A T7 RNA polymerase mutant and its preparation method and application

Abstract

The present invention discloses a T7 RNA polymerase mutant and its preparation method and application, belonging to the technical field of nucleic acid tool enzymes. The amino acid sequence of the T7 RNA polymerase mutant is shown in SEQ ID NO.1, and the 640th glycine in the amino acid sequence of the wild-type T7 RNA polymerase is replaced by tryptophan. Compared with the RNA polymerase of the wild-type T7 bacteriophage, the T7 RNA polymerase mutant has higher catalytic activity and better thermal stability. Compared with the RNA synthesis level of the relevant polymerase on the market, the RNA yield is increased by 30-50%. The present invention also provides a separation and purification method of a T7 RNA polymerase mutant, and by optimizing the purification process, the sample of the prepared T7 RNA polymerase does not contain DNase and RNase contamination.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid tool enzymes, and in particular to a T7 RNA polymerase mutant and a preparation method and application thereof. Background Art

[0002] With the rapid development of science and technology and biotechnology, a large number of RNA-related research and applications have been carried out, which has posed a high challenge to RNA synthesis. Currently, RNA in vitro synthesis includes two methods: chemical synthesis and enzymatic synthesis. Chemical synthesis is suitable for synthesizing RNA with a length of dozens of nucleotides. As the length of RNA increases, the production cost will rise sharply. When the nucleotides reach more than 100, chemical synthesis is no longer applicable. Usually, protein-encoding mRNA contains thousands of nucleotides, so enzymatic synthesis is currently the only method for preparing long-chain RNA.

[0003] Bacteriophage T7 RNA polymerase (T7 RNA Polymerase, T7RNAP) was first isolated from Escherichia coli cells infected by bacteriophage T7 in 1970. It is one of the simplest enzymes that catalyze RNA synthesis. T7 RNA polymerase has a high promoter specificity and will only transcribe DNA or DNA replicas located downstream of the T7 promoter in the T7 bacteriophage. In recent years, the T7 RNA polymerase transcription system has also played an important role in synthetic biology. T7 RNAP is widely used in in vitro RNA synthesis and in vivo protein expression (bacterial high expression system). Therefore, the construction, expression and purification process of high-activity and high-stability T7 RNA polymerase has become one of the research hotspots.

[0004] Using protein engineering technology to perform site-directed mutagenesis on T7 RNA polymerase to obtain mutants with improved functions is one of the means of developing T7 RNA polymerase products. In the prior art, patent document CN102177236A discloses that the amino acid residues at at least one position of glutamine at position 786, lysine at position 179, and valine at position 685 in the amino acid sequence of wild-type T7 RNA polymerase are replaced by other amino acids, thereby improving its thermal stability and specific activity. For another example, patent document CN 107460177A discloses that arginine at position 632 in the amino acid sequence of wild-type T7 RNA polymerase is replaced by cysteine, and transcription activity is improved.

[0005] Based on the expression system of T7 RNAP, researchers have developed two chassis strains, Corynebacterium glutamicum MB001 and Escherichia coli BL21 (DE3). How to separate and purify T7 RNA polymerase with high yield, high purity and good activity from the fermentation products of chassis strains is a problem that technicians in this field need to solve.

[0006] With the rapid development of purification process research, there are more and more purification methods for both small molecule monomers and large molecule proteins. The recombinant protein purification process needs to pay attention to the following issues: 1) If the recombinant protein is expressed intracellularly, the structure and activity of the target protein should be protected during the cell disruption process. 2) Each step of separation and purification will cause a certain amount of protein and protein activity loss, but too few separation steps cannot meet a certain purity standard; 3) The reagents and related operations used in each separation and purification link have different application ranges for different proteins; 4) T7 RNA polymerase samples obtained by conventional separation and purification methods usually have DNase and RNase contamination. Therefore, exploring a suitable purification method for a specific target protein is also a problem that needs to be solved in the development of high-activity T7 RNA polymerase products. Summary of the invention

[0007] The purpose of the present invention is to replace the amino acids of wild-type T7 RNA polymerase by genetic engineering modification technology to obtain a T7 RNA polymerase mutant with significantly improved enzyme activity and thermal stability, and to prepare a T7 RNA polymerase product free of DNase and RNase contamination by optimizing the separation and purification process conditions.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The invention adopts a genetic engineering method to replace the glycine (G) at position 640 in the amino acid sequence of the wild-type T7 RNA polymerase with tryptophan (W) to obtain a T7 RNA polymerase mutant G640W with an amino acid sequence as shown in SEQ ID NO.1.

[0010] Compared with the wild-type T7 phage RNA polymerase, the thermal stability and specific activity of the T7 RNA polymerase mutant G640W were greatly improved.

[0011] The T7 RNA polymerase G640W mutant of the present invention can be obtained by making a recombinant protein using gene recombination technology. When obtaining it using gene recombination technology, the mutant gene of T7 RNA polymerase is introduced into the host using appropriate genetic engineering technology, and the target protease is obtained by inducing expression.

[0012] The present invention provides a gene encoding the T7 RNA polymerase mutant, and the nucleotide sequence is shown in SEQ ID NO.2.

[0013] The invention utilizes gene site-directed mutagenesis technology to mutate the 1918th G in the coding sequence (SEQ ID NO.4) of the wild-type T7 RNA polymerase into T, and replaces the original codon GGG encoding the 640th glycine into TGG.

[0014] In addition, sequences useful for enzyme purification can be added to the produced T7 RNA polymerase mutant gene, such as using a signal peptide to form an extracellular secretory enzyme; or adding a histidine hexamer-containing tag sequence at the end to facilitate the subsequent purification process.

[0015] The host used for expression can be various cultured cells such as plant cells, animal cells, insect cells, etc. For the expression of T7 RNA polymerase gene, the present invention selects Escherichia coli as the host cell.

[0016] The T7 RNA polymerase mutant gene in the present invention is inserted into an appropriate vector and then introduced into the above-mentioned large intestine host cell. Usually, there are many types of expression vectors. When selecting a vector, it is necessary to consider whether the elements necessary for transcription after the insertion of the target gene in the selected vector can play the corresponding function, and it can be appropriately selected according to the type of host.

[0017] The present invention also provides a recombinant vector comprising the coding gene. Preferably, the expression vector uses plasmid pET-28a, pET-30a, but is not limited thereto.

[0018] The present invention also provides a recombinant genetic engineering bacterium comprising the recombinant vector. Preferably, the expression host is Escherichia coli BL21.

[0019] By culturing the transformed Escherichia coli containing the target gene in a suitable component culture medium under conditions where the introduced DNA construct can be expressed, the T7 RNA polymerase of the present invention is induced to express. The T7 RNA polymerase of the present invention is obtained by separation and purification from the Escherichia coli cells after induced expression, and conventional methods for separation and purification of proteins can be used. For example, when the enzyme of the present invention is expressed in cells, after the culture is completed, the cells are centrifuged and recovered, suspended in an appropriate aqueous buffer, and then the cells are broken by lysozyme treatment or ultrasonic crusher to obtain a cell-free extract. The cell-free extract is further centrifuged to obtain a supernatant, and the supernatant is purified by conventional protein separation and purification methods. The main purification methods include salting-out (ammonium sulfate precipitation), molecular sieves, affinity chromatography, and ion exchange chromatography.

[0020] The samples of T7 RNA polymerase obtained by conventional separation and purification methods are usually contaminated by DNase and RNase. The present invention flexibly combines and uses relevant purification process methods to achieve rapid separation of the target protein, and optimizes the relevant purification process. The prepared T7 RNA polymerase samples do not contain DNase and RNase contamination.

[0021] Specifically, the present invention provides a method for separating and purifying a T7 RNA polymerase mutant free of DNase and RNase contamination from an intracellular induced expression product of Escherichia coli, the method comprising the following steps:

[0022] (1) Transforming a recombinant expression vector containing a gene fragment having a nucleotide sequence as shown in SEQ ID NO.2 into an Escherichia coli host cell to obtain a recombinant genetically engineered bacterium, wherein the recombinant expression vector has a His tag coding sequence;

[0023] (2) After fermentation and cultivation of the recombinant genetically engineered bacteria, the cells are collected by centrifugation, a cell lysis solution is added, the cells are ultrasonically disrupted, the supernatant is collected by centrifugation, and the affinity chromatography loading solution is obtained by filtration;

[0024] (3) loading the affinity chromatography loading solution onto an affinity chromatography column filled with Ni-IDA filler, first eluting impurities with affinity chromatography washing solution, then eluting with affinity chromatography eluent at an ultraviolet detection wavelength of 280 nm until a peak begins to appear, collecting the eluate, and dialyzing with a storage solution to obtain an ion exchange chromatography loading solution;

[0025] (4) loading the ion exchange chromatography loading solution onto an ion exchange chromatography column filled with DEAE-650M filler, first eluting impurities with an ion exchange chromatography washing solution, and then eluting with an ion exchange chromatography eluent at a UV detection wavelength of 280 nm until a peak begins to appear, collecting the eluate to obtain the T7 RNA polymerase mutant;

[0026] In step (1), a recombinant genetic engineering bacterium for expressing a T7 RNA polymerase mutant is constructed. The original plasmid of the recombinant expression vector is pET-28a(+). The expressed recombinant protein carries a 6×His tag, and the protein separation and purification can be performed by affinity chromatography using nickel chelating resin.

[0027] In step (2), the recombinant genetically engineered bacteria are induced to express the recombinant protein intracellularly, and the cells are lysed to release the recombinant protein for separation and purification.

[0028] The fermentation culture comprises: expanding the activated recombinant genetic engineering bacteria to OD 600 =0.4-0.6, add IPTG with a final concentration of 0.1 mM, and culture with shaking at 25°C and 180 rpm for 18-20 h.

[0029] The present invention can effectively avoid the degradation of the target protein by optimizing the formula of the cell lysis solution and combining the ultrasonic process, thereby retaining the activity of the target protein and removing more foreign proteins.

[0030] The cell lysis solution comprises: 20-50 mM Tris-HCl, 10-50 mM NaCl, 3-10 mM EDTA, 3%-10% glycerol, 10-20 μg / mL lysozyme, 0.005%-0.010% sodium deoxycholate, 3-8 mM dithiothreitol (DTT), 0.1-0.5 mM benzamidine, 20-80 μg / mL phenylmethylsulfonyl fluoride (PMSF), 10-40 μg / mL bacitracin, 5-15 mM ammonium sulfate, and pH=7.5-8.0.

[0031] Ultrasonic disruption was performed under ice bath conditions, and the temperature of the bacterial solution was controlled below 10°C during the entire bacterial disruption process. The ultrasonic power was 400W, ultrasonication was performed for 2s, stop for 4s, and treatment was performed for 20min.

[0032] Preferably, the composition of the cell lysis solution includes: 50mM Tris-HCl, 50mM NaCl, 5mM EDTA, 5% glycerol, 12.5μg / mL lysozyme, 0.008% sodium deoxycholate, 5mM DTT, 0.3mM benzamidine, 50μg / mL PMSF, 20μg / mL bacitracin, 8mM ammonium sulfate, pH=7.9.

[0033] In step (3), affinity purification is performed using the histidine hexamer tag added to the G640W mutant T7 RNA polymerase. The recombinant protein is bound to the affinity chromatography column filler, and the impurities are first washed away using an affinity chromatography washing solution, and then the target fraction is eluted using an affinity chromatography elution solution.

[0034] The affinity chromatography washing solution comprises: 20-60mM phosphate buffer, 0.2-0.5M NaCl, 30mM imidazole, pH=7.5-8.0.

[0035] The affinity chromatography eluent comprises: 20-60 mM phosphate buffer, 0.2-0.5 M NaCl, 100 mM imidazole, pH=7.5-8.0.

[0036] Preferably, before loading, the chromatography column is first rinsed with nuclease-free water, and then the column is balanced with affinity chromatography buffer A; after loading, the column is first balanced with affinity chromatography buffer A. The composition of the affinity chromatography buffer A includes: 20-60mM phosphate buffer, 0.2-0.5M NaCl, 3-10mM imidazole, pH = 7.5-8.0.

[0037] Preferably, the loading solution is diluted with affinity chromatography buffer A. The cell disruption supernatant is diluted with affinity chromatography buffer A and then filtered to obtain affinity chromatography loading solution.

[0038] More preferably, the affinity chromatography buffer A comprises: 50 mM phosphate buffer, 0.5 M NaCl, 3 mM imidazole, pH = 7.5; the affinity chromatography wash solution comprises: 50 mM phosphate buffer, 0.5 M NaCl, 30 mM imidazole, pH = 7.5; the affinity chromatography eluate comprises: 50 mM phosphate buffer, 0.5 M NaCl, 100 mM imidazole, pH = 7.5.

[0039] After the affinity chromatography is completed, ion exchange chromatography is used for further separation and purification. Before the ion exchange chromatography, the fractions collected by the affinity chromatography are replaced with a buffer solution using dialysis.

[0040] The storage solution used for dialysis comprises: 30-50mM Tris-HCl, 4-8mM EDTA, 0.1%-0.2% Triton X-100, 45-55% glycerol, pH=7.5-8.0; the storage solution is used for dialysis to maintain the dissolution stability of the target protein.

[0041] Preferably, the storage solution comprises: 50 mM Tris-HCl, 5 mM EDTA, 0.1% Triton X-100, 50% glycerol, pH=8.

[0042] In step (4), the composition of the ion exchange chromatography washing solution includes: 20-80mM Tris-HCl, 100mMNaCl, 0.1%-0.3% Triton X-100, 3%-8% glycerol, pH=7.5-8.0;

[0043] The composition of the ion exchange chromatography eluent includes: 20-80mM Tris-HCl, 200mM NaCl, 0.1%-0.3% Triton X-100, 3%-8% glycerol, pH=7.5-8.0.

[0044] Preferably, before loading the sample, the chromatography column is first rinsed with nuclease-free water and then balanced with ion exchange chromatography buffer A; after loading the sample, the ion exchange chromatography buffer A is first used for balance; the composition of the ion exchange chromatography buffer A includes: 20-80mM Tris-HCl, 10-50mM NaCl, 0.1%-0.3% Triton X-100, 3%-8% glycerol, pH = 7.5-8.0.

[0045] More preferably, the composition of ion exchange chromatography buffer A includes: 50mM Tris-HCl, 20mM NaCl, 0.1% Triton X-100, 5% glycerol, pH = 7.9; the composition of ion exchange chromatography wash solution includes: 50mM Tris-HCl, 100mM NaCl, 0.1% Triton X-100, 5% glycerol, pH = 7.9; the composition of ion exchange chromatography eluent includes: 50mMTris-HCl, 200mM NaCl, 0.1% Triton X-100, 5% glycerol, pH = 7.9.

[0046] The target protein prepared by the separation and purification process of the present invention has high purity and is free of DNase and RNase contamination.

[0047] The present invention also provides the use of the T7 RNA polymerase mutant in in vitro transcription. The T7 RNA polymerase mutant can also be used for the synthesis of non-coding RNA or mRNA for non-therapeutic purposes.

[0048] The present invention has the following beneficial effects:

[0049] (1) The present invention provides a T7 RNA polymerase mutant, which is obtained by replacing the glycine at position 640 in the amino acid sequence of the wild-type T7 RNA polymerase with tryptophan. Compared with the wild-type T7 bacteriophage RNA polymerase, the T7 RNA polymerase mutant has higher catalytic activity and better thermal stability, and compared with the RNA synthesis level of related polymerases currently on the market, the RNA yield is increased by 30-50%.

[0050] (2) The present invention provides a method for separating and purifying a T7 RNA polymerase mutant. By optimizing the purification process, the prepared T7 RNA polymerase sample does not contain DNase and RNase contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the pET-28a(+) plasmid map.

[0052] Figure 2 This is the map of pET-28a(+)-T7RNAP plasmid.

[0053] Figure 3 This is the map of pET-28a(+)-G640W-T7RNAP plasmid.

[0054] Figure 4 The SDS-PAGE results of the expression product after IDA column chromatography, wherein lane 1 is the protein marker, lane 2 is the loading solution, lane 3 is the washing solution, and lanes 4-7 are the chromatography eluents.

[0055] Figure 5 The SDS-PAGE results of the expression product after IDA column chromatography and then DEAE-650M column chromatography, wherein lane 1 is the protein marker and lane 2 is the chromatography eluent.

[0056] Figure 6 The results of the Nickase residue detection in the purified product are shown in Figure 1, where lanes 1-2 are the Nickase-free control group, and lanes 3-4 are T7RNAP-G640W.

[0057] Figure 7 The results of Endonuclease residue detection in the purified product, where lanes 1-2 are Endonuclease non-contaminated control groups, and lanes 3-4 are T7RNAP-G640W.

[0058] Figure 8 The results of the detection of Exonuclease residues in the purified products, where lanes 1-2 are the Exonuclease-free control group, and lanes 3-4 are T7RNAP-G640W.

[0059] Fig. 9 The results of RNase residue detection in the purified product, where lanes 1-2 are the RNase-free control group, and lanes 3-4 are T7RNAP-G640W.

[0060] Fig.10 These are the results of in vitro transcription products of T7RNAP-G640W mutant and wild-type T7RNAP, where lanes 1-2 are T7RNAP-G640W, and lanes 3-4 are wild-type T7RNAP.

[0061] Fig.11 The results are as follows: detection of T7 RNAP in vitro transcription products by agarose gel nucleic acid electrophoresis, wherein lane 1 is RNAMarker, lanes 2-3 are commercially available product 1, and lanes 4-5 are experimental group T7 RNAP-G640W.

[0062] Fig.12 This is the result of detecting the in vitro transcription product of T7RNAP by agarose gel nucleic acid electrophoresis in Example 3, wherein lanes 1-2 are the experimental group T7RNAP-G640W, lanes 3-4 are commercially available product 1, and lanes 5-6 are commercially available product 2. DETAILED DESCRIPTION

[0063] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0064] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0065] Example 1: Construction of G640W mutant T7RNAP engineering bacteria

[0066] 1. Construction of wild-type T7RNAP expression vector

[0067] Using the wild-type T7 RNAP gene fragment (nucleotide sequence as shown in SEQ ID NO.4) as a template, the DNA fragment containing the T7 RNA polymerase gene was amplified by PCR and then PCR amplified with the pET-28a(+) plasmid ( Figure 1 )connect.

[0068] The specific method is as follows:

[0069] (1) A PCR reaction was performed using an artificial plasmid containing a fragment of the wild-type T7 RNAP gene (SEQ ID NO. 4) as a template using the following reagent composition and reaction conditions.

[0070] The primers for PCR reaction are as follows:

[0071] Primer connection F (SEQ ID NO.5):

[0072] 5'-AGTCTCGAGATGAACACGATTAACATCGCTAAGAACGAC-3';

[0073] Primer connected to R (SEQ ID NO.6):

[0074] 5'-ATAAGCGGCCGCTTACGGCGAACGCGAAGTCCGACT-3'.

[0075] The PCR reaction system (total reaction volume: 50 μL) was composed of the following reagents: 1 μL of Primer ligation F at a concentration of 10 μM, 1 μL of Primer ligation R at a concentration of 10 μM, 5 μL of 10× Taq Buffer, 3 μL of MgCl2 at a concentration of 25 μM, 1 μL of dNTP (Shanghai Biotech), 2.5 μL of DNA template, 0.5 μL of Taq enzyme (Shanghai Biotech), and 36 μL of ddH2O.

[0076] PCR reaction system conditions: Use a thermal cycler (manufactured by BIO-RAD), heat at 94°C for 3 minutes, then repeat the temperature cycle of 94°C, 30 seconds; 58°C, 30 seconds; 72°C, 1 minute 35 times, then heat at 72°C for 10 minutes and store at 4°C.

[0077] (2) The PCR product was purified using a PCR purification kit (Shanghai Biotechnology Co., Ltd.).

[0078] (3) The purified PCR product was digested with restriction enzymes Xho I (NEB) and Not I (NEB), and reacted with pET-28a(+) digested with restriction enzymes Xho I (NEB) and Not I (NEB) using T4 ligase at 4°C for 30 minutes.

[0079] (4) The reaction mixture of (3) was used to transform Escherichia coli TOP10 strain, and selection was performed on LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose, 1% agar, 50 μg / mL kanamycin sulfate (McLean), pH = 7.4). Colonies grown after overnight culture at 37°C were picked, and plasmids were extracted after amplification. The correct plasmid was identified by PCR reaction as pET-28a(+)-T7RNAP (such as Figure 2 ).

[0080] 2. Construction of G640W mutant T7RNAP expression vector

[0081] According to the following process, a site-directed mutation was introduced into the T7RNAP gene of the pET-28a(+)-T7RNAP plasmid prepared in step 1, so that the G at position 1918 of the wild-type T7RNAP gene nucleotide sequence (SEQ ID NO.4) was mutated to T, thereby obtaining the G640W mutant T7RNAP.

[0082] (1) Site-directed mutagenesis PCR was performed using pET-28a(+)-T7RNAP as a template plasmid and the following reagent composition and reaction conditions.

[0083] The primers for PCR reaction are as follows:

[0084] Primer mutation F (SEQ ID NO.7):

[0085] 5'-CTTACTGGTCCAAAGAGTTCGGCTTCC-3';

[0086] Primer mutation R (SEQ ID NO.8):

[0087] 5'-CTTTGGACCAGTAAGCCAGCGTCATGACTG-3'.

[0088] The PCR reaction system (total reaction volume: 50 μL) was composed of the following reagents: 1 μL of Primer mutant F at a concentration of 10 μM, 1 μL of Primer mutant R at a concentration of 10 μM, 5 μL of 10× Taq Buffer, 3 μL of MgCl2 at a concentration of 25 μM, 1 μL of dNTP (Shanghai Bioengineering), 2.5 μL of DNA template, 0.5 μL of Taq enzyme (Shanghai Bioengineering), and 36 μL of ddH2O.

[0089] PCR reaction conditions: Use a thermal cycler (manufactured by BIO-RAD) to heat at 94°C for 3 minutes, then repeat the temperature cycle of 94°C, 30 seconds, 55°C, 30 seconds, and 72°C, 2 minutes and 41 seconds 35 times, then heat at 72°C for 10 minutes and store at 4°C.

[0090] After the PCR reaction, 10 units of restriction enzyme DpnI (Shanghai Biotechnology) were added and digested at 37°C for 1 hour. The plasmid was transformed into E. coli TOP10 according to the conventional method and extracted and sequenced using the plasmid extraction and purification kit (Shanghai Biotechnology). The plasmid with the correct sequencing result was pET-28a(+)-G640W-T7RNAP plasmid ( Figure 3 ), the nucleotide sequence of the G640W mutant T7RNAP is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.1, that is, the glycine at position 640 of the wild-type T7RNAP amino acid sequence (SEQ ID NO.3) is substituted with tryptophan.

[0091] 3. Construction of G640W mutant T7RNAP engineered bacteria

[0092] (1) The pET-28a(+)-G640W-T7RNAP plasmid was transformed into Escherichia coli BL21 according to conventional methods and selected on LB agar medium (1% peptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose, 1% agar, 50 μg / mL kanamycin sulfate (McLean), pH = 7.4). The colonies grown after overnight culture at 37°C were picked and identified as G640W mutant T7RNAP engineered bacteria by colony PCR.

[0093] (2) The G640W mutant T7 RNAP engineered bacteria obtained in (1) were inoculated into 100 mL of LB liquid culture medium and cultured in a 500 mL conical flask at 37° C. and 220 rpm with shaking for 18-20 h.

[0094] (3) The bacterial cells obtained in (2) were prepared into a glycerol stock solution of G640W mutant T7 RNAP engineered bacteria according to conventional methods and stored at -80°C.

[0095] Example 2: Expression and purification of G640W mutant T7 RNAP

[0096] 1. Inducible expression of G640W mutant T7 RNAP

[0097] (1) In a 500 mL conical flask, 100 mL of LB liquid medium was inoculated with 400 μL of the glycerol stock solution of the G640W mutant T7 RNAP engineered bacteria obtained in Example 1 and cultured overnight at 37° C. and 220 rpm with shaking.

[0098] (2) In a 2000 mL conical flask, 500 mL of LB liquid medium was inoculated with 1.0 mL of the bacterial solution cultured in (1), and cultured at 37°C and 220 rpm.

[0099] (3) After culturing for about 2.5 hours (OD 600 value is about 0.4-0.6), add 500 μL of 0.1 M IPTG (isopropyl-β-thiogalactoside) to each bottle, and culture with shaking at 25°C and 180 rpm for another 18-20 h.

[0100] (4) After the culture is completed, the cells are centrifuged at 4°C and 8000 rpm for 15 minutes to recover the cells. If the cells are not immediately disrupted, they are stored at -20°C.

[0101] 2. Isolation and purification of G640W mutant T7 RNAP

[0102] (1) The recovered bacterial cells were added with an appropriate amount of cell lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 5% (v / v) glycerol, 12.5 μg / mL lysozyme (BBI), 0.008% sodium deoxycholate (McLean), 5 mM DTT (Beyotime), 0.3 mM benzamidine (Inokai), 50 μg / mL PMSF (BBI), 20 μg / mL bacitracin (BBI), 8 mM ammonium sulfate (Aladdin), pH = 7.9), and ultrasonically disrupted using an ultrasonic cell disruptor (Shinshiba).

[0103] The ultrasonic conditions are as follows: place the beaker containing the bacterial suspension to be lysed in a 0°C ice-water mixture, insert the ultrasonic probe into the appropriate position of the bacterial liquid, set the ultrasonic power to 400W, ultrasonicate for 2s, stop for 4s, and ultrasonicate for 20min. During the ultrasonic process, the liquid temperature is always kept below 10°C.

[0104] After the ultrasound, the supernatant was centrifuged at 4°C and 10,000 rpm for 10 min, diluted with TED-A (50 mM PB, 0.5 M NaCl, 3 mM imidazole, pH = 7.5), filtered through a 0.45 μm water filter membrane, and temporarily stored at 4°C overnight for affinity purification using nickel chelating resin.

[0105] (2) The target protein was purified by affinity purification and ion exchange purification using the histidine hexamer tag added to the G640W mutant T7 RNA polymerase using the following method.

[0106] (2-1) A column filled with 30 mL of IDA filler (made by Xi'an Lanxiao) was fixedly connected to a low-pressure chromatography system (Bio-Lab100, made by Jiangsu Hanbang), and first rinsed with Nuclease-free water at 2 mL / min for 10 CV. It was then balanced with TED-A (50 mM PB, 0.5 M NaCl, 3 mM imidazole, pH = 7.5) until the baseline was stable, at a flow rate of 3 mL / min for 8-10 CV.

[0107] Load the sample solution obtained in (1) at a flow rate of 0.5-1.0 mL / min. After loading, rinse with TED-A at 2 mL / min for 12 CV until the baseline (UV, cd) is stable. Then elute the related impurities with 3% TED-B (50 mM PB, 0.5 M NaCl, 1 M imidazole, pH = 7.5) for 10 CV. Then elute with 10% TED-B (50 mM PB, 0.5 M NaCl, 1 M imidazole, pH = 7.5) until the peak begins at UV = 280 nm in the low-pressure chromatography system, and collect in separate tubes, 0.5 CV per tube.

[0108] Take 20uL of sample from each tube, add 5×SDS loading buffer, and incubate at 100℃ for 10min. Perform SDS-PAGE electrophoresis at 180V for 30min. Observe the purity of the sample after staining with SDS-PAGE fast staining and decolorization instrument (GenScript). The results are as follows: Figure 4 shown.

[0109] (2-2) Based on the protein purity shown by SDS-PAGE in (2-1), the TED column chromatograms were combined and dialyzed twice at 4°C for about 6 hours each time using 10 volumes of storage buffer (50 mM Tris-HCL, 5 mM EDTA, 0.1% (v / v) Triton X-100, 50% (v / v) glycerol, pH = 8).

[0110] (2-3) A column filled with 30 mL of DEAE-650M filler (TOYOPEARL) was fixedly connected to a low-pressure chromatography system (Jiangsu Hanbang) and flushed with Nuclease free water at 2 mL / min for 10 CV. DEAE-A (50 mM Tris-HCl, 20 mM NaCl, 0.1% Triton X-100, 5% glycerol, pH = 7.9) was equilibrated until the baseline (UV, cd) was stable, at a flow rate of 1-2 mL / min for about 10 CV.

[0111] The dialysate obtained in (2-2) was diluted 5 times with DEAE-A and then loaded, 0.5-1.0 mL / min. DEAE-A (50 mM Tris-HCl, 20 mM NaCl, 0.1% Triton X-100, 5% glycerol, pH = 7.9) was washed, 1-2 mL / min, 20 CV. Then 10% DEAE-B (50 mM Tris-HCl, 1 M NaCl, 0.1% Triton X-100, 5% glycerol, pH = 7.9) was used to wash away related impurities, 1-2 mL / min, 10 CV. Then 20% DEAE-B (50 mM Tris-HCl, 1 M NaCl, 0.1% Triton X-100, 5% glycerol, pH = 7.9) was used for elution, 1-2 mL / min, and the samples were collected in different tubes when the peak started at UV = 280 nm, 0.5 CV per tube.

[0112] Take 20 μL of sample from each tube, add 5×SDS loading buffer, and incubate at 100°C for 10 min. Perform SDS-PAGE electrophoresis at 180V for 30 min. Observe the purity of the sample after staining with SDS-PAGE fast staining and decolorization instrument (GenScript). The results are as follows: Figure 5 shown.

[0113] (2-4) Use 10 times the volume of storage buffer (50mM Tris-HCL, 5mM EDTA, 0.1% TritonX-100, 50% glycerol, pH=8) and dialyze twice at 4°C, each time for about 8 hours. Detection Example 1: RNase and DNase contamination detection

[0114] (1) Protein concentration determination The protein concentration of the G640W mutant T7 RNA polymerase in the dialysate obtained in Example 2 (2-4) was determined by spectrophotometry using a modified Bradford protein concentration determination kit (Shanghai Biotechnology Co., Ltd.) and was 0.43 mg / mL.

[0115] (2) Nickase residue detection: 4 μL of the sample to be tested and 1 μL of 0.5 μg / μL pBR322 DNA (NEB) were added to 10 μL of the reaction system, incubated at 37°C for 4 h, and then detected by agarose gel electrophoresis. The results are as follows Figure 6 As shown, the DNA bands in agarose gel electrophoresis did not change.

[0116] (3) Endonuclease residue detection: 4 μL of the sample to be tested and 1 μL of 0.35 μg / μL λDNA (NEB) were added to the 10 μL reaction system, incubated at 37°C for 4 h, and then detected by agarose gel electrophoresis. The results are as follows Figure 7 As shown, the DNA bands in agarose gel electrophoresis did not change.

[0117] (4) Exonucleas residue detection: 4 μL of the sample to be tested and 1 μL of λDNA-HindⅢdigest (NEB) at a concentration of 0.5 μg / μL were added to the 10 μL reaction system, incubated at 37°C for 4 h, and then detected by agarose gel electrophoresis. The results are as follows Figure 8 As shown, the DNA bands in agarose gel electrophoresis did not change.

[0118] (5) RNase residual detection: 4 μL of the sample to be tested and 1 μL of 1 μg / μL MS2 RNA (Roche) were added to the 10 μL reaction system, incubated at 37°C for 4 h, and then detected by agarose gel electrophoresis. The results are as follows Fig. 9 As shown, the agarose gel electrophoresis bands did not change.

[0119] The RNase and DNase residues of the G640W mutant T7 RNA polymerase sample isolated and purified in Example 2 were detected. Figure 6-Figure 9 It was judged that the samples obtained by this preparation method were not contaminated by relevant RNase and DNase enzymes.

[0120] Test Example 2: Activity Assay

[0121] Activity determination was performed by measuring the amount of RNA generated in an in vitro transcription reaction, and the template DNA used was a DNA having a T7 promoter sequence that specifically recognizes T7 RNA polymerase. The one used here was obtained by PCR amplification using a linear double-stranded DNA (SEQ ID NO. 9) containing a T7 promoter sequence as a template.

[0122] 1. Using SEQ ID NO.9 as the transcription template, the following reagent composition and reaction conditions were used to perform in vitro transcription reaction.

[0123] Reaction system (total reaction liquid volume: 20 μL), reagent composition: Nuclease free water 4.6 μL, 10×reaction buffer 2 μL, RNase Inhibitor 1 μL, inorganic pyrophosphatase (100 U / μL, Yisheng) 0.4 μL, dNTP (100 mM ATP, CTP, GTP, UTP, Shanghai Biotechnology) 2 μL each, DNA template (sequence SEQ ID NO.9) 2 μL, T7 RNAP enzyme 2 μL.

[0124] The experimental group used the G640W mutant T7 RNA polymerase of Example 2, and the control group used the wild-type T7 RNA polymerase, with an enzyme concentration of 0.3-0.5 mg / mL, and the experiment was repeated twice.

[0125] Reaction conditions: Incubate the reaction at 37°C for 2 hours.

[0126] The product was sampled for agarose gel electrophoresis. Fig.10 shown.

[0127] The amount of RNA produced was quantitatively detected using the commercially available (XR) RNA Quantification Kit for Qubit 4 (manufactured by Thermofisher) and Qubit 4 (manufactured by Thermofisher). The data are shown in Table 1.

[0128] Table 1. Amounts of RNA generated by in vitro transcription reactions

[0129]

[0130] Depend on Fig.10 As can be seen from Table 1, compared with the transcription activity of wild-type T7 RNAP, the in vitro transcription catalytic activity of the T7 RNAP-G640W mutant is significantly higher than that of the wild-type T7 RNAP.

[0131] 2. In a clean bench, add the corresponding reagents according to the reaction system in step 1, including T7RNAP (the experimental group uses the G640W mutant T7RNA polymerase of Example 2, and the control group uses the commercially available T7RNA polymerase, repeated twice), mix and centrifuge, put into the PCR instrument, run at 37°C for 2 hours, add 1 μL of deoxyribonuclease I to each sample and incubate for 15 minutes. Sample the product for agarose gel electrophoresis, such as Fig.11 The results of RNA quantification are shown in Table 2.

[0132] Table 2. Activity test in vitro transcription reaction system and the amount of RNA generated

[0133]

[0134] According to the data in the above table, it can be concluded that the G640W mutant T7 RNA polymerase obtained by the present invention has good transcription catalytic activity. Compared with the traditional in vitro transcription reaction, it has a very high synthesis amount. Compared with the level of relevant kits on the market (150-180 μg of RNA synthesized per reaction (20 μL)), the RNA yield is increased by 30-50%.

[0135] Test Example 3: Thermal Stability Test

[0136] The thermal stability test was performed by measuring the amount of RNA generated in an in vitro transcription reaction at 46° C. The reaction system was the same as that in Test Example 2, and the reaction conditions were: incubation at 46° C. for 2 hours.

[0137] In the clean bench, add the corresponding reagents according to the reaction system in step 1 of detection example 2, including T7RNAP (the experimental group uses the G640W mutant T7RNA polymerase of Example 2, and the control group uses the wild-type T7RNA polymerase and commercially available T7RNA polymerase, repeated twice), mix and centrifuge, put into the PCR instrument, run at 46°C for 2 hours, add 1 μL of deoxyribonuclease I to each sample and incubate for 15 minutes. Sample the product for agarose gel electrophoresis, such as Fig.12 The results of RNA quantification are shown in Table 3.

[0138] Table 3. In vitro transcription reaction system and RNA amount generated in thermal stability test

[0139] RNA synthesis amount (μg) T7RNAP-G640W 168.07 T7RNAP-G640W 169.34 Wild-type T7 RNAP 65.36 Wild-type T7 RNAP 67.27 Commercially available products 1 126.06 Commercially available products 1 123.12 Commercially available products 2 108.05 Commercially available products 2 110.56

[0140] According to the data in the above table, it can be concluded that the thermal stability of the T7RNAP-G640W mutant is significantly higher than that of the wild-type T7RNAP. Under the temperature condition of 46°C, the G640W mutant T7RNA polymerase obtained by the present invention still retains a high in vitro transcription catalytic activity, which is higher than the relevant T7RNAP sold on the market.

Claims

1. A T7 RNA polymerase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the T7 RNA polymerase mutant according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

3. A recombinant vector comprising the coding gene as claimed in claim 2.

4. A recombinant genetically engineered bacterium comprising the recombinant vector as claimed in claim 3.

5. A method for preparing a T7 RNA polymerase mutant free of DNase and RNase contamination, characterized in that: The following steps are involved: (1) Transforming a recombinant expression vector containing a gene fragment having a nucleotide sequence as shown in SEQ ID NO.2 into an Escherichia coli host cell to obtain a recombinant genetically engineered bacterium, wherein the recombinant expression vector has a His tag coding sequence; (2) After fermentation and cultivation of the recombinant genetically engineered bacteria, the cells are collected by centrifugation, a cell lysis solution is added, the cells are ultrasonically disrupted, the supernatant is collected by centrifugation, and the affinity chromatography loading solution is obtained by filtration; The cell lysis solution comprises: 20-50 mM Tris-HCl, 10-50 mM NaCl, 3-10 mM EDTA, 3%-10% glycerol, 10-20 μg / mL lysozyme, 0.005%-0.010% sodium deoxycholate, 3-8 mM dithiothreitol, 0.1-0.5 mM benzamidine, 20-80 μg / mL phenylmethylsulfonyl fluoride, 10-40 μg / mL bacitracin, 5-15 mM ammonium sulfate, pH=7.5-8.0; (3) loading the affinity chromatography loading solution onto an affinity chromatography column filled with Ni-IDA filler, first eluting impurities with affinity chromatography washing solution, then eluting with affinity chromatography eluent at an ultraviolet detection wavelength of 280 nm until a peak begins to appear, collecting the eluate, and dialyzing with a storage solution to obtain an ion exchange chromatography loading solution; The affinity chromatography washing solution comprises: 20-60 mM phosphate buffer, 0.2-0.5 M NaCl, 30 mM imidazole, pH = 7.5-8.0; The affinity chromatography eluent comprises: 20-60 mM phosphate buffer, 0.2-0.5 M NaCl, 100 mM imidazole, pH = 7.5-8.0; The storage solution comprises: 30-50 mM Tris-HCl, 4-8 mM EDTA, 0.1%-0.2% Triton X-100, 45-55% glycerol, pH=7.5-8.0; (4) loading the ion exchange chromatography loading solution onto an ion exchange chromatography column filled with DEAE-650M filler, first eluting impurities with an ion exchange chromatography washing solution, and then eluting with an ion exchange chromatography eluent at a UV detection wavelength of 280 nm until a peak begins to appear, collecting the eluate to obtain the T7 RNA polymerase mutant; The composition of the ion exchange chromatography washing solution includes: 20-80mM Tris-HCl, 100mM NaCl, 0.1%-0.3% Triton X-100, 3%-8% glycerol, pH=7.5-8.0; The composition of the ion exchange chromatography eluent includes: 20-80mM Tris-HCl, 200mM NaCl, 0.1%-0.3% Triton X-100, 3%-8% glycerol, pH=7.5-8.

0.

6. The preparation method according to claim 5, characterized in that: In step (2), the cell disruption supernatant is diluted with affinity chromatography buffer A and then filtered to obtain an affinity chromatography loading solution; The affinity chromatography buffer A comprises: 20-60 mM phosphate buffer, 0.2-0.5 M NaCl, 3-10 mM imidazole, pH=7.5-8.

0.

7. The preparation method according to claim 6, characterized in that: In step (3), before loading the sample, the chromatography column is first rinsed with nuclease-free water, and then the column is equilibrated with affinity chromatography buffer A; after loading the sample, the column is first equilibrated with affinity chromatography buffer A, and then impurities are eluted with affinity chromatography washing solution, and then at a UV detection wavelength of 280 nm, affinity chromatography eluent is used to elute until a peak begins to appear, and the eluate is collected.

8. The preparation method according to claim 5, characterized in that: In step (4), before loading the sample, the chromatography column is first rinsed with nuclease-free water, and then the column is balanced with ion exchange chromatography buffer A; after loading the sample, the column is first balanced with ion exchange chromatography buffer A, and then the impurities are eluted with ion exchange chromatography washing solution, and then the ion exchange chromatography eluent is used to elute at an ultraviolet detection wavelength of 280 nm until the peak begins to appear, and the eluate is collected; The composition of the ion exchange chromatography buffer A includes: 20-80mM Tris-HCl, 10-50mM NaCl, 0.1%-0.3% Triton X-100, 3%-8% glycerol, pH=7.5-8.

0.

9. The preparation method according to claim 5, characterized in that: The original plasmid of the recombinant expression vector is pET-28a(+), and the fermentation culture includes: expanding the activated recombinant genetic engineering bacteria to OD 600 =0.4-0.6, add IPTG with a final concentration of 0.1 mM, and culture with shaking at 25°C and 180 rpm for 18-20 h.

10. Use of the T7 RNA polymerase mutant according to claim 1 in in vitro transcription.

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