A method for isolating and purifying sarcoplasmic reticulum calcium pump vesicles

Abstract

The application discloses a sarcoplasmic reticulum calcium pump vesicle separation and purification method and relates to the technical field of biology.The sarcoplasmic reticulum calcium pump vesicle separation and purification method provided by the application combines differential centrifugation, sucrose discontinuous density gradient centrifugation and gel exclusion chromatography in sequence, can accurately separate and purify the sarcoplasmic reticulum calcium pump vesicle from skeletal muscle, and the vesicle is complete in structure, full in shape, clear in outline and uniform in size, which is favorable for high-resolution cryo-EM structural analysis of the sarcoplasmic reticulum calcium pump protein SERCA1, and lays a good foundation for the study of the mechanism of the enzyme catalyzing ATP hydrolysis, calcium transfer from the cytoplasm to the sarcoplasmic reticulum lumen and muscle excitation and contraction.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for separating and purifying sarcoplasmic reticulum calcium pump vesicles. Background Technology

[0002] The sarcoplasmic reticulum of skeletal muscle controls the concentration of free calcium in the sarcoplasm, thereby mediating muscle contraction and relaxation. The calcium pump is an important protein pump that maintains the difference in calcium ion concentration between the inside and outside of the cell. SERCA is a transmembrane 10-way cation-transporting ATPase with a tetramer structure. Its N2 and C2 ends are located in the cytoplasm, resembling a spherical structure. It actively pumps cytoplasmic calcium into the sarcoplasmic reticulum and is also known as the "calcium pump."

[0003] SERCA1 (a calcium pump) is primarily found in skeletal muscle cells, located on the sarcoplasmic reticulum membrane within the cells, and regulates the concentration difference of calcium ions across the sarcoplasmic reticulum. Muscle contraction occurs when calcium ions are transported out of the sarcoplasmic reticulum; muscle relaxation occurs when calcium ions are transported into the sarcoplasmic reticulum. SERCA1 transports calcium ions from the cytoplasm to the sarcoplasmic reticulum, triggering muscle relaxation. Due to the intracellular membrane system and its respective transport activities, studies of ion transport within whole muscle cells are complex.

[0004] The ability to isolate and purify the calcium pumps in the sarcoplasmic reticulum is of great biological significance for studying the structure of calcium pumps in the sarcoplasmic reticulum of skeletal muscle and the mechanism of calcium ion transport. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to separate and purify the calcium pump on the sarcoplasmic reticulum.

[0006] To address the above problems, the present invention proposes the following technical solution:

[0007] A method for isolating and purifying sarcoplasmic reticulum calcium pump vesicles includes the following steps:

[0008] Step 1: Take skeletal muscle tissue, remove connective tissue, and cut into minced meat;

[0009] Step 2: Mix the minced meat with the lysis solution and homogenize to obtain a homogenate; wherein the mass-to-volume ratio of minced meat to lysis solution is 0.1-2.0 g: 3 ml;

[0010] Step 3: Centrifuge the homogenate at 2–8°C and 1,000–5,000 g for 10–30 min to obtain supernatant S1; filter supernatant S1 and centrifuge the filtrate at 2–8°C and 10,000–30,000 g for 10–30 min to obtain supernatant S2; filter supernatant S2 and centrifuge the filtrate at 2–8°C and 100,000–300,000 g for 30–90 min to obtain precipitate P3;

[0011] Step 4: Resuspend the precipitate P3 in buffer solution to obtain resuspension R1; add 0.8-1.8M discontinuous density sucrose solution and resuspension R1 to the superposition tube in sequence, and centrifuge for 1.5-3.0h at a temperature of 2-8℃ and a centrifugal force of 100,000-200,000g.

[0012] Step 5: Remove 2 / 5 to 1 / 2 of the solution from the superposition tube from top to bottom and discard it. The remaining solution in the superposition tube is concentrated in a 30-60 kDa retention concentration tube and then further separated by gel size exclusion chromatography. The eluted fraction between 6.0 and 12.0 mL is the sarcoplasmic reticulum calcium pump vesicle.

[0013] A further technical solution is that, in step two, the lysis solution contains 20-30 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 6.8-7.2), 0.2-0.3 M sucrose, and 0.2-0.6 mM dithiothreitol.

[0014] A further technical solution is that, in step one, the volume of the minced meat is less than 2mm. 3 .

[0015] A further technical solution is that, in step three, the supernatant S1 is filtered with 2 to 6 layers of gauze; the supernatant S2 is filtered with 2 to 6 layers of gauze.

[0016] A further technical solution is that, in step four, the buffer solution contains 20-30 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 6.8-7.2), 0.5-0.8 M potassium chloride, 0.2-0.4 M sucrose, and 0.2-0.6 mM dithiothreitol.

[0017] A further technical solution is that, in step four, the sucrose solution is prepared from sucrose and a buffer solution, wherein the buffer solution contains 0.1–0.3 M potassium chloride, 4–6 mM adenine nucleoside triphosphate, 4–6 mM magnesium chloride, 1–3 mM calcium chloride, 1–3 mM ethylene glycol diethyl ether diaminetetraacetic acid, 45–55 mM potassium phosphate, and pH 7.2–7.6.

[0018] A further technical solution is that, in step four, the discontinuous density sucrose solution includes sucrose solutions with concentrations of 0.8M, 1.1M, 1.3M, and 1.6M.

[0019] A further technical solution is that, in step four, the precipitate P3 is resuspended in a buffer solution, and homogenization and ultrasonic disruption are also performed to obtain a resuspension R1.

[0020] A further technical solution is that, in step five, the chromatography column used in the gel size exclusion chromatography is a Superose 610 / 300GL.

[0021] A further technical solution is that, in step five, the buffer solution used in the gel exclusion chromatography contains 15-25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid at pH 7.2-7.5 and 90-110 mM sodium chloride.

[0022] Compared with the prior art, the technical effects achieved by the present invention include:

[0023] The present invention provides a method for isolating and purifying sarcoplasmic reticulum calcium pump vesicles. Utilizing differential centrifugation and discontinuous density gradient centrifugation combined with gel exclusion chromatography, this method accurately isolates and purifies sarcoplasmic reticulum calcium pump vesicles from skeletal muscle. The vesicles are structurally intact, well-formed, clearly defined, and uniform in size. This facilitates high-resolution cryo-electron microscopy for structural analysis of the sarcoplasmic reticulum calcium pump protein SERCA1. It lays a solid foundation for studying the mechanism by which this enzyme catalyzes ATP hydrolysis and the transfer of calcium from the cytoplasm to the sarcoplasmic reticulum lumen, and its involvement in muscle excitation and contraction. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic flowchart of the method for separating and purifying sarcoplasmic reticulum calcium pump vesicles provided in Embodiment 1 of the present invention;

[0026] Figure 2 The SDS-PAGE gel electrophoresis result of precipitate P3 obtained in step three of Example 1 of this invention;

[0027] Figure 3 This refers to the SDS-PAGE gel electrophoresis results of density gradient centrifugation samples taken in step four of Example 1 of this invention;

[0028] Figure 4 The results of gel size exclusion chromatography and SDS-PAGE gel electrophoresis in step five of Example 1 of this invention;

[0029] Figure 5 This is an electron micrograph of the sarcoplasmic reticulum calcium pump vesicles of component A4 obtained in Example 1 of the present invention;

[0030] Figure 6 This is an electron micrograph of the sarcoplasmic reticulum calcium pump vesicles of component A5 obtained in Example 1 of the present invention;

[0031] Figure 7 This is an electron micrograph of the sarcoplasmic reticulum calcium pump vesicle of component A6 obtained in Example 1 of the present invention. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0034] It should also be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0035] This invention provides a method for isolating and purifying sarcoplasmic reticulum calcium pump vesicles, comprising the following steps:

[0036] Step 1: Take skeletal muscle tissue, remove connective tissue, and cut into minced meat.

[0037] In this embodiment of the invention, the skeletal muscle from which connective tissue has been removed is cut into small square pieces of 0.5cm × 0.5cm × 0.5cm using surgical scissors, placed on ice, and moved to the next step as soon as possible.

[0038] Step 2: Mix the minced meat with the lysis solution and homogenize to obtain a homogenate; wherein the mass-volume ratio of minced meat to lysis solution is 0.1-2.0g:3ml.

[0039] In this embodiment, the process also includes washing the above-mentioned minced meat two to three times with pre-cooled ultrapure water in a 4°C cold room, then pouring the washed minced meat into a homogenizer, adding lysis solution, mixing and homogenizing for 30 seconds, pausing for 30 seconds, and repeating 5 times to obtain a homogenate.

[0040] Step 3: Centrifuge the homogenate at 2–8°C and 1,000–5,000 g for 10–30 min to obtain supernatant S1; filter supernatant S1 and centrifuge the filtrate at 2–8°C and 10,000–30,000 g for 10–30 min to obtain supernatant S2; filter supernatant S2 and centrifuge the filtrate at 2–8°C and 100,000–300,000 g for 30–90 min to obtain precipitate P3.

[0041] In this embodiment, differential centrifugation is used to separate the target product. After the first centrifugation, the cell nuclei and large cell debris in the homogenate are precipitated. After the second centrifugation, the mitochondria are removed. After the third centrifugation, the endoplasmic reticulum, Golgi apparatus, cell membrane and other components in the precipitate are resuspended and further purified by discontinuous sucrose density gradient.

[0042] Step 4: Resuspend the precipitate P3 in buffer solution to obtain resuspension R1; add 0.8-1.8M discontinuous density sucrose solution and resuspension R1 to the supernatant tube in sequence, and centrifuge for 1.5-3.0h at a temperature of 2-8℃ and a centrifugal force of 100,000-200,000g.

[0043] Since the precipitate P3 obtained after differential centrifugation still contains impurities, the separation and purification method for precipitate P3 needs to be adjusted. In this embodiment, a discontinuous sucrose density gradient method is used to further purify precipitate P3.

[0044] In this embodiment, after resuspending the precipitate P3 in the buffer solution, it is necessary to further sonicate it to improve the purification efficiency.

[0045] In this embodiment, the sucrose solution is prepared from sucrose and a buffer solution containing 0.1–0.3 M potassium chloride, 4–6 mM adenine nucleoside triphosphate, 4–6 mM magnesium chloride, 1–3 mM calcium chloride, 1–3 mM ethylene glycol diethyl ether diaminetetraacetic acid, 45–55 mM potassium phosphate, and pH 7.2–7.6.

[0046] Step 5: Remove 2 / 5 to 1 / 2 of the solution from the superposition tube from top to bottom and discard it. The remaining solution in the superposition tube is concentrated in a 30-60 kDa retention concentration tube and then further separated by gel size exclusion chromatography. The eluted fraction between 6.0 and 12.0 mL is the sarcoplasmic reticulum calcium pump vesicle.

[0047] It should be noted that mass spectrometry identification of the separated calcium pump vesicles confirmed that the molecular weight of the sarcoplasmic reticulum calcium pump vesicles was 118 kDa. Due to the purification process involving differential centrifugation and density gradient centrifugation, the solution concentrated in the concentration tube in step five contained sarcoplasmic reticulum calcium pump vesicles and a small amount of impurities. Further separation by gel size exclusion chromatography, combined with SDS-PAGE gel electrophoresis analysis, confirmed that the bands with molecular weights between 100-140 kDa were sarcoplasmic reticulum calcium pump vesicles, with corresponding elution fractions between 6.0 mL and 12.0 mL, preferably between 6.4 and 10.0 mL.

[0048] To improve the purity and uniformity of calcium pump vesicles, this embodiment uses gel exclusion chromatography to further separate the target sample after density gradient centrifugation.

[0049] The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles provided by this invention utilizes differential centrifugation and discontinuous density gradient centrifugation, combined with gel exclusion chromatography, to accurately separate and purify sarcoplasmic reticulum calcium pump vesicles from skeletal muscle. The vesicles have intact structures, full shapes, clear outlines, and uniform sizes.

[0050] Example 1

[0051] This embodiment provides a method for the isolation and purification of sarcoplasmic reticulum calcium pump vesicles. The flowchart is shown below. Figure 1 The following is a detailed description of each step:

[0052] 1. Take 100g of skeletal muscle, remove the connective tissue, and cut the skeletal muscle tissue into small pieces of 0.5cm×0.5cm×0.5cm square using surgical scissors. Place them on ice and proceed to the next step of fragmentation as soon as possible.

[0053] 2. In a 4°C cold room, wash the tissue two to three times with pre-cooled ultrapure water. Pour the tissue into a homogenizer and add tissue lysis buffer (25mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid pH 7.0, 0.29M sucrose, 0.5mM dithiothreitol) to the 300mL mark. Homogenize for 30s, pause for 30s, and repeat 5 times.

[0054] 3. Add the skeletal muscle tissue lysis solution to a 50 mL BD centrifuge tube, centrifuge at 3,000 × g for 20 min at 4 °C to allow cell nuclei and large cell debris to settle, obtaining precipitate P1, and retain the supernatant S1.

[0055] 4. After filtering the supernatant S1 through four layers of gauze, the filtrate is centrifuged a second time at 20,000×g for 20 min at 4℃ to remove the precipitate P2 and obtain the supernatant S2.

[0056] 5. After filtering the supernatant S2 through four layers of gauze, the filtrate is centrifuged for the third time at 200,000×g for 1 hour at 4℃. The supernatant S3 is discarded to obtain precipitate P3.

[0057] Samples of the supernatant S1, precipitate P1, supernatant S2, precipitate P2, supernatant S3, and precipitate P3 were taken and subjected to SDS-PAGE gel electrophoresis. The results are shown in the figure. Figure 2 The results showed that precipitate P3 contained few contaminating protein bands, and the protein molecular weights were mainly concentrated in the 100-140 kDa range, consistent with the molecular weight of the target protein of this invention, sarcoplasmic reticulum calcium pump vesicle (SERCA1). The obtained precipitate P3 was further purified in the following steps.

[0058] 6. Resuspend the precipitate P3 in buffer solution A (25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, pH 7.0, 0.65 M potassium chloride, 0.29 M sucrose, 0.5 mM dithiothreitol), homogenize for 30 s, pause for 30 s, repeat 5 times. Then sonicate on ice for 3 s, pause for 8 s, for a total of 3 min to obtain resuspended solution R1.

[0059] 7. Using an ultracentrifuge tube, slowly add 2 mL of resuspended solution R1 to a sucrose solution with a discontinuous density gradient from top to bottom: 0.8 M, 1.1 M, 1.3 M, and 1.6 M. Add 2.5 mL of each density of sucrose solution and centrifuge at 25000×g for 2 h at 4℃.

[0060] In this embodiment, the sucrose solution is prepared as follows:

[0061] Prepare a buffer solution containing 0.15M potassium chloride, 5mM adenine nucleoside triphosphate, 5mM magnesium chloride, 2mM calcium chloride, 2mM ethylene glycol diethyl ether diaminetetraacetic acid, 50mM potassium phosphate, pH 7.4.

[0062] To prepare a 0.8M sucrose solution: Dissolve 0.8M sucrose in a buffer solution.

[0063] To prepare a 1.1M sucrose solution: Dissolve 1.1M sucrose in a buffer solution.

[0064] To prepare a 1.3M sucrose solution: Dissolve 1.3M sucrose in a buffer solution.

[0065] To prepare a 1.6M sucrose solution: Dissolve 1.6M sucrose in a buffer solution.

[0066] 8. After centrifugation, sort the ultracentrifugation tubes from top to bottom, 1 mL per tube, for a total of 12 tubes. Take 10 μL of each sample from each of the 12 tubes, add 5× loading buffer, mix thoroughly, heat at 95℃ for 10 minutes, cool to room temperature, centrifuge at 12000×g for 5 minutes, and then perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results are as follows... Figure 3 As shown.

[0067] The results showed that the components in tubes 7-12 were the purest and contained the most protein, indicating that after density gradient centrifugation, the target protein was located in the middle and lower layers of the supercritical tube.

[0068] 9. Combine the fractions from tubes 7 to 12, concentrate to a final volume of 1 mL using a 50 kDa concentration tube, and further separate the target protein using Superose 610 / 300GL size exclusion chromatography (molecular sieve). The molecular sieve buffer used was 20 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 7.4) and 100 mM sodium chloride. The molecular sieve elution results are as follows: Figure 4 As can be seen, there is only one highest UV absorption peak in elution volumes between 6.0 mL and 12.0 mL, and no other impurity peaks were observed as the elution volume increased, indicating that the final eluted components obtained in this example have high purity. Further SDS-PAGE gel electrophoresis analysis was performed on eluted components A2, A3, A4, A5, A6, A7, A8, A9, and A10, and the molecular weights of the bands were between 100 and 140 kDa.

[0069] The bands A4, A5, and A6 of the above components were cut out and sent for mass spectrometry identification. The product was confirmed to be calcium ATPase1 (SERCA1), which is a calcium pump (sarcoplasmic reticulum calcium pump vesicle) with a molecular weight of 118 kDa.

[0070] Negative staining of components A4, A5, and A6 was evaluated using a 120kV transmission electron microscope. Figure 5-7 As shown, the calcium pump vesicles obtained by the method of this invention are in good condition, with intact structure, full shape, clear outline, uniform size, and few impurities.

[0071] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0072] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for isolating and purifying sarcoplasmic reticulum calcium pump vesicles, characterized in that, The sarcoplasmic reticulum calcium pump vesicles have a molecular weight between 100-140 kDa and include the following steps: Step 1: Take skeletal muscle tissue, remove connective tissue, and cut into minced meat; Step 2: Mix the minced meat with the lysis solution and homogenize to obtain a homogenate; wherein the mass-to-volume ratio of minced meat to lysis solution is 0.1~2.0g:3ml; Step 3: Centrifuge the homogenate at 2-8℃ and 1,000-5,000g for 10-30 min to obtain supernatant S1; filter supernatant S1 and centrifuge the filtrate at 2-8℃ and 10,000-30,000g for 10-30 min to obtain supernatant S2; filter supernatant S2 and centrifuge the filtrate at 2-8℃ and 100,000-300,000g for 30-90 min to obtain precipitate P3; Step 4: Resuspend the precipitate P3 in buffer solution to obtain resuspension R1; add 0.8~1.8 M discontinuous density sucrose solution and resuspension R1 to the superposition tube in sequence, and centrifuge for 1.5~3.0 h at a temperature of 2~8℃ and a centrifugal force of 100,000~200,000g. Step 5: Remove 2 / 5 to 1 / 2 of the solution from the superposition tube from top to bottom and discard it. The remaining solution in the superposition tube is concentrated by a 30 to 60 kDa retention concentration tube and then further separated by gel size exclusion chromatography. The eluted fraction between 6.0 and 12.0 mL is the sarcoplasmic reticulum calcium pump vesicle. In step four, the sucrose solution is prepared from sucrose and a buffer solution containing 0.1-0.3 M potassium chloride, 4-6 mM adenine nucleoside triphosphate, 4-6 mM magnesium chloride, 1-3 mM calcium chloride, 1-3 mM ethylene glycol diethyl ether diaminetetraacetic acid, 45-55 mM potassium phosphate, and pH 7.2-7.

6. The chromatography column used in the gel size exclusion chromatography method is a Superose 6 10 / 300 GL.

2. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles as described in claim 1, characterized in that, In step two, the lysis buffer contains 20-30 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 6.8-7.2), 0.2-0.3 M sucrose, and 0.2-0.6 mM dithiothreitol.

3. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles as described in claim 1, characterized in that, In step one, the volume of the minced meat is less than 2 mm. 3 .

4. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles as described in claim 1, characterized in that, In step three, the supernatant S1 is filtered with 2 to 6 layers of gauze; the supernatant S2 is filtered with 2 to 6 layers of gauze.

5. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles as described in claim 1, characterized in that, In step four, the buffer solution contains 20-30 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 6.8-7.2), 0.5-0.8 M potassium chloride, 0.2-0.4 M sucrose, and 0.2-0.6 mM dithiothreitol.

6. The method for isolating and purifying sarcoplasmic reticulum calcium pump vesicles as described in claim 1, characterized in that, In step four, the discontinuous density sucrose solutions include 0.8 M, 1.1 M, 1.3 M, and 1.6 M sucrose solutions.

7. The method for isolating and purifying sarcoplasmic reticulum calcium pump vesicles as described in claim 1, characterized in that, In step four, the precipitate P3 is resuspended in a buffer solution, and homogenization and ultrasonic disruption are also performed to obtain a resuspension R1.

8. The method for isolating and purifying sarcoplasmic reticulum calcium pump vesicles as described in claim 1, characterized in that, In step five, the buffer solution used in the gel exclusion chromatography contains 15-25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid at pH 7.2-7.5 and 90-110 mM sodium chloride.

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