Preparation method of thermostable T7 RNA polymerase and thermostable T7 RNA polymerase

Abstract

The invention provides a preparation method of thermal-stability T7RNA polymerase and the thermal-stability T7RNA polymerase, belongs to the field of biology, and can solve the problems that an existing T7RNA polymerase preparation process is complex, the expression culture period is long, multi-step purification treatment is needed, liquid change treatment needs to be carried out independently before storage, and the production cost is high. The method comprises the following steps: transferring plasmids containing a T7RNA polymerase gene sequence into expression host bacteria M15, culturing, screening, cloning, adding glycerol for strain preservation, and establishing a strain three-level library; the method comprises the following steps: taking glycerol bacteria, carrying out streak resuscitation on a flat plate, selecting monoclone, inoculating the monoclone into an LB culture medium, culturing, carrying out enlarged culture, carrying out IPTG induced expression, centrifugally collecting thalli, cleaning, adding a lysis buffer solution, resuspending, carrying out ultrasonic cell disruption, centrifuging, and collecting supernate; purifying by nickel column affinity chromatography, removing impure protein by an impure washing buffer solution, eluting target protein, purifying by matching with anions and cations, and collecting the purified protein; and mixing the purified protein with a storage buffer solution, and subpackaging and storing. The method can be applied to rapid preparation of the thermal-stability T7RNA polymerase.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for rapidly preparing a thermostable T7 RNA polymerase and the thermostable T7 RNA polymerase. Background Art

[0002] T7 RNA polymerase (T7 RNAP) is a single-subunit RNA polymerase with a molecular weight of about 99 kDa, which was first isolated from Escherichia coli cells infected with bacteriophage T7 in 1970. T7 RNA polymerase is one of the simplest and most widely studied RNA polymerases that catalyze RNA synthesis, and has advantages such as high transcriptional efficiency and strong elongation ability, and is widely used in in vitro synthesis of RNA and in vivo protein expression in bacterial high-expression systems. Currently, there are conventional and heat-resistant types on the market: the conventional T7 RNA polymerase can efficiently transcribe templates in vitro at 37°C; while the heat-resistant T7 RNA polymerase can perform in vitro transcription at higher temperatures, improving the transcriptional efficiency of RNAs with higher GC content and the synthesis ability of long fragments, while reducing the formation of dsRNA by-products and reducing the immunogenicity of the synthesized RNA. With the continuous development of mRNA drug research, the broad application prospects of mRNA technology are becoming increasingly clear. And with the popularization of related technologies, the demand for T7 RNA polymerase is gradually increasing, and the preparation process of T7 RNA polymerase directly affects the quality, activity, yield and application effect of T7 RNA polymerase.

[0003] The existing preparation process of T7 RNA polymerase is complex, the expression and culture period is relatively long, and multiple purification treatments are required (generally, 2-3 purification methods need to be combined to obtain a protein with better purity), and a separate liquid change treatment is required before storage, resulting in high production costs, which to a certain extent limits the wide application of T7 RNA polymerase.

[0004] Therefore, developing a method for preparing T7 RNA polymerase that is fast, simple, efficient and high-yield is an urgent problem to be solved at present. Summary of the Invention

[0005] Aiming at the technical problems of the existing complex preparation process of T7 RNA polymerase, relatively long expression and culture period, multiple purification treatments required, a separate liquid change treatment required before storage, and high production costs, the present invention provides a method for preparing T7 RNA polymerase that is fast, simple, efficient and high-yield.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing thermostable T7 RNA polymerase, comprising the following steps: transferring a plasmid containing the T7 RNA polymerase gene sequence into the expression host bacterium M15, screening the fastest growing and highest expressing clone after cultivation, adding glycerol for strain preservation, and establishing a three-level strain bank; streaking and resuscitating the glycerol bacteria on a plate, picking monoclonal colonies and inoculating them into LB medium for cultivation, performing large-scale cultivation after primary seed cultivation, then performing IPTG induction expression, and centrifuging for the first time to collect the bacteria; washing the collected bacteria and resuspending them in lysis buffer, then performing ultrasonic cell disruption, and centrifuging for the second time to collect the supernatant, which is the protein lysate; purifying the protein lysate by nickel column affinity chromatography, removing impurity proteins with washing buffer, then performing target protein elution, subjecting the eluted protein to anion and cation purification, and collecting the purified protein; measuring the protein concentration of the purified protein, mixing it with storage buffer in a ratio of 1:1, aliquoting, and storing at -80°C. The present invention can obtain a protein with a purity of 80% or higher by one-step affinity chromatography. By assisting with weak cations for the second purification and optimizing the purification conditions as described above, the protein can be further purified and components such as imidazole that may affect the experiment can be removed. The purity of the purified protein is as high as 99%. The entire purification process is easy to operate, low in cost, fast, and high in yield.

[0007] In some of these embodiments, before the IPTG induction expression step, pick monoclonal colonies and inoculate them into 4 - 5 mL of LB medium, and culture overnight for 12 - 16 hours at 30 - 37°C and a rotation speed of 180 - 250 rpm. After primary seed cultivation, perform large-scale cultivation at an inoculation ratio of 1% - 2%, and culture for 2 - 3 hours until the OD is 0.6 - 0.8.

[0008] In some of these embodiments, during the IPTG induction expression step, the concentration of IPTG is 0.1 - 0.5 mM, the induction temperature is 25 - 35°C, the induction rotation speed is 150 - 200 rpm, and the induction time is 2 - 4 h. The present invention optimizes the expression culture conditions, shortens the expression time, and the expressed T7 RNA polymerase has a high and stable yield.

[0009] In some of these embodiments, the binding solution used in the step of purifying the protein lysate by nickel column affinity chromatography includes at least one of imidazole, glycerol, sodium chloride, Tris-HCl, and DTT; wherein, the concentration of imidazole is 0 - 50 mM, the concentration of glycerol is 0 - 10%, the concentration of sodium chloride is 100 - 500 mM, the concentration of Tris-HCl is 10 - 50 mM, the concentration of DTT is 0 - 1 mM, and the pH of the binding solution is 7.0 - 8.0.

[0010] In some of these embodiments, the washing buffer used in the step of removing impurity proteins includes at least one of imidazole, glycerol, sodium chloride, Tris-HCl, and DTT; wherein, the concentration of imidazole is 50-150 mM, the concentration of glycerol is 0-10%, the concentration of sodium chloride is 100-500 mM, the concentration of Tris-HCl is 10-50 mM, the concentration of DTT is 0-1 mM, and the pH of the washing buffer is 7.0-8.0.

[0011] In some of these embodiments, the eluent used in the step of eluting the target protein includes at least one of imidazole, glycerol, sodium chloride, Tris-HCl, and DTT; wherein, the concentration of imidazole is 150-500 mM, the concentration of glycerol is 0-10%, the concentration of sodium chloride is 100-500 mM, the concentration of Tris-HCl is 10-50 mM, the concentration of DTT is 0-1 mM, and the pH of the eluent is 7.0-8.0.

[0012] In some of these embodiments, the binding solution used in the anion and cation purification steps for the eluted protein includes at least one of sodium chloride, Tris-HCl, and DTT; wherein, the concentration of sodium chloride is 0-100 mM, the concentration of Tris-HCl is 10-50 mM, the concentration of DTT is 0-1 mM, and the pH of the binding solution is 6.0-8.5.

[0013] In some of these embodiments, it further includes the step of adding the stored protein to an in vitro transcription buffer for performance verification.

[0014] In some of these embodiments, the in vitro transcription buffer contains at least one of Tris-HCl, MgCl2, DTT, NaCl, and spermidine. Among them, the concentration of Tris-HCl is 40-400 mM, the concentration of MgCl2 is 10-30 mM, the concentration of DTT is 20-100 mM, the concentration of NaCl is 20-100 mM, and the concentration of spermidine is 5-10 mM. By optimizing the components and concentrations of the in vitro transcription buffer, the optimized in vitro transcription buffer has better effects than commercial products. Using the preparation method of the present invention, the rapid preparation of T7 RNA polymerase can be completed within one day.

[0015] On the other hand, the present invention also provides a thermostable T7 RNA polymerase prepared by the above preparation method.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows: In the preparation method of the thermostable T7 RNA polymerase of the present invention, by optimizing the expression culture conditions, the expression level of T7 RNA polymerase is rapidly increased; by optimizing the purification conditions, a protein with a purity of up to 80% or more can be obtained by one-step affinity chromatography, and with the assistance of anion and cation purification, the protein can be further purified and components such as imidazole that may affect the experiment can be removed. The purified protein has a purity of up to 99%, and the entire purification process is easy to operate, low-cost, fast and has a high yield; the components and concentrations of the in vitro transcription buffer used in the present invention have better effects than commercial products, and the rapid preparation of T7 RNA polymerase can be completed within one day. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the SDS-PAGE diagram of protein purification in Example 1 of the present invention;

[0018] Figure 2 It is the SDS-PAGE diagram of protein purification in Example 2 of the present invention;

[0019] In each drawing, lane 1 is the protein lysate; lane 2 is the flow-through of nickel column affinity chromatography; lane 3 is the washing of nickel column affinity chromatography purification; lane 4 is the purified protein of nickel column affinity chromatography; lane 5 is the protein Marker; lane 6 is the flow-through of cation column. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] In order to understand the features and technical content of the embodiments of the present invention in more detail, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the following described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments of the present invention. In the following technical description, for the convenience of explanation, many details are provided to fully understand the disclosed embodiments. However, one or more embodiments can still be implemented without these details. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection requested by the present invention.

[0021] The embodiments of the present invention provide a preparation method of thermostable T7 RNA polymerase. By optimizing the preparation process of T7 RNA polymerase, the preparation efficiency of T7 RNA polymerase is improved, which specifically includes the following steps:

[0022] S1. Transfer the plasmid containing the T7 RNA polymerase gene sequence into the expression host bacterium M15. After the plate is cultured overnight at 35-37 °C for 12-16 hours, pick multiple monoclonal colonies for screening, select the clone with the fastest growth rate and the highest expression level, add glycerol for strain preservation, and establish a three-level strain bank;

[0023] S2. Take a glycerol stock and streak it on a plate for resuscitation. Pick a single colony and inoculate it into 4 - 5 mL of LB medium. Incubate overnight for 12 - 16 hours at 30 - 37°C with a rotation speed of 180 - 250 rpm. After primary seed culture, perform subculture at an inoculation ratio of 1% - 2%. Culture for 2 - 3 hours until the OD reaches 0.6 - 0.8, then perform IPTG induction expression. Among them, the IPTG concentration is 0.1 - 0.5 mM, the induction temperature is 25 - 35°C, the induction rotation speed is 150 - 200 rpm, and the induction time is 2 - 4 h. After the induction expression ends, centrifuge at 6000 - 8000 rpm for 5 - 10 min, and collect and wash the thalli.

[0024] S3. Weigh the thalli collected in step S2, and resuspend them by adding 0.5 - 1 mL of lysis buffer per gram of thalli. The resuspended thalli are subjected to ultrasonic cell disruption using an ultrasonic cell disruptor to release intracellular proteins and impurities. Centrifuge at 12000 - 13000 rpm at 4 - 8°C, and collect the supernatant. The collected supernatant is the protein lysate.

[0025] S4. Purify the protein lysate by nickel column affinity chromatography, remove the impurity proteins with the washing buffer, and then perform the elution of the target protein. Purify the eluted protein with anion and cation purification, and collect the purified protein.

[0026] Among them, the binding solution in the step of purifying the protein lysate by nickel column affinity chromatography includes one or more of the following components: imidazole, glycerol, sodium chloride, Tris - HCl, DTT. Among them, the concentration of imidazole is 0 - 50 mM, the concentration of glycerol is 0 - 10%, the concentration of sodium chloride is 100 - 500 mM, the concentration of Tris - HCl is 10 - 50 mM, the concentration of DTT is 0 - 1 mM, and the pH of the binding solution is 7.0 - 8.0; Set the concentration gradients of imidazole to be 0, 10, 20, 30, 40, 50 mM respectively, set the concentration gradients of glycerol to be 0, 5%, 10% respectively, set the concentration gradients of sodium chloride to be 100, 200, 300, 400, 500 mM respectively, set the concentration gradients of Tris - HCl to be 10, 25, 50 mM respectively, set the concentration gradients of DTT to be 0, 0.5, 1 mM respectively, and set the pH gradients to be 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 respectively. Conduct cross - experiments with different concentrations of the above components to confirm that the binding condition with the most binding of the target protein and the least binding of the impurity proteins is the optimal binding condition: the concentration of imidazole is 20 mM, the concentration of glycerol is 5%, the concentration of sodium chloride is 400 mM, the concentration of Tris - HCl is 50 mM, the concentration of DTT is 1 mM, and the pH is 7.8.

[0027] In the step of removing impurity proteins with the washing buffer, the washing buffer includes one or more of the following components: imidazole, glycerol, sodium chloride, Tris-HCl, DTT. Among them, the concentration of imidazole is 50 - 150 mM, the concentration of glycerol is 0 - 10%, the concentration of sodium chloride is 100 - 500 mM, the concentration of Tris-HCl is 10 - 50 mM, the concentration of DTT is 0 - 1 mM, and the pH of the washing buffer is 7.0 - 8.0; by setting concentration gradients for imidazole and pH and conducting cross-combination experiments, the optimal washing conditions are confirmed to be an imidazole concentration of 100 mM, a glycerol concentration of 5%, a sodium chloride concentration of 400 mM, a Tris-HCl concentration of 50 mM, a DTT concentration of 0.5 mM, and a pH of 7.8, removing impurity proteins to the greatest extent;

[0028] In the step of eluting the target protein, the eluent includes one or more of the following components: imidazole, glycerol, sodium chloride, Tris-HCl, DTT. Among them, the concentration of imidazole is 150 - 500 mM, the concentration of glycerol is 0 - 10%, the concentration of sodium chloride is 100 - 500 mM, the concentration of Tris-HCl is 10 - 50 mM, the concentration of DTT is 0 - 1 mM, and the pH of the eluent is 7.0 - 8.0; by setting concentration gradients for imidazole and pH and conducting cross-combination experiments, the optimal elution conditions are confirmed to be an imidazole concentration of 300 mM, a glycerol concentration of 5%, a sodium chloride concentration of 400 mM, a Tris-HCl concentration of 50 mM, a DTT concentration of 0.5 mM, and a pH of 7.8. The target protein is eluted quickly and efficiently, and the eluted protein is identified by SDS-PAGE to show a purity of more than 80%;

[0029] For the eluted protein in combination with the anion and cation purification steps, the binding buffer includes one or more of the following components: sodium chloride, Tris-HCl, DTT. Among them, the concentration of sodium chloride is 0 - 100 mM, the concentration of Tris-HCl is 10 - 50 mM, the concentration of DTT is 0 - 1 mM, and the pH of the binding buffer is 6.0 - 8.5; set concentration gradients of sodium chloride at 0, 20, 40, 60, 80, 100 mM respectively, set concentration gradients of Tris-HCl at 10, 25, 50 mM respectively, set pH gradients at 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 respectively, and cross-combine these concentrations, and pass them through the anion and cation respectively to determine the conditions and chromatography methods for the target protein to flow through and the impurity proteins to bind to the column; after purification, the target protein flows through and the impurity proteins bind to the column, effectively removing impurities, and the purity of the purified protein is above 99%, and the purified protein is collected;

[0030] S5. Measure the protein concentration of the purified protein. The protein concentration is 0.1 - 0.5 μg / μL. Add the storage buffer (containing 50% glycerol and 0.5 - 1 mM DTT) in a 1:1 ratio, aliquot, and store at -80°C.

[0031] S6. Add 0.05 - 0.1 μg of the stored protein to the in vitro transcription buffer, which can completely transcribe the template, thus successfully preparing T7 RNA polymerase. The in vitro transcription buffer contains one or more of the following components: Tris-HCl, MgCl2, DTT, NaCl, spermidine. Among them, the concentration of Tris-HCl is 40 - 400 mM, the concentration of MgCl2 is 10 - 30 mM, the concentration of DTT is 20 - 100 mM, the concentration of NaCl is 20 - 100 mM, and the concentration of spermidine is 5 - 10 mM. By optimizing the optimal concentrations of each component, the optimal formula is confirmed as follows: the concentration of Tris-HCl is 300 mM, the concentration of MgCl2 is 25 mM, the concentration of DTT is 80 mM, the concentration of NaCl is 50 mM, and the concentration of spermidine is 5 mM.

[0032] In step S2 of the present invention, by optimizing the expression conditions, the cultivation and induction can be completed in 6 hours, shortening the expression time; the expressed T7 RNA polymerase has a high and stable yield, increasing the expression level.

[0033] After expression in Escherichia coli, the collected bacterial cells need to be disrupted. After cell ultrasonic disruption in step S3 of the present invention, there are relatively many impurities such as nucleic acids, miscellaneous proteins, and cell debris in the cells, making the purification process relatively complex and having a lot of miscellaneous proteins. In step S4 of the present invention, through nickel column affinity chromatography, miscellaneous proteins are specifically adsorbed. By exploring the buffer formula, adjusting the pH and imidazole concentration, the miscellaneous proteins can be removed to the greatest extent. The purity of the eluted protein is 80% - 85%. Through ion chromatography purification, the miscellaneous proteins are adsorbed on the column, and the target protein passes through. The purity of the collected protein is above 99%. The operation is simple, the yield is high, and there is no need to change the solution, and there is no need for complex methods such as dialysis and concentration. Glycerol, DTT, and EDTA can be directly added for storage, and the two-step purification can be completed in 2 hours, saving time and reducing losses.

[0034] To introduce the preparation method of the thermostable T7 RNA polymerase provided by the embodiments of the present invention more clearly and in detail, the following will be described in combination with specific embodiments.

[0035] Example 1

[0036] The preparation method of the thermostable T7 RNA polymerase in this example includes the following steps:

[0037] S1. Strain Preparation: Transfer the plasmid containing the T7 RNA polymerase gene sequence into the expression host strain M15. After the plate is incubated overnight at 37°C for 12 hours, pick 4 monoclonal colonies for screening. Select the clone with the fastest growth rate and the highest expression level, add 50% glycerol for strain preservation, and establish a three-level strain library;

[0038] S2. Expression Culture: Take a glycerol stock and streak it on a plate for resuscitation. Pick a monoclonal colony and inoculate it into 4 - 5 mL of LB medium. Incubate overnight at 37°C with a rotation speed of 250 rpm for 12 hours. After primary seed culture, perform scale-up culture. When OD reaches 0.6, perform IPTG induction expression. Among them, the IPTG concentration is 0.1 mM, the induction temperature is 30°C, the induction rotation speed is 200 rpm, and the induction time is 3.5 h. After the induction expression ends, centrifuge at 8000 rpm for 10 min, collect and wash the cells;

[0039] S3. Ultrasonic Cell Disruption: Weigh the cells collected in step S2, and resuspend them by adding 1 mL of lysis buffer per gram of cells. The resuspended cells are subjected to ultrasonic cell disruption in an ultrasonic cell disruptor at a power of 25%, pulse for 3 sec, pause for 3 sec, for 15 min to release intracellular proteins and impurities. Centrifuge at 12000 rpm for 30 min at a temperature of 4 - 8°C, and collect the supernatant. The collected supernatant is the protein lysate;

[0040] S4. Purification: Purify the protein lysate by nickel column affinity chromatography. The protein concentration is 1 mg / mL, and the incubation time is 30 min to specifically adsorb the target protein. Adjust the imidazole concentration, pH, and buffer formulation to wash away the impurity proteins and elute the target protein. SDS-PAGE identification shows that the purity is above 80%. Among them, the affinity chromatography binding buffer contains 20 mM imidazole, 5% glycerol, 400 mM sodium chloride, 50 mM Tris-HCl, 1 mM DTT, and the pH is 8.0; the imidazole concentration in the impurity washing buffer is 100 mM; the imidazole concentration in the elution buffer is 300 mM, and elute 2 - 3 column volumes to collect 90% of the protein;

[0041] The purified protein is further purified by cation exchange chromatography. The cation exchange chromatography binding buffer components include 100 mM sodium chloride, 50 mM Tris-HCl, 1 mM DTT, and the pH is 8.5. The loading concentration is 0.5 - 1 mg / mL. The target protein flows through, and the impurity proteins bind to the column, effectively removing impurities. The purity of the purified protein is above 99%, and collect the purified protein;

[0042] S5. Storage: Measure the protein concentration of the purified protein. When the protein concentration is 0.1 - 0.5 μg / μL, add 50% glycerol and 1 mM DTT in a 1:1 ratio, aliquot and store at -80°C;

[0043] S6. Performance verification: The components and contents of the in vitro transcription buffer are as follows: 300 mM Tris-HCl, 25 mM MgCl2, 80 mM DTT, 50 mM NaCl, 5 mM spermidine. Add 0.05 - 0.1 μg of the stored protein to the in vitro transcription buffer for performance verification. It can completely transcribe the template, and the T7 RNA polymerase is prepared.

[0044] Appendix Figure 1 It is the SDS-PAGE diagram of protein purification in Example 1. In the figure, lane 1 is the protein lysate, lane 2 is the flow-through of nickel column affinity chromatography, lane 3 is the washing of nickel column affinity chromatography purification, lane 4 is the purified protein of nickel column affinity chromatography, lane 5 is the protein Marker, and lane 6 is the flow-through of cation column. It can be seen from the figure that the purity of T7 RNA polymerase is relatively high after nickel column affinity chromatography purification. After cation purification, the protein purity is high, and there is only one target protein, indicating that the purification method of the present invention is simple and has high purity.

[0045] Example 2

[0046] The preparation method of the thermostable T7 RNA polymerase in this example includes the following steps:

[0047] S1. Strain preparation: Transfer the plasmid containing the T7 RNA polymerase gene sequence into the expression host strain M15. After the plate is incubated overnight at 37°C for 12 hours, pick 4 monoclonal colonies for screening. Select the clone with the fastest growth rate and the highest expression level, add 50% glycerol for strain preservation, and establish a three-level strain library;

[0048] S2. Expression culture: Take a tube of glycerol bacteria and streak it on the plate for resuscitation. Pick a monoclonal colony and inoculate it into 4 - 5 mL of LB medium. Incubate it overnight at 37°C and a rotation speed of 250 rpm for 12 hours. After primary seed culture, perform scale-up culture. After OD reaches 0.6, perform IPTG induction expression. Among them, the IPTG concentration is 0.1 mM, the induction temperature is 35°C, the induction rotation speed is 200 rpm, and the induction time is 3 h. After the induction expression is completed, centrifuge at 8000 rpm for 10 min, collect and wash the bacteria;

[0049] S3. Ultrasonic cell disruption: Weigh the bacteria collected in step S2, and resuspend it according to 1 mL of lysis buffer per gram of bacteria. The resuspended bacteria are subjected to ultrasonic cell disruption in an ultrasonic cell disruptor with a power of 25%, break for 3 sec, stop for 3 sec, for 15 min, to release intracellular proteins and impurities. Centrifuge at 12000 rpm at a temperature of 4 - 8°C for 30 min, and collect the supernatant. The collected supernatant is the protein lysate;

[0050] S4. Purification: The protein lysate was purified by nickel column affinity chromatography. The protein concentration was 1 mg / mL and the incubation time was 30 min to specifically adsorb the target protein. The imidazole concentration, pH and buffer formulation were adjusted to wash away the impurity proteins and elute the target protein. SDS-PAGE identification showed that the purity was over 80%. Among them, the affinity chromatography binding buffer contained 50 mM imidazole, 10% glycerol, 500 mM sodium chloride, 50 mM Tris-HCl, 1 mM DTT, and the pH was 8.0; the impurity washing buffer had an imidazole concentration of 150 mM; the elution buffer had an imidazole concentration of 500 mM, and 90% of the protein could be collected by eluting 2 - 3 column volumes.

[0051] The purified protein was further purified by cation exchange chromatography. The components of the cation exchange binding buffer included 60 mM sodium chloride, 25 mM Tris-HCl, and the pH was 7.0. The loading concentration was 0.5 - 1 mg / mL. The target protein flowed through while the impurity proteins bound to the column, effectively removing the impurities. The purity of the purified protein was over 99%, and the purified protein was collected.

[0052] S5. Storage: The protein concentration of the purified protein was measured. The protein concentration was 0.1 - 0.5 μg / μL. 50% glycerol and 1 mM DTT were added in a 1:1 ratio, and after aliquoting, it was stored at -80 °C.

[0053] S6. Performance verification: The components and contents of the in vitro transcription buffer were as follows: 40 mM Tris-HCl, 10 mM MgCl2, 20 mM DTT, 20 mM NaCl, 5 mM spermidine. 0.05 - 0.1 μg of the stored protein was added to the in vitro transcription buffer for performance verification, and it was able to completely transcribe the template, and the T7 RNA polymerase was successfully prepared.

[0054] Appendix Figure 2 Figure 1 is the SDS-PAGE diagram of protein purification in Example 2. In the figure, lane 1 is the protein lysate, lane 2 is the flow-through of nickel column affinity chromatography, lane 3 is the impurity washing of nickel column affinity chromatography purification, lane 4 is the purified protein of nickel column affinity chromatography, lane 5 is the protein Marker, and lane 6 is the flow-through of the cation column. It can be seen from the figure that the T7 RNA polymerase has a relatively high purity after nickel column affinity chromatography purification. After cation exchange purification, the protein purity is high and there is only one target protein band, indicating that the purification method of the present invention is simple and has a high purity.

[0055] Finally, it should be noted that: The present invention is not limited to the above-listed embodiments. The above description is only a preferred and feasible embodiment of the present invention. Moreover, the above-described embodiments are only used to illustrate the technical solution of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, evolutions, and improvements made by those of ordinary skill in the art to the technical solution of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a thermostable T7 RNA polymerase, characterized in that: The following steps are involved: The plasmid containing the T7 RNA polymerase gene sequence was transferred into the expression host bacteria M15. After cultivation, the clones with the fastest growth rate and the highest expression level were selected and added to glycerol for strain preservation, and a three-level strain library was established; Take the glycerol bacteria and streak them on the plate to revive them. Pick a single clone and inoculate it into LB medium for culture. After primary seed culture, expand the culture, induce expression with IPTG, and collect the bacteria by centrifugation for the first time. After washing the collected bacteria, add lysis buffer to resuspend them, and then perform ultrasonic cell disruption. After the second centrifugation, collect the supernatant to obtain the protein lysis solution. The protein lysate is purified by nickel column affinity chromatography, impurity proteins are removed by washing buffer, and then the target protein is eluted, the eluted protein is purified with anions and cations, and the purified protein is collected; The protein concentration of the purified protein was measured, and the protein was mixed with the storage buffer at a ratio of 1:1 and packaged, and stored at -80°C.

2. The method for preparing the thermostable T7 RNA polymerase according to claim 1, characterized in that: Before the IPTG-induced expression step, pick a single clone and inoculate it into 4-5 mL LB medium. Culture it overnight at 30-37°C and 180-250 rpm for 12-16 hours. After primary seed culture, expand the culture at an inoculation ratio of 1%-2% and culture it for 2-3 hours until the OD reaches 0.6-0.

8.

3. The method for preparing the thermostable T7 RNA polymerase according to claim 1, characterized in that: In the IPTG-induced expression step, the concentration of IPTG is 0.1-0.5 mM, the induction temperature is 25-35° C., the induction speed is 150-200 rpm, and the induction time is 2-4 h.

4. The method for preparing the thermostable T7 RNA polymerase according to claim 1, characterized in that: The binding solution used in the nickel column affinity chromatography purification step includes at least one of imidazole, glycerol, sodium chloride, Tris-HCl, and DTT; Wherein, the concentration of imidazole is 0-50mM, the concentration of glycerol is 0-10%, the concentration of sodium chloride is 100-500mM, the concentration of Tris-HCl is 10-50mM, the concentration of DTT is 0-1mM, and the pH of the binding solution is 7.0-8.

0.

5. The method for preparing the thermostable T7 RNA polymerase according to claim 1, characterized in that: The washing buffer used in the step of removing impurity proteins comprises at least one of imidazole, glycerol, sodium chloride, Tris-HCl, and DTT; Wherein, the concentration of imidazole is 50-150mM, the concentration of glycerol is 0-10%, the concentration of sodium chloride is 100-500mM, the concentration of Tris-HCl is 10-50mM, the concentration of DTT is 0-1mM, and the pH of the washing buffer is 7.0-8.

0.

6. The method for preparing the thermostable T7 RNA polymerase according to claim 1, characterized in that: The eluent used in the step of eluting the target protein includes at least one of imidazole, glycerol, sodium chloride, Tris-HCl, and DTT; Wherein, the concentration of imidazole is 150-500mM, the concentration of glycerol is 0-10%, the concentration of sodium chloride is 100-500mM, the concentration of Tris-HCl is 10-50mM, the concentration of DTT is 0-1mM, and the pH of the eluent is 7.0-8.

0.

7. The method for preparing the thermostable T7 RNA polymerase according to claim 1, characterized in that: The eluted protein is combined with a binding solution used in the anion and cation purification steps including at least one of sodium chloride, Tris-HCl, and DTT; Among them, the concentration of sodium chloride is 0-100mM, the concentration of Tris-HCl is 10-50mM, the concentration of DTT is 0-1mM, and the pH of the binding solution is 6.0-8.

5.

8. The method for preparing the thermostable T7 RNA polymerase according to claim 1, characterized in that: It also includes a step of adding the stored protein to an in vitro transcription buffer to perform performance verification.

9. The method for preparing the thermostable T7 RNA polymerase according to claim 8, characterized in that: The in vitro transcription buffer comprises at least one of Tris-HCl, MgCl2, DTT, NaCl, and spermidine; Among them, the concentration of Tris-HCl is 40-400mM, the concentration of MgCl2 is 10-30mM, the concentration of DTT is 20-100mM, the concentration of NaCl is 20-100mM, and the concentration of spermidine is 5-10mM.

10. A thermostable T7 RNA polymerase, characterized in that The thermostable T7 RNA polymerase is prepared by the preparation method of any one of claims 1 to 9.

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