Preparation method of recombinant human transmembrane channel-like protein

Abstract

The invention discloses a preparation method of a recombinant human transmembrane channel-like protein, which comprises the following steps: 1, optimizing a gene sequence of a human I-type transmembrane channel-like protein to obtain an optimized target gene sequence; 2, protein expression; and 3, recombining the human I-type transmembrane channel-like protein and purifying the recombinant human I-type transmembrane channel-like protein. According to the invention, the high-yield and high-quality preparation of the recombinant human transmembrane channel-like protein sample can be realized, so that the requirements of various applications such as antibody preparation, targeted drug design, biological sensing chip manufacturing and the like on the basis of the protein at the downstream can be met.

Description

Technical Field

[0001] The invention relates to a recombinant human transmembrane channel-like protein, in particular to a preparation method of the recombinant human transmembrane channel-like protein. Background Art

[0002] Human type I transmembrane channel-like proteins are mainly distributed in the inner ear hair cells and are responsible for converting the mechanical vibration stimulation of sound waves into electrical signals. Genetic mutations related to them can cause familial hereditary deafness. Due to its distribution, sequence composition and other reasons, large-scale, high-quality recombinant expression and purification preparation of type I transmembrane channel-like proteins, especially human-derived proteins, have not yet been achieved at this stage, which greatly restricts downstream research and production applications. Specifically, the existing technology has the following disadvantages: (1) Although a small amount of type I transmembrane channel-like proteins can be obtained through tissue samples using in situ extraction technology, it is limited to such proteins from animal sources; due to ethical and tissue source issues, human type I transmembrane channel-like proteins cannot be prepared by this technology. (2) Type I transmembrane channel-like protein samples obtained by other technologies cannot meet various downstream applications in terms of sample yield, quality (purity, conformation and size uniformity, stability) and other indicators due to the lack of process optimization and innovation. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing a recombinant human transmembrane channel-like protein. The present invention can achieve high-yield and high-quality preparation of recombinant human transmembrane channel-like protein samples, thereby meeting the downstream application requirements of antibody preparation, targeted drug design, biosensing chip manufacturing, etc. based on the protein.

[0004] The technical solution of the present invention is a method for preparing a recombinant human transmembrane channel-like protein, comprising the following steps:

[0005] The first step is to optimize the gene sequence of human type I transmembrane channel-like protein to obtain the optimized target gene sequence;

[0006] The second step, protein expression, includes the following specific steps:

[0007] 2.1. Construct the optimized target gene sequence into any type of expression vector containing CMV promoter to obtain transfected cells;

[0008] 2.2. After the transfected cells are suspended and cultured for a certain period of time, sodium butyrate is added to the set working concentration, and the suspension culture is continued for a certain period of time, and the cell pellet is collected by centrifugation;

[0009] The third step is to recombinant human type I transmembrane channel-like protein and purify it, including the following specific steps:

[0010] 3.1. Freeze the collected cell pellet for more than 12 hours, take it out and thaw it, then add a lysis buffer containing protease inhibitors; then lyse the cells, and use ultracentrifugation to centrifuge the lysed sample (the above cell lysate) to obtain the precipitate;

[0011] 3.2. Resuspend the precipitate in a purification buffer containing protease inhibitors and FC12 surfactant for incubation, and then obtain the supernatant by ultracentrifugation;

[0012] 3.3. Incubate the supernatant obtained in the previous step with the purified filler for a period of time, then add 15-20 times the volume of the purified filler column with washing buffer to remove impurities, add 1.5-3 times the volume of the purified filler column with elution buffer containing competitive eluent, elute and collect the target protein;

[0013] 3.4. After concentrating the target protein obtained in step 3.3, further purification is performed.

[0014] In the aforementioned method for preparing a recombinant human transmembrane channel-like protein, in the first step, the operation of optimizing the human type I transmembrane channel-like protein gene sequence is: removing the N-terminal 1-148 amino acid sequence of the human type I transmembrane channel-like protein gene sequence.

[0015] In the aforementioned method for preparing a recombinant human transmembrane channel-like protein, in step 2.1, the process of preparing and constructing the expression vector is to prepare 1.5 mg of a plasmid containing the optimized target gene and 0.65 mg of plasmids containing the gene sequences of chaperone protein A and chaperone protein B, and mix the above three plasmids;

[0016] HEK293 cells were grown in suspension culture to a cell density of 2.5-3*10 6 / mL, cells per 0.9L;

[0017] The three mixed plasmids were transiently transfected into cultured HEK293 cells using the PEI method.

[0018] In the aforementioned method for preparing a recombinant human transmembrane channel-like protein, in step 2.2, the transfected cells are suspended and cultured at 37°C, 130rpm, and 8% CO2 for 24 hours, sodium butyrate is added to a working concentration of 10mM, and the suspension culture is continued at 30°C, 130rpm, and 8% CO2 for 36 hours, and the cell pellet is collected by centrifugation.

[0019] In the aforementioned method for preparing a recombinant human transmembrane channel-like protein, the mass ratio between cells and lysis buffer containing protease inhibitors is 1:5; the lysis buffer containing protease inhibitors includes 2mM EDTA, 20mM Hepes, 200mM KCl, 2mM TCEP and 10% glycerol, and the pH of the lysis buffer containing protease inhibitors is 8.0.

[0020] In the aforementioned method for preparing a recombinant human transmembrane channel-like protein, the specific process of step 3.2 is: resuspending the precipitate in 40 mL of a purification buffer containing a protease inhibitor and 1% FC12 surfactant, incubating at 4° C. for 2 hours, and obtaining the supernatant by ultracentrifugation;

[0021] The purification buffer further comprises 20 mM Hepes, 200 mM KCl, 3 mM CaCl2·2H2O, 2 mM TCEP, 10% glycerol and 5 mM neomycin, and the pH of the purification buffer is 8.0.

[0022] In the aforementioned method for preparing a recombinant human transmembrane channel-like protein, the specific process of step 3.3 is: incubate the supernatant obtained in the previous step with the purified filler at 4°C for 2 hours, add 15-20 times the volume of the purified filler column with a washing buffer to remove impurities, and then add 1.5-3 times the volume of the purified filler column with an elution buffer containing a competitive eluent to elute and collect the target protein.

[0023] In the aforementioned method for preparing a recombinant human transmembrane channel-like protein, the washing buffer comprises 20mM Hepes, 200mM KCl, 3mM CaCl2·2H2O, 2mM TCEP, 10% glycerol, 5mM neomycin and 0.01% GDN, and the pH of the washing buffer is 8.0;

[0024] The elution buffer included 20 mM Hepes, 200 mM KCl, 3 mM CaCl2·2H2O, 2 mM TCEP, 10% glycerol, 5 mM neomycin and 0.01% GDN, and the pH of the elution buffer was 8.0.

[0025] In the aforementioned method for preparing a recombinant human transmembrane channel-like protein, in step 3.4, an ultrafiltration tube or ultrafiltration facility with a molecular weight cutoff of 30 kDa is selected for concentration, and further purification is performed by gel filtration.

[0026] In step 3.4, a purification buffer is used in the gel filtration method to maintain the stability of the properties of the protein sample. The purification buffer includes 20 mM Hepes, 200 mM KCl, 3 mM CaCl2·2H2O, 2 mM TCEP, 10% glycerol, 5 mM neomycin, 0.01% GDN, pH = 8.0.

[0027] Compared with the prior art, the present invention first optimizes the gene sequence of human type I transmembrane channel-like protein, then reasonably optimizes and improves the expression process of the target protein such as transfection conditions and expression conditions, and finally purifies the target protein (by specifically selecting the additives of the buffer and their dosage), that is, obtaining a human type I transmembrane channel-like protein sample through recombinant expression and purification, which has the characteristics of being simple, easy to replicate, and easy to expand production, and the preparation materials and instruments are also obtained.

[0028] At the same time, the human type I transmembrane channel-like protein obtained after the transformation has higher sample stability and higher yield compared with its wild type; the human type I transmembrane channel-like protein obtained by the present application through the purification process has the characteristics of high yield, high purity, high conformational uniformity, and high particle uniformity. Therefore, it is more suitable for frontier fields such as structural exploration, antibody preparation, and biosensing chip preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a polyacrylamide gel electrophoresis result diagram of the human type I transmembrane channel-like protein of the present invention (Coomassie brilliant blue staining);

[0030] Figure 2 It is a peak diagram of gel filtration detection of affinity-purified human type I transmembrane channel-like protein samples; wherein A is a peak diagram of the wild type (no auxiliary protein added and no auxiliary molecule added), B is a peak diagram of N-terminal truncation (no auxiliary protein added and no auxiliary molecule added), C is a peak diagram of N-terminal truncation (addition of auxiliary protein and no addition of auxiliary molecule), and D is a peak diagram of N-terminal truncation (addition of auxiliary protein and addition of auxiliary molecule);

[0031] Figure 3 This is the result of negative staining detection of human type I transmembrane channel-like protein. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0033] Embodiment: A method for preparing a recombinant human transmembrane channel-like protein comprises the following steps: (1) gene design and optimization.

[0034] The gene sequence of human type I transmembrane channel-like protein was designed and optimized, and the sequence is shown in the gene sequence table below. The main purpose is to remove the 1-148 amino acid sequence at its N-terminus.

[0035] Experimental results show that this optimization can obtain human type I transmembrane channel-like proteins with higher stability and higher yield.

[0036] (2) Expression process of recombinant human type I transmembrane channel-like protein.

[0037] 2-1. The modified target gene sequence is constructed into any type of expression vector containing CMV promoter. During expression preparation, HEK293 cells in suspension culture are grown to a cell density of 2.5-3*10 6 / mL, for every 0.9L of cells, 1.5mg of plasmid containing the target gene (transmembrane channel-like protein gene sequence) and 0.65mg of plasmids containing chaperone protein A and chaperone protein B gene sequences need to be prepared. After the above three plasmids are mixed, they are transiently transfected into HEK293 cells by PEI method. The method of transfection of plasmids is not limited here.

[0038] 2-2. The transfected HEK293 cells were suspended and cultured at 37°C, 130 rpm, and 8% CO2 for 24 hours, sodium butyrate was added to a working concentration of 10 mM, and the suspension culture was continued at 30°C, 130 rpm, and 8% CO2 for 36 hours. The cell pellets were collected by centrifugation.

[0039] (3) Purification process of recombinant human type I transmembrane channel-like protein.

[0040] 3-1. Freeze the collected cell pellet at -80℃ for more than 12 hours. After thawing, add lysis buffer (2mM EDTA, 20mM Hepes, 200mM KCl, 2mMTCEP, 10% glycerol, pH=8.0) containing protease inhibitors at a cell:solution mass ratio of 1:5. Cells can be lysed by ultrasound or high-pressure crushing. Keep the operation at 4℃. The solution and ambient temperature during the lysis process must not exceed 10℃. To ensure that the cells have been fully lysed, microscopic examination after sampling can be used as a quality control step. After lysis, the sample is ultracentrifuged (186000g, 4℃, 90min) to obtain the precipitate.

[0041] 3-2. Resuspend the precipitate in 40 mL of purification buffer (20 mM Hepes, 200 mM KCl, 3 mM CaCl2·2H2O, 2 mM TCEP, 10% glycerol, 5 mM neomycin, pH = 8.0) containing protease inhibitors and 1% FC12 surfactant, incubate at 4°C for 2 hours, and obtain the supernatant by ultracentrifugation (186000g, 4°C, 30 min).

[0042] 3-3. Incubate the supernatant obtained in the previous step with the purified filler at 4°C for 2 hours, add 15-20 times the volume of the purified filler column with washing buffer (20mM Hepes, 200mM KCl, 3mM CaCl2·2H2O, 2mM TCEP, 10% glycerol, 5mM neomycin, pH=8.0, 0.01% GDN) to remove impurities, add 1.5-3 times the volume of the purified filler column with elution buffer containing a competitive eluent (20mM Hepes, 200mM KCl, 3mM CaCl2·2H2O, 2mM TCEP, 10% glycerol, 5mM neomycin, pH=8.0, 0.01% GDN), elute and collect the target protein.

[0043] 3-4. After concentrating the target protein obtained in step 3.3, further purify it.

[0044] When concentrating, use ultrafiltration tubes or ultrafiltration facilities with a molecular weight cutoff of 30kDa, and use gel filtration for further purification. Superdex 200 or gel filtration fillers with similar separation range can be used, combined with protein chromatography equipment, to complete gel filtration chromatography, select samples with the correct peak position, collect and concentrate. Taking superdex 20010 / 300 filler as an example, the peak position of the target protein sample is about 11.5mL.

[0045] In step 3.4, a purification buffer is used in the gel filtration method to maintain the stability of the properties of the protein sample. The purification buffer includes 20 mM Hepes, 200 mM KCl, 3 mM CaCl2·2H2O, 2 mM TCEP, 10% glycerol, 5 mM neomycin, 0.01% GDN, pH = 8.0.

[0046] The above preparation process is simple and easy to scale up, and the preparation materials and instruments are also available. Compared with its wild type, the modified human type I transmembrane channel-like protein has higher sample stability and higher yield. The human type I transmembrane channel-like protein obtained by this purification process has the characteristics of high yield, high purity, high conformational uniformity, and high particle uniformity. Therefore, it is more suitable for cutting-edge fields such as structural exploration, antibody preparation, and biosensing chip preparation.

[0047] The human type I transmembrane channel-like protein was tested by polyacrylamide gel electrophoresis, and the results (Coomassie brilliant blue staining) were as follows: Figure 1 As shown;

[0048] Sample source: After affinity purification, further gel filtration chromatography is performed to take samples at the correct peak position.

[0049] The unmodified target protein (wild type) and the optimized target protein (N-terminal truncation) were recombinantly expressed in HEK293 cells. The affinity-purified target protein was further purified by gel filtration, and the sample with the correct peak position was taken for the detection shown in the figure. The red arrow indicates the band position of the target protein. It can be seen that the wild type has two obvious degradation bands (blue arrows).

[0050] The experimental results show that the expression yield of the optimized target protein (N-terminal truncation) can reach about 1-1.5 mg / L cell. After affinity chromatography combined with gel filtration chromatography, the yield of purified protein is about 0.2-0.4 mg / L, and the purity is greater than 95% ( Figure 1 ), and compared with the wild type, there is no obvious degradation band, and the protein stability is better.

[0051] After gel filtration of affinity-purified human type I transmembrane channel-like protein samples, the peak shape was detected, such as Figure 2 As shown. Human type I transmembrane channel-like protein is a dimer in the correctly folded state, wrapped by the surfactant of the buffer. Both the high polymer (protein aggregates) and non-dimer forms are "junk" proteins that may be inactive. The theoretical molecular weight of the wild-type monomer is about 100kDa, and the theoretical molecular weight of the N-terminal truncation monomer is 74.4kDa. Therefore, for the correct form of the dimer target protein, the wild-type should be located closer to the peak (~12.5mL) than the N-terminal truncation. Figure 2 A in Figure 2 On the other hand, the ratio of protein aggregates to target protein (which can be quantified by peak height or peak area) is also an important quality control criterion. The lower the ratio of protein aggregates, the better. Figure 2 In D, the theoretical peak position of the optimized target protein complex (N-terminal truncation) is about 11.6mL (superdex200, 10 / 300), as shown by the red arrow. The sample here was confirmed to be a type I transmembrane channel-like protein by Coomassie brilliant blue staining, Western-blot and mass spectrometry. The peak is sharp and symmetrical, indicating that the target protein has a good conformation in the solution. The green arrow shows the peak of the excessively added auxiliary protein that is not bound to the target protein.

[0052] The human type I transmembrane channel-like protein prepared in this application was negatively stained (0.03 mg / mL, uranyl acetate staining, 120 kV electron microscope), and the results were as follows Figure 3 As shown in the figure, the dimer particles of human type I transmembrane channel-like protein samples are uniform in size and are rod-shaped (about 15-20 nm when viewed from the side) or spherical (about 8-12 nm when viewed from the top).

Claims

1. A method for preparing a recombinant human transmembrane channel-like protein, characterized in that: The following steps are involved: The first step is to optimize the gene sequence of human type I transmembrane channel-like protein to obtain the optimized target gene sequence; The second step, protein expression, includes the following specific steps: 2.

1. Construct the optimized target gene sequence into any type of expression vector containing CMV promoter to obtain transfected cells; 2.

2. After the transfected cells are suspended and cultured for a certain period of time, sodium butyrate is added to the set working concentration, and the suspension culture is continued for a certain period of time, and the cell pellet is collected by centrifugation; The third step is to recombinant human type I transmembrane channel-like protein and purify it, including the following specific steps: 3.

1. Freeze the collected cell pellet for more than 12 hours, take it out and thaw it, then add a lysis buffer containing protease inhibitors; then lyse the cells, centrifuge the cell lysate by ultracentrifugation and obtain the pellet; 3.

2. Resuspend the precipitate in a purification buffer containing protease inhibitors and FC12 surfactant for incubation, and then obtain the supernatant by ultracentrifugation; 3.

3. Incubate the supernatant obtained in the previous step with the purified filler for a period of time, then add 15-20 times the volume of the purified filler column with washing buffer to remove impurities, add 1.5-3 times the volume of the purified filler column with elution buffer containing competitive eluent, elute and collect the target protein; 3.

4. After concentrating the target protein obtained in step 3.3, further purification is performed.

2. The method for preparing a recombinant human transmembrane channel-like protein according to claim 1, characterized in that: In the first step, the operation of optimizing the human type I transmembrane channel-like protein gene sequence is to remove the 1-148 amino acid sequence at the N-terminus of the human type I transmembrane channel-like protein gene sequence.

3. The method for preparing a recombinant human transmembrane channel-like protein according to claim 1, characterized in that: In step 2.1, the process of preparing and constructing the expression vector is to prepare 1.5 mg of a plasmid containing the optimized target gene and 0.65 mg of plasmids containing the gene sequences of chaperone protein A and chaperone protein B, and mix the above three plasmids; HEK293 cells were grown in suspension culture to a cell density of 2.5-3*10 6 / mL, cells per 0.9L; The three mixed plasmids were transiently transfected into cultured HEK293 cells using the PEI method.

4. The method for preparing a recombinant human transmembrane channel-like protein according to claim 1, characterized in that: In step 2.2, the transfected cells were suspended and cultured at 37°C, 130 rpm, and 8% CO2 for 24 hours, sodium butyrate was added to a working concentration of 10 mM, and the suspension culture was continued at 30°C, 130 rpm, and 8% CO2 for 36 hours, and the cell pellet was collected by centrifugation.

5. The method for preparing a recombinant human transmembrane channel-like protein according to claim 1, characterized in that: The mass ratio between cells and a lysis buffer containing protease inhibitors is 1:5; the lysis buffer containing protease inhibitors includes 2mM EDTA, 20mM Hepes, 200mM KCl, 2mM TCEP and 10% glycerol, and the pH of the lysis buffer containing protease inhibitors is 8.

0.

6. The method for preparing a recombinant human transmembrane channel-like protein according to claim 1, characterized in that: The specific process of step 3.2 is as follows: resuspend the precipitate in 40 mL of purification buffer containing protease inhibitors and 1% FC12 surfactant, incubate at 4°C for 2 hours, and obtain the supernatant by ultracentrifugation; The purification buffer further comprises 20 mM Hepes, 200 mM KCl, 3 mM CaCl2·2H2O, 2 mM TCEP, 10% glycerol and 5 mM neomycin, and the pH of the purification buffer is 8.

0.

7. The method for preparing a recombinant human transmembrane channel-like protein according to claim 1, characterized in that: The specific process of step 3.3 is: incubate the supernatant obtained in the previous step with the purified filler at 4°C for 2 hours, add 15-20 times the volume of the purified filler column with washing buffer to remove impurities, and then add 1.5-3 times the volume of the purified filler column with elution buffer containing competitive eluent to elute and collect the target protein.

8. The method for preparing a recombinant human transmembrane channel-like protein according to claim 7, characterized in that: The washing buffer comprises 20 mM Hepes, 200 mM KCl, 3 mM CaCl2·2H2O, 2 mM TCEP, 10% glycerol, 5 mM neomycin and 0.01% GDN, and the pH of the washing buffer is 8.0; The elution buffer included 20 mM Hepes, 200 mM KCl, 3 mM CaCl2·2H2O, 2 mM TCEP, 10% glycerol, 5 mM neomycin and 0.01% GDN, and the pH of the elution buffer was 8.

0.

9. The method for preparing a recombinant human transmembrane channel-like protein according to claim 1, characterized in that: In step 3.4, an ultrafiltration tube or ultrafiltration device with a molecular weight cutoff of 30 kDa is used for concentration, and further purification is performed by gel filtration.

10. The method for preparing a recombinant human transmembrane channel-like protein according to claim 9, characterized in that: In step 3.4, a purification buffer is used in the gel filtration method to maintain the stability of the properties of the protein sample. The purification buffer in this step includes 20 mM Hepes, 200 mM KCl, 3 mM CaCl2·2H2O, 2 mM TCEP, 10% glycerol, 5 mM neomycin, 0.01% GDN, pH = 8.0.

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