Preparation method of nanopore membrane protein

Abstract

The invention discloses a preparation method of a nanopore membrane protein, and relates to the field of nanopore detection technology protein preparation, and the preparation method comprises the following steps: 1, customizing a synthesized protein coding gene, and constructing plasmid DNA in a corresponding vector; 2, transferring the plasmid DNA vector into an escherichia coli expression strain by using a heat shock method, and carrying out induced culture for expression and purification of a target protein; step 3, ultrasonically crushing the thalli, and then purifying by magnetic beads to obtain membrane protein; and 4, carrying out electrophoresis characterization and nanopore forming and translocation detection. According to the method, a method combining crushing and magnetic bead purification is adopted, complex nickel column purification is not needed, the manual operation time is shortened, the single-batch preparation cost is reduced, the preparation efficiency and quality of the membrane protein are greatly improved, mass production of the membrane protein can be achieved, and the purity of the protein is improved.

Description

Technical Field

[0001] The invention belongs to the field of protein preparation using nanopore detection technology, and in particular relates to a method for preparing nanopore membrane protein. Background Art

[0002] In recent years, nanopore membrane proteins have shown great potential for application in scientific research and medicine as sensors, sequencers or single-molecule reactors. These nanopore membrane proteins can accurately identify and manipulate single biomolecules, thus making it possible to achieve efficient biological detection and drug development.

[0003] In the preparation process of nanopore membrane proteins, liquid chromatography technology (such as nano-liquid chromatography) plays a key role. This technology can effectively separate the target protein from complex biological samples by precisely controlling the flow and separation of the sample in the chromatographic column. However, the application of liquid chromatography technology usually relies on expensive protein chromatographs (FPLC) and corresponding nickel columns. The FPLC system drives the mobile phase through a high-pressure pump to adsorb, desorb and separate the sample in the chromatographic column. The nickel column uses the affinity of transition metal nickel with specific amino acids (such as histidine) in the protein to achieve specific adsorption and separation of the target protein. Although this combination can prepare high-purity nanopore membrane proteins, it still has many shortcomings. For example, the FPLC system usually takes a long time to complete a protein separation process, and its low-throughput characteristics limit its ability to process a large number of samples; and the FPLC system and its supporting nickel column are expensive, which increases the cost of preparing nanopore membrane proteins; the operation of the FPLC system is relatively complicated and requires professional technicians to operate and maintain. Once the nickel column purification process is operated incorrectly, it may cause protein contamination and affect the preparation quality. Therefore, the present invention provides a method for optimizing purification by combining ultrasonic disruption of bacteria with magnetic bead purification to solve the above problems. Summary of the invention

[0004] In view of the defects and problems of the prior art, such as high cost, complicated operation, insufficient preparation quality and efficiency, the present invention provides a method for preparing nanopore membrane protein. The present invention adopts a method combining crushing and magnetic bead purification, does not require complicated nickel column purification, can effectively reduce the time of manual operation and the cost of single batch preparation, greatly improves the preparation efficiency and quality of membrane protein, can realize mass production of membrane protein, and improves the purity of protein.

[0005] The solution adopted by the present invention to solve the technical problem is: a method for preparing a nanopore membrane protein, comprising the following steps: Step 1: Custom-synthesize a specific protein-coding gene and construct plasmid DNA on the corresponding vector, adding a hexahistidine tag to the C-terminus of the gene for nickel affinity purification; Step 2: Use the heat shock method to transfer the plasmid DNA containing the protein coding gene into the Escherichia coli expression strain and induce the culture; use 0.5-1mM isopropyl β-D-thiogalactoside (IPTG) as an inducer at 16-20°C for 6-8 hours to induce expression; Step 3: Collect the induced expression bacteria, use ultrasound to break the bacteria to release the target protein, and then heat the cell lysate at 65℃~90℃, and purify the target membrane protein with His-tag using magnetic beads; Step 4: Characterize the purified protein by SDS-PAGE electrophoresis to verify the purity and size of the protein; use a patch clamp detection system to detect the pore formation of the nanopores formed by the purified protein, and detect the translocation of small molecule biological substances through the nanopores.

[0006] Furthermore, in step 1, one of the commonly used nanopore membrane proteins among αHL, MspA, Omp family, FraC, CsgG, FhuA or Aerolysin can be selected; and a hexahistidine tag is added to the C-terminus of the nanopore membrane protein located on the outer surface of the pore vestibule.

[0007] Furthermore, in the step 1, the recombinant plasmid uses a pET series vector as an expression vector.

[0008] Furthermore, the broken bacterial cells in step 3 were dissolved with breaking buffer (4 M Urea, 50 mM Tris, 300 mM NaCl, pH 8.0), and the cells were ultrasonically broken in an ice bath at a power of 60 W for 20 min, with 2 s of ultrasonication and a pause of 6 s as one cycle; after the ultrasonication, the metal bath was heated at 90° C. for 10 min, and then centrifuged at 12000 rpm, 4° C. for 40 min, and then the supernatant was collected for magnetic bead purification.

[0009] Furthermore, the magnetic beads in step 3 are Ni 2+Magnetic beads, after adding the supernatant to the magnetic beads, shake on a rotating mixer at room temperature (RT) for 60 min; after magnetic separation, discard the supernatant; then add 1 mL of wash buffer A (0.5 M NaCl, 20 mM HEPES, 5 mM imidazole, 0.5% (w / v) Genapol X-80, pH = 8.0); resuspend the beads at room temperature and shake on a rotating mixer (RT) for 5 min; after magnetic separation, discard the supernatant; after magnetic separation, discard the supernatant; then add 100 µL of elution buffer B1 (0.5 M NaCl, 20 mM HEPES, 219.5 mM imidazole, 0.5% (w / v) Genapol X-80, pH = 8.0); resuspend the beads and shake again at room temperature (RT) for 5 min; after magnetic separation, discard the supernatant; finally, add 100 µL of elution buffer B2 (0.5 M NaCl, 20 mM HEPES, 500 mM imidazole, 0.5% (w / v) Genapol X-80, pH = 8.0); the tube was shaken on a rotating mixer at room temperature (RT) for 5 min; after magnetic separation, the supernatant was collected; the supernatant contained the target nanopore membrane protein and could be used for all downstream nanopore measurements.

[0010] Furthermore, in step 4, the purified protein was characterized by SDS-PAGE electrophoresis, as follows: prepare 12% SDS-PAGE, Tris-Gly electrophoresis buffer, load 10 µL, concentrate gel 80 V 20 min, separate gel 120 V, 60 min, after the gel electrophoresis, Coomassie brilliant blue staining for 20 min, and take pictures using white light after decolorization.

[0011] Furthermore, the nanopore formation detection in step 4 is a patch clamp detection system: two half-pools of equal volume are separated in the fluid pool, namely the Cis end and the Trans end, with a 150 μm channel hole between the two, a certain amount of conductive buffer and amphiphilic lipid solution are added to the half-pool, thereby forming a phospholipid bilayer near the channel hole, and a certain amount of prepared membrane protein is added to the Cis end to autonomously form a nanopore single channel; the two half-pools are respectively connected to separate Ag / AgCl electrodes and electrically connected to the amplifier Axopatch 200B to form a closed loop; the Axopatch 200B patch clamp can record and amplify the ion current signal generated by a single molecule passing through the nanopore; the translocation and docking signals use a 5kHz quadrupole Bessel low-pass filter with a sampling frequency of 25kHz, and the entire device is placed in a Faraday cage containing a copper box to minimize the coupling of external electromagnetic noise.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The preparation method of the nanopore membrane protein provided by the present invention adopts a method combining ultrasonic disruption of bacteria and magnetic bead purification for purification optimization, thereby significantly reducing the cost and complexity of preparing the nanopore membrane protein. The present invention adds a hexa-histidine tag (His-tag) to the C-terminus of the nanopore membrane protein located on the outer surface of the pore vestibule without interfering with the assembly and sensing characteristics of the membrane protein. Due to the addition of the hexa-histidine tag (His-tag), the purification of the membrane protein can be carried out using charged nickel-coated magnetic beads, thereby avoiding FPLC.

[0013] Furthermore, the present invention takes into account the thermal stability of membrane proteins. The resuspended cells containing nanopore membrane proteins are thoroughly heated before purifying cations, thereby effectively dissolving the cells to increase the yield. In addition, heat treatment can also effectively eliminate those thermally unstable contaminating proteins.

[0014] The supernatant collected after magnetic bead purification in the method of the present invention contains the desired nanopore membrane protein, and according to the gel electrophoresis characterization results, these proteins are pure enough to be used for all downstream nanopore measurements without any further purification. Compared with traditional FPLC, the method using ultrasound and magnetic bead purification is simpler, reduces manual operation time, is easy to operate and implement, and thus improves the preparation efficiency of membrane proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the nanopore detection principle of the present invention.

[0016] Figure 2 This is the result of SDS-PAGE protein purification; Lane M: Protein Marker; Lane 1: supernatant after fragmentation.

[0017] Figure 3 Figure 2 is a representative current signal generated when ssDNA passes through the MspA nanopore under a bias voltage of 100 mV. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0019] Example 1: Preparation of nanopore membrane protein This embodiment provides a method for preparing a nanopore membrane protein, wherein the nanopore membrane protein is one of commonly used pore-forming membrane proteins such as αHL, MspA, Omp family, FraC, CsgG, FhuA, Aerolysin, etc. In this embodiment, MspA is taken as an example, and the method for preparing a nanopore membrane protein specifically comprises the following steps: (1) Synthesis of membrane protein MspA plasmid DNA The coding gene of MspA was customized and synthesized, and its amino acid residue sequence is shown in SEQ ID NO.1; (SEQ ID NO.1) And they were constructed in pET-30a (+) plasmid DNA. At the C-terminus of each gene, located on the outer surface of the pore vestibule, a hexahistidine tag (His-tag) was added for nickel affinity purification, that is, by adding a hexahistidine tag (His-tag), the purification of membrane proteins can be performed using charged nickel-coated magnetic beads.

[0020] (2) Plasmid culture, expression and purification The heat shock method was used to transfer 100 ng of plasmid DNA encoding MspA into 100 µL of Escherichia coli BL21 (DE3) competent cells. The transformation steps were as follows: heat shock at 42°C for 90 s, standing on ice for 2 min, then spreading on solid LB medium (LB BorthAgar) plates (100 µg / mL kanamycin sulfate), and culturing in a 37°C incubator overnight.

[0021] (3) Take a single colony and add it to a 50 mL culture tube containing 15 mL LB (Luria-Bertani) and add 3 mg / mL kanamycin sulfate. Shake the tube (175 rpm) at 37°C for 5 hours. Then induce with 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) and shake at 16°C (175 rpm) overnight. Then, collect the cells by centrifugation (4000 rpm, 20°C, 20 min).

[0022] (4) Ultrasonic disruption of bacteria The collected bacterial cells were dissolved in the disruption buffer (4 M Urea, 50 mM Tris, 300 mM NaCl, pH 8.0), and the cells were disrupted by ultrasound in an ice bath at a power of 400 W for 15 min (ultrasound for 2 s, pause for 6 s as one cycle). After the ultrasound, the metal bath was heated at 90°C for 10 min, and then centrifuged at 13,000 rpm, 4°C, for 40 min, and the supernatant was collected.

[0023] (5) Magnetic bead purification Ni 2+ Pretreatment of magnetic beads: Ni 2+ The magnetic beads were treated with PBS (pH 7.4) containing 20% ​​ethanol solution. Before using the magnetic beads, 100 μL of the bead solution was transferred to a 1.5 mL centrifuge tube. The supernatant was discarded after magnetic separation. 1 mL of LE buffer (50 mM NaH2PO4, 300 mM NaCl, pH = 8.0) was added to the magnetic beads, vortexed for 30 seconds, and the supernatant was discarded after magnetic separation. Wash with LE buffer 3 times, and finally discard the LE buffer. After pretreatment, the magnetic beads are ready for use.

[0024] The supernatant of the bacterial lysate was collected and pretreated Ni 2+ Magnetic beads (bacteria solution: magnetic beads = 20:1), shake on a room temperature rotating mixer (RT) for 60 min. After magnetic separation, discard the supernatant. Then add 1 mL of washing buffer (0.5 M NaCl, 20 mM HEPES, 5 mM imidazole, 0.5% (w / v) Genapol X-80, pH = 8.0).

[0025] Resuspend the beads at room temperature and shake on a rotating mixer for 5 min at RT. After magnetic separation, discard the supernatant. Then add 100 µL of elution buffer B (0.5 M NaCl, 20 mM HEPES, 219.5 mM imidazole, 0.5% (w / v) Genapol X-80, pH=8.0). Resuspend the beads and shake for another 5 min at room temperature (RT). After magnetic separation, discard the supernatant. Finally, add 100 µL of elution buffer C (0.5 M NaCl, 20 mM HEPES, 500 mM imidazole, 0.5% (w / v) Genapol X-80, pH=8.0). Shake the tube for 5 min at room temperature (RT). After magnetic separation, collect the supernatant. Based on the gel electrophoresis characterization results, the collected supernatant contains the desired nanopore membrane protein (MspA octamer form) and can be used for all downstream nanopore measurements without any further purification.

[0026] (6) SDS-PAGE detection Prepare 12% SDS-PAGE gel, Tris-Gly electrophoresis buffer, load 10 µL, concentrate gel 80 V for 20 min, separate gel 120 V for 60 min, stain with Coomassie brilliant blue for 20 min after gel electrophoresis, take photos with white light after destaining, and the results are as follows: Figure 2 shown.

[0027] Example 2: Construction of a single-channel nanopore detection system for nanopore formation detection See also Figure 1 , two half-cells of equal volume were separated in the fluid pool, one for the Cis end and the other for the Trans end, and a 150μm channel hole was set between the two. 10μL of pentane-hexadecane mixture was dripped on both sides of the 150μm channel hole, and then 20μL of phospholipid pentane solution was added to the two half-cells of the fluid pool using the pulling method to form a lipid bilayer. MspA protein with a final concentration of 2.2 ng / mL was added to the cis end to form a single nanopore channel. The two half-cells were connected to separate Ag / AgCl electrodes and electrically connected to the amplifier Axopatch 200B. The cis end was set to ground, and the bias voltage applied from the trans end was +100mV to form a closed loop; the Axopatch 200B patch clamp can record and amplify the ion current signal generated by a single molecule passing through the nanopore. The current signal was recorded using a 25 kHz sampling frequency and a 5 kHz low-pass filter, and the nanopore test experiments were performed at 24 ± 1 °C using a buffer solution (1 M KC1, 500 mM MgCl2.6H2O, 25 mM HEPES buffer, pH = 8.0).

[0028] Example 3: ssDNA translocation through the MspA nanopore ssDNA was synthesized by Bio-Industry, dissolved in 100 μM TE buffer, and stored at -20°C. After a single channel was formed, ssDNA was added to the cis end at a final concentration of 1 μM. A bias voltage was applied using a patch clamp, and the current signal was recorded in real time. Data was recorded using a digital acquisition Digidata 1550B. The results are shown in Figure 2. Figure 3 The whole experimental system was placed in a Faraday cage to shield the noise. The raw data were analyzed using Clamfit 11.4.

[0029] The present invention adopts a purification optimization method combining ultrasonic disruption of bacteria with magnetic bead purification, and adds a hexahistidine tag (His-tag) to the C-terminus of the nanopore membrane protein, so that the purification of the membrane protein can be carried out using charged nickel-coated magnetic beads, thereby avoiding FPLC. The present invention effectively reduces the time of manual operation and the cost of single batch preparation, improves the preparation efficiency and quality of membrane proteins, and can achieve mass production of membrane proteins and improve the purity of proteins.

[0030] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a nanopore membrane protein, characterized in that: The following steps are involved: Step 1: Custom-synthesize a specific protein-coding gene and construct plasmid DNA on the corresponding vector, adding a hexahistidine tag to the C-terminus of the gene for nickel affinity purification; Step 2: Use the heat shock method to transfer the plasmid DNA containing the protein coding gene into the Escherichia coli expression strain and induce the culture; use 0.5-1mM isopropyl β-D-thiogalactoside (IPTG) as an inducer at 16-20°C for 6-8 hours to induce expression; Step 3: Collect the induced expression bacteria, use ultrasound to break the bacteria to release the target protein, and then heat the cell lysate at 65℃~90℃, and purify the target membrane protein with His-tag using magnetic beads; Step 4: Characterize the purified protein by SDS-PAGE electrophoresis to verify the purity and size of the protein; use a patch clamp detection system to detect the pore formation of the nanopores formed by the purified protein, and detect the translocation of small molecule biological substances through the nanopores.

2. The method for preparing a nanopore membrane protein according to claim 1, characterized in that: In the step 1, one of the commonly used nanopore membrane proteins can be selected from αHL, MspA, Omp family, FraC, CsgG, FhuA or Aerolysin; a hexahistidine tag is added to the C-terminus of the nanopore membrane protein located on the outer surface of the pore vestibule.

3. The method for preparing a nanopore membrane protein according to claim 1, characterized in that: In the step 1, the recombinant plasmid uses the pET series vector as the expression vector.

4. The method for preparing a nanopore membrane protein according to claim 1, characterized in that: In the step 3, the bacterial cells were broken by dissolving with breaking buffer, 4 M Urea, 50 mM Tris, 300 mM NaCl, pH 8.0, and ultrasonically breaking the cells in an ice bath at a power of 60 W for 20 min, ultrasonically for 2 s, and pausing for 6 s as one cycle; after the ultrasonication, the metal bath was heated at 90°C for 10 min, and then centrifuged at 12000 rpm, 4°C for 40 min, and then the supernatant was collected for magnetic bead purification.

5. The method for preparing a nanopore membrane protein according to claim 1, characterized in that: The magnetic beads in step 3 are Ni 2+ Magnetic beads, after adding the supernatant to the magnetic beads, shake on a rotating mixer at room temperature for 60 minutes; after magnetic separation, discard the supernatant; then add 1 mL of wash buffer A; resuspend the beads at room temperature and shake up and down on a rotating mixer for 5 minutes; after magnetic separation, discard the supernatant; after magnetic separation, discard the supernatant; then add 100µL of elution buffer B1; resuspend the beads and shake on a rotating mixer at room temperature for another 5 minutes; after magnetic separation, discard the supernatant; finally, add 100µL of elution buffer B2 and shake the tube at room temperature for 5 minutes; after magnetic separation, collect the supernatant; the supernatant contains the target nanopore membrane protein and can be used for all downstream nanopore measurements.

6. The method for preparing a nanopore membrane protein according to claim 1, characterized in that: In step 4, the purified protein was characterized by SDS-PAGE electrophoresis as follows: 12% SDS-PAGE, Tris-Gly electrophoresis buffer, 10 µL loading volume, 80 V for 20 min for the concentrated gel, 120 V for 60 min for the separation gel, Coomassie brilliant blue staining for 20 min after the gel electrophoresis, and photographed using white light after decolorization.

7. The method for preparing a nanopore membrane protein according to claim 1, characterized in that: The nanopore formation detection in step 4 is a patch clamp detection system: two half-pools of equal volume are separated in the fluid pool, namely the Cis end and the Trans end, with a 150 μm channel hole between the two, a certain amount of conductive buffer and amphiphilic lipid solution are added to the half-pool, so as to form a phospholipid bilayer near the channel hole, and the prepared membrane protein is added to the Cis end to form a single nanopore channel autonomously; the two half-pools are respectively connected to separate Ag / AgCl electrodes and electrically connected to the amplifier Axopatch 200B to form a closed loop; the Axopatch 200B patch clamp can record and amplify the ion current signal generated by a single molecule passing through the nanopore; the translocation and docking signals use a 5kHz quadrupole Bessel low-pass filter with a sampling frequency of 25kHz, and the entire device is placed in a Faraday cage containing a copper box to reduce the coupling of external electromagnetic noise.

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