres in ultrapure water.

[0032] The extraction of mitochondria from tissue in step 2 comprises the following steps: Step S1: Weigh 50 ml of target tissue and add PBS to wash at least once; Step S2: Cut the target tissue for extraction, add 497uL of homogenate, mix well, and use a glass homogenizer to break the cells to obtain tissue homogenate; Step S3: weigh 595 g of tissue homogenate and centrifuge at 3.5°C for 5 min; Step S4: Take 200uL of the supernatant from the tissue homogenate into a centrifuge tube; Step S5: Add 1 mg of peptide-modified microspheres to the centrifuge tube and incubate at 3.5°C for 2 h; Step S6: centrifuge the tissue homogenate and polypeptide-modified beads in the centrifuge tube, discard the supernatant after centrifugation, retain the precipitate, and then add 195uL of buffer to the centrifuge tube for washing, so that mitochondria can be obtained.

[0033] In the step A2, the reducing agent is sodium cyanoborohydride.

[0034] In the step A2, the reducing agent is added and the reaction is carried out at 20° C. for 12 hours. In the step A3, the glycine aqueous solution is added and the reaction is carried out at 20° C. for 1.5 hours. Embodiment five:

[0035] The microsphere surface modification polypeptide in step 1 comprises the following steps: Step A1: Take 5.3 mg of microspheres containing sulfate groups and aldehyde groups, and add 500 uL of 0.4 mg / ml mitochondrial protein peptide aqueous solution; Step A2: After mixing evenly, add 5 ml of reducing agent and react for 13 hours. The N-terminal amino group of the mitochondrial protein peptide reacts with the aldehyde group on the microsphere to undergo aminomethylation reaction, thereby bonding the peptide to the surface of the microsphere; Step A3: Add 500uL of 1mol / L glycine aqueous solution, react for 2.5h, and wash 5 times with ultrapure water to obtain GLy-blocked polypeptide-modified microspheres, and store the obtained GLy-blocked polypeptide-modified microspheres in ultrapure water.

[0036] The extraction of mitochondria from tissue in step 2 comprises the following steps: Step S1: Weigh 100 ml of target tissue and add PBS to wash at least once; Step S2: Cut the target tissue for extraction, add 503uL of homogenate, mix well, and use a glass homogenizer to break the cells to obtain tissue homogenate; Step S3: Weigh 600±5 g of tissue homogenate and centrifuge at 4.5°C for 10 min; Step S4: Take 200uL of the supernatant from the tissue homogenate into a centrifuge tube; Step S5: Add 1 mg of peptide-modified microspheres to the centrifuge tube and incubate at 4.5°C for 3 h; Step S6: centrifuge the tissue homogenate and polypeptide-modified beads in the centrifuge tube, discard the supernatant after centrifugation, retain the precipitate, and then add 205uL of buffer to the centrifuge tube for washing, so that mitochondria can be obtained.

[0037] In the step A2, the reducing agent is sodium cyanoborohydride.

[0038] In the step A2, the reducing agent is added and the reaction is carried out at 25° C. for 13 hours. In the step A3, the glycine aqueous solution is added and the reaction is carried out at 25° C. for 2.5 hours.

[0039] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A mitochondrial membrane protein peptide, characterized in that: The amino acid sequence of the mitochondrial membrane protein peptide is: NH2-Gly-Gly-Gly-Phe-(D)Arg-Phe-Lys-Phe-(D)Arg-Phe-Lys(Ac)-CON-H2.

2. A method for isolating mitochondria using the mitochondrial membrane protein peptide according to claim 1, characterized in that: The mitochondrial isolation method comprises the following steps: Step 1: Modify the mitochondrial protein peptide on the surface of the microspheres to obtain GLy-blocked peptide-modified microspheres, and store the peptide-modified microspheres in ultrapure water; Step 2: Use GLy-blocked polypeptide-modified microspheres to extract mitochondria from the target tissue to obtain separated mitochondria.

3. A mitochondrial isolation method according to claim 2, characterized in that: The microsphere surface modification polypeptide in step 1 comprises the following steps: Step A1: Take 5±0.3 mg of microspheres containing sulfate groups and aldehyde groups, and add 500 uL of 0.4 mg / ml mitochondrial protein peptide aqueous solution; Step A2: After mixing evenly, add 5 ml of reducing agent and react for 12-13 hours. The N-terminal amino group of the mitochondrial protein peptide reacts with the aldehyde group on the microsphere to undergo aminomethylation reaction, thereby bonding the peptide to the surface of the microsphere; Step A3: Add 500uL of 1mol / L glycine aqueous solution, react for 2±0.5h, and wash with ultrapure water 3-5 times to obtain GLy-blocked polypeptide-modified microspheres, and store the obtained GLy-blocked polypeptide-modified microspheres in ultrapure water.

4. A mitochondrial isolation method according to claim 3, characterized in that: The extraction of mitochondria from tissue in step 2 comprises the following steps: Step S1: Weigh 50-100 ml of target tissue and add PBS to wash at least once; Step S2: Cut the target tissue for extraction, add 500±3uL of homogenate, mix well, and use a glass homogenizer to break the cells to obtain tissue homogenate; Step S3: Weigh 600±5 g of tissue homogenate and centrifuge at 4±0.5°C for 5-10 min; Step S4: Take 200uL of the supernatant from the tissue homogenate into a centrifuge tube; Step S5: Add 1 mg of peptide-modified microspheres to the centrifuge tube and incubate at 4±0.5°C for 2-3 hours; Step S6: centrifuge the tissue homogenate and polypeptide-modified beads in the centrifuge tube, discard the supernatant after centrifugation, retain the precipitate, and then add 200±5uL of buffer to the centrifuge tube for washing, so that mitochondria can be obtained.

5. A mitochondrial isolation method according to claim 4, characterized in that: In the step A2, the reducing agent is sodium cyanoborohydride.

6. A mitochondrial isolation method according to claim 5, characterized in that: In the step A2, after adding the reducing agent, the reaction is carried out at 20-25° C. for 12-13 hours. In the step A3, after adding the glycine aqueous solution, the reaction is carried out at 20-25° C. for 2±0.5 hours.

7. A method for isolating mitochondria according to claim 6, characterized in that: The 500uL homogenate added in step S2 contains 10mmol / L potassium dihydrogen phosphate and 136mmol / L potassium chloride, and the pH value of the homogenate is 7.

25.

8. A mitochondrial isolation method according to claim 7, characterized in that: The 200uL buffer added in step S6 contains 10mmol / L potassium dihydrogen phosphate and 136mmol / L potassium chloride, and the pH value of the buffer is 7.25.

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