Separation and purification method of sarcoplasmic reticulum calcium pump vesicles

Abstract

The invention discloses a separation and purification method of sarcoplasmic reticulum calcium pump vesicles, and relates to the technical field of biology. According to the separation and purification method of the sarcoplasmic reticulum calcium pump vesicles, differential centrifugation, sucrose discontinuous density gradient centrifugation and gel exclusion chromatography are sequentially combined, the sarcoplasmic reticulum calcium pump vesicles can be accurately separated and purified from skeletal muscles, and the vesicles are complete in structure, full in shape, clear in outline and uniform in size. The structural analysis of the sarcoplasmic reticulum calcium pump protein SERCA1 by a high-resolution freezing electron microscope is facilitated. A good foundation is laid for researching the mechanism that the enzyme catalyzes ATP hydrolysis, calcium is transferred from cytoplasm to a sarcoplasmic reticulum cavity and participates in muscle excitation and contraction.

Description

Technical Field

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

[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 inside and outside the cell. SERCA is a cation transporting ATPase that spans the membrane 10 times. Its structure is a tetramer, and both the N2 end and the C2 end are located in the cytoplasm. It is a spherical structure that can actively pump cytoplasmic calcium into the sarcoplasmic reticulum, and is also called a "calcium pump."

[0003] SERCA1 (calcium pump) is mainly present in skeletal muscle cells, located on the membrane of the sarcoplasmic reticulum within skeletal muscle cells, regulating the concentration difference of calcium ions inside and outside the sarcoplasmic reticulum. When calcium ions are transported out of the sarcoplasmic reticulum, the muscle contracts; when calcium ions are transported into the sarcoplasmic reticulum, the muscle relaxes. SERCA1 transports calcium ions from the cytoplasm to the sarcoplasmic reticulum, triggering muscle relaxation. The study of sarcolemmal ion transport using whole muscle cells is complicated by the intracellular membrane system and its respective transport activities.

[0004] How to isolate and purify the calcium pump on the sarcoplasmic reticulum is of great biological significance for studying the calcium pump structure of the sarcoplasmic reticulum of skeletal muscle and the calcium ion transport mechanism. Summary of the invention

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

[0006] In order to solve the above problems, the present invention proposes the following technical solutions:

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

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

[0009] Step 2: Mixing and homogenizing the minced meat and the lysate to obtain a homogenate; wherein the mass volume ratio of the minced meat to the lysate is 0.1-2.0 g:3 ml;

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

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

[0012] Step 5: Take out 2 / 5 to 1 / 2 of the volume of the solution in the ultracentrifuge tube from top to bottom and discard it. The remaining solution in the ultracentrifuge tube is concentrated by a 30-60 kDa cutoff concentration tube and further separated by gel exclusion chromatography. The elution component between 6.0 and 12.0 mL is the sarcoplasmic reticulum calcium pump vesicles.

[0013] A further technical solution is that in step 2, the lysate contains 20-30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 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 1, the volume of the minced meat is less than 2 mm 3 .

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

[0016] A further technical solution is that in step 4, the buffer contains 20-30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 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] Its further technical scheme is that in the step 4, the sucrose solution is prepared from sucrose and a buffer solution, and the buffer solution contains 0.1-0.3M potassium chloride, 4-6mM adenine nucleoside triphosphate, 4-6mM magnesium chloride, 1-3mM calcium chloride, 1-3mM ethylene glycol diethyl ether diamine tetraacetic acid, 45-55mM potassium phosphate, and pH 7.2-7.6.

[0018] A further technical solution is that in step 4, the discontinuous density sucrose solution includes a sucrose solution with a concentration of 0.8M, a sucrose solution with a concentration of 1.1M, a sucrose solution with a concentration of 1.3M, and a sucrose solution with a concentration of 1.6M.

[0019] A further technical solution is that in the step 4, the precipitate P3 is resuspended in a buffer solution, and homogenization and ultrasonic crushing 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 exclusion chromatography is Superose 610 / 300GL.

[0021] A further technical solution is that in step five, the buffer used in the gel exclusion chromatography method contains 15-25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 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 separation and purification method of sarcoplasmic reticulum calcium pump vesicles provided by the present invention utilizes the methods of 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, and the vesicles have complete structure, full shape, clear outline and uniform size. It is conducive to the structural analysis of sarcoplasmic reticulum calcium pump protein SERCA1 by high-resolution cryo-electron microscopy. It lays a good foundation for studying the mechanism of the enzyme catalyzing ATP hydrolysis and calcium transfer from the cytoplasm to the sarcoplasmic reticulum cavity, and participating in muscle excitation and contraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0025] Figure 1 A schematic diagram of the process of separating and purifying sarcoplasmic reticulum calcium pump vesicles provided in Example 1 of the present invention;

[0026] Figure 2 The SDS-PAGE gel electrophoresis result of the precipitate P3 obtained in step 3 of Example 1 of the present invention;

[0027] Figure 3 The SDS-PAGE gel electrophoresis result of density gradient centrifugation sampling in step 4 of Example 1 of the present invention;

[0028] Figure 4 The results of gel exclusion chromatography and SDS-PAGE gel electrophoresis in step 5 of Example 1 of the present invention;

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

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

[0031] Figure 7 This is an electron microscopic image of the sarcoplasmic reticulum calcium pump vesicles of the A6 component obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] 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. Obviously, the embodiments described below 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.

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

[0034] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0035] The embodiment of the present invention provides a method for separating and purifying sarcoplasmic reticulum calcium pump vesicles, comprising the following steps:

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

[0037] In the embodiment of the present invention, the skeletal muscle with the connective tissue removed was cut into small pieces of 0.5 cm×0.5 cm×0.5 cm square using surgical scissors, placed on ice, and the next step was performed as soon as possible.

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

[0039] In this embodiment, the minced meat is washed two to three times with pre-cooled ultrapure water in a 4°C cold room, and then the washed minced meat is poured into a homogenizer, a lysis solution is added, and the mixture is homogenized for 30 seconds, and then paused for 30 seconds, and the mixture is repeated 5 times to obtain a homogenate.

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

[0041] This embodiment uses differential centrifugation to separate the target product. After the first centrifugation, the cell nuclei and large cell fragments in the homogenate can be precipitated; after the second centrifugation, the mitochondria are removed; after the third centrifugation, the endoplasmic reticulum, Golgi body, 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 a buffer solution to obtain a resuspension R1; add a 0.8-1.8 M discontinuous density sucrose solution and the resuspension R1 to the ultracentrifuge tube in sequence, and centrifuge for 1.5-3.0 h at a temperature of 2-8° C. and a centrifugal force of 100,000-200,000 g.

[0043] Since the precipitate P3 obtained after differential centrifugation still contains impurities, it is necessary to adjust the separation and purification method of the precipitate P3. In this embodiment, the precipitate P3 is further purified using a discontinuous sucrose density gradient method.

[0044] In this embodiment, after the precipitate P3 is resuspended in the buffer solution, it needs to be further ultrasonically disrupted to improve the purification efficiency.

[0045] The sucrose solution in this embodiment is prepared from sucrose and a buffer solution, wherein the buffer solution contains 0.1-0.3M potassium chloride, 4-6mM adenosine triphosphate, 4-6mM magnesium chloride, 1-3mM calcium chloride, 1-3mM ethylene glycol diethyl ether diamine tetraacetic acid, 45-55mM potassium phosphate, and pH 7.2-7.6.

[0046] Step 5: Take out 2 / 5 to 1 / 2 of the volume of the solution in the ultracentrifuge tube from top to bottom and discard it. The remaining solution in the ultracentrifuge tube is concentrated by a 30-60 kDa cutoff concentration tube and further separated by gel exclusion chromatography. The elution component between 6.0 and 12.0 mL is the sarcoplasmic reticulum calcium pump vesicles.

[0047] It should be noted that, by mass spectrometry identification of the separated calcium pump vesicles, it was finally confirmed that the molecular weight of the sarcoplasmic reticulum calcium pump vesicles was 118 kDa. Due to the purification by the aforementioned differential centrifugation and density gradient centrifugation, the solution concentrated in the concentrator in step 5 contains sarcoplasmic reticulum calcium pump vesicles and a small amount of impurities. The components obtained by further separation by gel exclusion chromatography, combined with SDS-PAGE gel electrophoresis analysis, can be confirmed that the components with a molecular weight of 100-140 kDa are sarcoplasmic reticulum calcium pump vesicles, and the corresponding elution components are between 6.0 mL and 12.0 mL, and the preferred elution components are between 6.4 and 10.0 mL.

[0048] In order to improve the purity and homogeneity of calcium pump vesicles, in this embodiment, the target sample after density gradient centrifugation is further separated by gel exclusion chromatography.

[0049] The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles provided by the present invention utilizes differential centrifugation and discontinuous density gradient centrifugation methods combined with gel exclusion chromatography, and can accurately separate and purify sarcoplasmic reticulum calcium pump vesicles from skeletal muscle, and the vesicles have a complete structure, a full shape, a clear outline, and a uniform size.

[0050] Example 1

[0051] This embodiment provides a method for separating and purifying sarcoplasmic reticulum calcium pump vesicles, and the steps are shown in the flowchart. 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 crushing 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, add tissue lysis buffer (25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 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 lysate to a 50 mL BD centrifuge tube and centrifuge at 3,000 × g for 20 min at 4°C to precipitate the cell nuclei and large cell debris to obtain the precipitate P1 and keep the supernatant S1.

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

[0056] 5. After the supernatant S2 is filtered through four layers of gauze, the filtrate is centrifuged for the third time at 200,000×g for 1 h at 4°C, and the supernatant S3 is discarded to obtain the precipitate P3.

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

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

[0059] 7. Use an ultracentrifuge tube to slowly add 2 mL of resuspension R1 to a discontinuous density gradient sucrose solution with a concentration gradient of 0.8 M, 1.1 M, 1.3 M, and 1.6 M from top to bottom, 2.5 mL of each density sucrose solution, and centrifuge at 25,000 × g for 2 h at 4°C.

[0060] In this embodiment, the preparation method of sucrose solution is as follows:

[0061] A buffer solution was prepared containing 0.15 M potassium chloride, 5 mM adenosine triphosphate, 5 mM magnesium chloride, 2 mM calcium chloride, 2 mM ethylene glycol diethyl ether diamine tetraacetic acid, 50 mM potassium phosphate, pH 7.4.

[0062] Prepare 0.8 M sucrose solution: Dissolve 0.8 M sucrose in buffer solution.

[0063] Prepare 1.1 M sucrose solution: Dissolve 1.1 M sucrose in buffer solution.

[0064] Prepare 1.3 M sucrose solution: Dissolve 1.3 M sucrose in buffer solution.

[0065] Prepare 1.6 M sucrose solution: Dissolve 1.6 M sucrose in buffer solution.

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

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

[0068] 9. Combine the components from tubes 7 to 12, concentrate to a final volume of 1 mL using a 50 kDa cutoff concentrator, and further separate the target protein using Superose 610 / 300GL gel exclusion chromatography (molecular sieve) with a molecular sieve buffer of 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid pH 7.4, 100 mM sodium chloride. The results of molecular sieve elution are shown in the following table. Figure 4 It can be seen that there is only one highest ultraviolet absorption peak between 6.0mL and 12.0mL of elution volume, and no other impurity peaks are seen with the increase of elution volume, indicating that the components finally eluted in this embodiment have high purity. SDS-PAGE gel electrophoresis analysis of eluted components A2, A3, A4, A5, A6, A7, A8, A9 and A10 shows that the molecular weight of the band is between 100-140kDa.

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

[0070] The three components A4, A5 and A6 were negatively stained and evaluated using a 120 kV transmission electron microscope, e.g. Figure 5-7 It shows that the calcium pump vesicles obtained by the method of the present invention are in good condition, have complete structure, full shape, clear outline, uniform size and few impurities.

[0071] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0072] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for separating and purifying sarcoplasmic reticulum calcium pump vesicles, characterized in that: The following steps are involved: Step 1: Take skeletal muscle tissue, remove connective tissue, and cut into minced meat; Step 2: Mixing and homogenizing the minced meat and the lysate to obtain a homogenate; wherein the mass volume ratio of the minced meat to the lysate is 0.1-2.0 g:3 ml; Step 3: centrifuge the homogenate at a temperature of 2 to 8°C and a centrifugal force of 1,000 to 5,000 g for 10 to 30 min to obtain supernatant S1; filter the supernatant S1, and centrifuge the filtrate at a temperature of 2 to 8°C and a centrifugal force of 10,000 to 30,000 g for 10 to 30 min to obtain supernatant S2; filter the supernatant S2, and centrifuge the filtrate at a temperature of 2 to 8°C and a centrifugal force of 100,000 to 300,000 g for 30 to 90 min to obtain precipitate P3; Step 4: resuspend the precipitate P3 in a buffer to obtain a resuspension R1; add a 0.8-1.8M discontinuous density sucrose solution and the resuspension R1 to the ultracentrifuge tube in sequence, and centrifuge for 1.5-3.0 hours at a temperature of 2-8°C and a centrifugal force of 100,000-200,000g; Step 5: Take out 2 / 5 to 1 / 2 of the volume of the solution in the ultracentrifuge tube from top to bottom and discard it. The remaining solution in the ultracentrifuge tube is concentrated by a 30-60 kDa cutoff concentration tube and further separated by gel exclusion chromatography. The elution component between 6.0 and 12.0 mL is the sarcoplasmic reticulum calcium pump vesicles.

2. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles according to claim 1, characterized in that: In the step 2, the lysate contains 20-30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at 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 according to claim 1, characterized in that: In step 1, the volume of the minced meat is less than 2 mm 3 .

4. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles according to claim 1, characterized in that: In the step 3, the supernatant S1 is filtered through 2 to 6 layers of gauze; and the supernatant S2 is filtered through 2 to 6 layers of gauze.

5. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles according to claim 1, characterized in that: In step 4, the buffer contains 20-30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 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 separating and purifying sarcoplasmic reticulum calcium pump vesicles according to claim 1, characterized in that: In the step 4, the sucrose solution is prepared from sucrose and a buffer solution, wherein the buffer solution contains 0.1-0.3M potassium chloride, 4-6mM adenine nucleoside triphosphate, 4-6mM magnesium chloride, 1-3mM calcium chloride, 1-3mM ethylene glycol diethyl ether diamine tetraacetic acid, 45-55mM potassium phosphate, and pH 7.2-7.

6.

7. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles according to claim 6, characterized in that: In the step 4, the discontinuous density sucrose solution includes a sucrose solution with a concentration of 0.8 M, a sucrose solution with a concentration of 1.1 M, a sucrose solution with a concentration of 1.3 M, and a sucrose solution with a concentration of 1.6 M.

8. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles according to claim 1, characterized in that: In the step 4, the precipitate P3 is resuspended in a buffer solution, and homogenization and ultrasonic disruption are performed to obtain a resuspension R1.

9. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles according to claim 1, characterized in that: In the step 5, the chromatography column used in the gel exclusion chromatography is Superose 610 / 300GL.

10. The method for separating and purifying sarcoplasmic reticulum calcium pump vesicles according to claim 1, characterized in that: In the step 5, the buffer used in the gel exclusion chromatography method contains 15-25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 7.2-7.5, and 90-110 mM sodium chloride.

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