CN116179507B

CN116179507B - A T7 RNA polymerase mutant and its preparation method and application

Info

Publication number: CN116179507B
Application number: CN202310269396.9A
Authority: CN
Inventors: 韦威; 姚红; 李胤直; 徐钰婷
Original assignee: Core Biomedical Technology Hangzhou Co ltd
Current assignee: Core Biomedical Technology Hangzhou Co ltd
Priority date: 2023-03-20
Filing date: 2023-03-20
Publication date: 2025-01-24
Anticipated expiration: 2043-03-20
Also published as: CN116179507A

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

T7 RNA polymerase mutant and preparation method and application thereof

Technical Field

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

Background

With rapid development of science and technology and biotechnology, research and application of RNA related are greatly developed, which presents a high challenge for RNA synthesis, and at present, RNA in vitro synthesis includes two methods of chemical synthesis and enzymatic synthesis, wherein the chemical synthesis is suitable for synthesizing RNA with a length of several tens of nucleotides, and with the increase of the length of RNA, the production cost is rapidly increased. When the number of nucleotides reaches more than one hundred, chemical synthesis is no longer applicable, and mRNA encoding a protein typically contains thousands of nucleotides, so enzymatic synthesis is currently the only method for preparing long-chain RNA.

Phage T7RNA Polymerase (T7 RNA Polymerase, T7 RNAP) was first isolated in 1970 from phage T7-infected E.coli cells, one of the simplest enzymes catalyzing RNA synthesis. T7RNA polymerase has a high degree of promoter specificity and will only transcribe DNA or DNA copies of the T7 phage downstream of the T7 promoter. In recent years, the T7RNA polymerase transcription system plays an important role in synthesis biology, and T7RNAP is widely applied to in vitro synthesis of RNA and in vivo protein expression (bacterial high expression system). Therefore, the research of the construction, expression and purification process of the T7RNA polymerase with high activity and high stability becomes one of research hotspots.

Site-directed mutagenesis of T7 RNA polymerase using protein engineering techniques to obtain mutants with improved function is one of the means for the development of T7 RNA polymerase products. In the prior art, patent document CN102177236a discloses that the heat stability and specific activity of wild-type T7 RNA polymerase are improved by substituting at least one amino acid residue of glutamine at position 786, lysine at position 179 and valine at position 685 in the amino acid sequence of the wild-type T7 RNA polymerase with other amino acids. For example, patent document CN 107460177a discloses that the transcription activity is improved by substituting cysteine for arginine at position 632 in the amino acid sequence of wild-type T7 RNA polymerase.

Based on the expression system of T7RNAP, researchers developed two chassis strains of Corynebacterium glutamicum MB001 and Escherichia coli BL21 (DE 3). How to separate and purify T7RNA polymerase with high yield, high purity and good activity from fermentation products of chassis strains is a problem to be solved by the technicians in the field.

With the rapid development of purification process research, the purification methods of small molecular monomers or macromolecular proteins are more and more, and the recombinant protein purification process needs to pay attention to the following problems that 1) if the recombinant protein is expressed in cells, the structure and activity of the target protein are protected in the process of cell disruption treatment. 2) The steps of separation and purification of each step can cause a certain amount of protein and loss of protein activity, but the separation steps are too few and cannot reach a certain purity standard, 3) the application range of the reagent and related operation used in each separation and purification link to different proteins is different, 4) DNase and RNase pollution usually exist in the sample of the T7 RNA polymerase obtained by the conventional separation and purification method. Thus, exploring a suitable set of purification methods for a particular target protein is also a problem that currently needs to be addressed in developing high-activity T7 RNA polymerase products.

Disclosure of Invention

The invention aims to replace amino acid of wild T7RNA polymerase by using a genetic engineering technology to obtain a T7RNA polymerase mutant with obviously improved enzyme activity and thermal stability, and preparing a T7RNA polymerase product without DNase and RNase pollution by optimizing separation and purification process conditions.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention adopts a genetic engineering method to replace 640 glycine (G) in the amino acid sequence of wild type T7RNA polymerase with tryptophan (W) to obtain a T7RNA polymerase mutant G640W with the amino acid sequence shown as SEQ ID NO. 1.

Compared with the RNA polymerase of the wild type T7 phage, the thermal stability and specific activity of the T7RNA polymerase mutant G640W are greatly improved.

The T7RNA polymerase G640W mutant of the present invention may be obtained by using a recombinant protein produced by a gene recombination technique, and when the mutant gene of the T7RNA polymerase is obtained by using a gene recombination technique, the mutant gene is introduced into a host by using an appropriate genetic engineering technique, and induced expression is performed to obtain the target protease.

The invention provides a coding gene for coding the T7 RNA polymerase mutant, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

The invention uses gene site-directed mutagenesis technology to mutate 1918G of the coding sequence (SEQ ID NO. 4) of wild T7RNA polymerase into T and replace the codon GGG originally encoding 640 th glycine with TGG.

In addition, sequences useful for purification of enzymes, for example, extracellular secretion type enzymes can be formed by using signal peptides, or tag sequences containing histidine hexamers can be added to the ends of the T7RNA polymerase mutant genes, so that the development of a post-purification process is facilitated.

The host used for expression may be various cultured cells such as plant cells, animal cells, insect cells, etc., and E.coli is selected as the host cell according to the present invention for T7RNA polymerase gene expression.

The T7RNA polymerase mutant gene of the present invention is inserted into an appropriate vector and then introduced into the above-mentioned E.coli host cell, and usually the expression vector is of a large variety, and the selection of the vector requires consideration of the elements necessary for transcription of the selected vector after insertion of the target gene, and can be appropriately selected depending on the host type.

The invention also provides a recombinant vector containing the coding gene. Preferably, the expression vector adopts plasmids pET-28a and pET-30a, but is not limited to the above.

The invention also provides a recombinant genetically engineered bacterium containing the recombinant vector. Preferably, the expression host is E.coli BL21.

The T7RNA polymerase of the present invention can be obtained by culturing E.coli containing the gene of interest after transformation in a suitable component medium under conditions that allow expression of the introduced DNA construct. The T7RNA polymerase of the invention is obtained by separating and purifying from E.coli cells after induced expression, and conventional methods for separating and purifying proteins can be adopted. For example, when the enzyme of the present invention is expressed in cells, after completion of the culture, the cells are centrifuged and collected, suspended in an appropriate aqueous buffer, and then the cells are disrupted by lysozyme treatment or an ultrasonic disrupter to obtain a cell-free extract. Further, the cell-free extract was subjected to centrifugation to obtain a supernatant, and the supernatant was purified by a conventional method for separating and purifying proteins. The main purification methods are salting out (ammonium sulfate precipitation), molecular sieves, affinity chromatography, ion exchange chromatography, etc.

The samples of T7RNA polymerase obtained by conventional separation and purification methods are usually contaminated by DNase and RNase. The invention flexibly combines related purification process methods to realize rapid separation of target proteins, optimizes related purification processes, and ensures that the prepared sample of the T7RNA polymerase does not contain DNase and RNase pollution.

Specifically, the invention provides a method for separating and purifying a T7 RNA polymerase mutant without DNase and RNase pollution from an intracellular induction expression product of escherichia coli, which comprises the following steps:

(1) Transferring a recombinant expression vector containing a gene fragment with a nucleotide sequence shown as SEQ ID NO.2 into an escherichia coli host cell to obtain recombinant genetic engineering bacteria, wherein the recombinant expression vector has a His tag coding sequence;

(2) Centrifugally collecting thalli after fermenting and culturing the recombinant genetically engineered bacteria, adding cell lysate, ultrasonically crushing, centrifugally taking supernatant, and filtering to obtain affinity chromatography loading liquid;

(3) Loading the affinity chromatography loading liquid to an affinity chromatography column filled with Ni-IDA filler, eluting impurities with the affinity chromatography eluting liquid, eluting with the affinity chromatography eluting liquid at 280nm ultraviolet detection wavelength until the peak starts, collecting the eluting liquid, and dialyzing with a storage liquid to obtain the ion exchange chromatography loading liquid;

(4) Loading the ion exchange chromatography loading liquid to an ion exchange chromatography column filled with DEAE-650M filler, eluting impurities by using ion exchange chromatography eluting liquid, and then eluting by using ion exchange chromatography eluting liquid until the eluting liquid starts to peak under the ultraviolet detection wavelength of 280nm, and collecting the eluting liquid to prepare the T7 RNA polymerase mutant;

In the step (1), a recombinant genetically engineered bacterium for expressing the T7 RNA polymerase mutant is constructed. The original plasmid of the recombinant expression vector is pET-28a (+). The expressed recombinant protein is provided with a 6 XHis tag, and the protein separation and purification can be performed by utilizing nickel chelate resin for affinity chromatography.

In the step (2), the recombinant genetically engineered bacteria are induced to express recombinant proteins in cells, and the recombinant proteins are released by cell lysis for separation and purification.

The fermentation culture comprises the steps of carrying out amplification culture on the activated recombinant genetically engineered bacteria until OD ₆₀₀ = 0.4-0.6, adding IPTG with the final concentration of 0.1mM, and carrying out shaking culture for 18-20h at 25 ℃ and 180 rpm.

According to the invention, the degradation of the target protein can be effectively avoided by optimizing the formula of the cell lysate and combining an ultrasonic process, the activity of the target protein is maintained, and more impurity proteins are removed.

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

Ultrasonic crushing is carried out under ice bath condition, and the temperature of bacterial liquid in the whole bacterial breaking process is controlled below 10 ℃. Ultrasonic power 400W, ultrasonic power 2s, ultrasonic power 4s, and ultrasonic power 20min.

Preferably, the composition of the cell lysate comprises 50mM Tris-HCl,50mM NaCl,5mM EDTA,5% glycerol, 12.5. Mu.g/mL lysozyme, 0.008% sodium deoxycholate, 5mM DTT,0.3mM benzamidine, 50. Mu.g/mL PMSF, 20. Mu.g/mL bacitracin, 8mM ammonium sulfate, pH=7.9.

In step (3), affinity purification was performed using a histidine hexamer tag added to the G640W mutant T7RNA polymerase. The recombinant protein is combined on the filler of the affinity chromatographic column, firstly, the impurity is washed by adopting the affinity chromatographic impurity-washing liquid, and then the target fraction is eluted by removing the affinity chromatographic eluent.

The affinity chromatography impurity-washing liquid comprises 20-60mM phosphate buffer solution, 0.2-0.5M NaCl,30mM imidazole and pH=7.5-8.0.

The composition of the affinity chromatography eluent comprises 20-60mM phosphate buffer, 0.2-0.5M NaCl,100mM imidazole and pH=7.5-8.0.

Preferably, before loading, the chromatographic column is washed with nuclease-free water and then is balanced by the affinity chromatography buffer A, and after loading, the chromatographic column is balanced by the affinity chromatography buffer A. The affinity chromatography buffer A comprises 20-60mM phosphate buffer, 0.2-0.5M NaCl,3-10mM imidazole and pH=7.5-8.0.

Preferably, the loading solution is diluted with affinity chromatography buffer a. Diluting the cell disruption supernatant with an affinity chromatography buffer A, and filtering to obtain an affinity chromatography loading liquid.

More preferably, the composition of the affinity chromatography buffer A comprises 50mM phosphate buffer, 0.5M NaCl,3mM imidazole and pH=7.5, the composition of the affinity chromatography wash buffer comprises 50mM phosphate buffer, 0.5M NaCl,30mM imidazole and pH=7.5, and the composition of the affinity chromatography eluent comprises 50mM phosphate buffer, 0.5M NaCl,100mM imidazole and pH=7.5.

After the affinity chromatography is finished, further separating and purifying by utilizing ion exchange chromatography, and before the ion exchange chromatography, replacing the fraction collected by the affinity chromatography by adopting a dialysis method with a buffer solution.

The composition of the stock solution used for dialysis comprises 30-50mM Tris-HCl,4-8mM EDTA,0.1% -0.2% Triton X-100,45-55% glycerol and pH=7.5-8.0, and the stock solution is used for dialysis to be favorable for maintaining the dissolution stability of target proteins.

Preferably, the composition of the stock solution comprises 50mM Tris-HCl,5mM EDTA,0.1%Triton X-100,50% glycerol, pH=8.

In the step (4), the ion exchange chromatography impurity washing liquid comprises 20-80mM Tris-HCl,100mM NaCl,0.1% -0.3% Triton X-100,3% -8% glycerol, and pH=7.5-8.0;

The ion exchange chromatography eluent comprises 20-80mM Tris-HCl,200mM NaCl,0.1% -0.3% Triton X-100,3% -8% glycerol and pH=7.5-8.0.

Preferably, before loading, the chromatographic column is washed by nuclease-free water and then balanced by ion exchange chromatography buffer A, after loading, the ion exchange chromatography buffer A is used for balancing, and the composition of the ion exchange chromatography buffer A comprises 20-80mM Tris-HCl,10-50mM NaCl, 0.1-0.3% Triton X-100, 3-8% glycerol and pH=7.5-8.0.

More preferably, the composition of ion exchange chromatography buffer A comprises 50mM Tris-HCl,20mM NaCl,0.1% Triton X-100,5% glycerol, pH=7.9, the composition of ion exchange chromatography wash comprises 50mM Tris-HCl,100mM NaCl,0.1% Triton X-100,5% glycerol, pH=7.9, and the composition of ion exchange chromatography eluate comprises 50mM Tris-HCl,200mM NaCl,0.1% Triton X-100,5% glycerol, pH=7.9.

The target protein prepared by the separation and purification process has high purity and does not contain DNase and RNase pollution.

The invention also provides application of the T7RNA polymerase mutant in-vitro transcription. The T7RNA polymerase mutants may also be applied for non-coding RNA or mRNA synthesis for non-therapeutic purposes.

The invention has the beneficial effects that:

(1) The invention provides a T7RNA polymerase mutant, which is prepared by substituting 640 th glycine in an amino acid sequence of wild type T7RNA polymerase with tryptophan. Compared with the RNA polymerase of wild type T7 phage, the T7RNA polymerase mutant has higher catalytic activity and better thermal stability, and compared with the RNA synthesis level of related polymerase in the current market, the RNA yield is improved by 30-50%.

(2) The invention provides a method for separating and purifying a T7RNA polymerase mutant, wherein a sample of the prepared T7RNA polymerase does not contain DNase and RNase pollution by optimizing a purification process.

Drawings

FIG. 1 shows a pET-28a (+) plasmid map.

FIG. 2 shows a pET-28a (+) -T7RNAP plasmid map.

FIG. 3 is a map of pET-28a (+) -G640W-T7RNAP plasmid.

FIG. 4 shows SDS-PAGE results after column chromatography of the expression product IDA, wherein lane 1 is a protein Marker, lane 2 is a loading solution, lane 3 is a wash solution, and lanes 4-7 are chromatographic eluents.

FIG. 5 shows SDS-PAGE results of the expression product after IDA column chromatography and DEAE-650M column chromatography, wherein lane 1 is a protein Marker and lane 2 is a chromatographic eluate.

FIG. 6 shows the result of Nickase residue detection in purified product, wherein lanes 1-2 are Nickase, no-contamination control, and lanes 3-4 are T7RNAP-G640W.

FIG. 7 shows the result of endonucleolytic residual assay in purified product, wherein lanes 1-2 are endonucleolytic non-contaminating control and lanes 3-4 are T7RNAP-G640W.

FIG. 8 shows the result of Exonuclease residue detection in purified product, wherein lanes 1-2 are Exonuclease, no-contamination control, and lanes 3-4 are T7RNAP-G640W.

FIG. 9 shows the results of the detection of Rnase residue in purified product, wherein lanes 1-2 are the Rnase non-contaminating control, and lanes 3-4 are T7RNAP-G640W.

FIG. 10 shows the results of in vitro transcription of T7RNAP-G640W mutant and wild type T7RNAP, wherein lanes 1-2 are T7RNAP-G640W and lanes 3-4 are wild type T7RNAP.

FIG. 11 shows the results of agarose gel nucleic acid electrophoresis for the detection of T7RNAP in vitro transcripts, wherein lanes 1 are RNA MARKER, lanes 2-3 are commercially available commodity 1, and lanes 4-5 are experimental group T7RNAP-G640W.

FIG. 12 shows the results of detecting T7RNAP in vitro transcription products by agarose gel nucleic acid electrophoresis in detection example 3, wherein lanes 1-2 are experimental group T7RNAP-G640W, lanes 3-4 are commercial product 1, and lanes 5-6 are commercial product 2.

Detailed Description

The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The test methods used in the examples described below are conventional methods unless otherwise specified, and the materials, reagents, etc., used are commercially available reagents and materials unless otherwise specified.

Example 1 construction of G640W mutant T7RNAP engineering bacteria

1. Construction of wild type T7RNAP expression vector

A DNA fragment containing the T7RNA polymerase gene was amplified by PCR using a wild type T7RNAP gene fragment (nucleotide sequence shown in SEQ ID NO. 4) as a template, and ligated with pET-28a (+) plasmid (FIG. 1).

The specific method comprises the following steps:

(1) PCR was performed using an artificial plasmid containing a fragment of the wild type T7RNAP gene (SEQ ID NO. 4) as a template, with the following reagent composition and reaction conditions.

The PCR reaction primers were as follows:

primer ligation F (SEQ ID NO. 5):

5’-AGTCTCGAGATGAACACGATTAACATCGCTAAGAACGAC-3’;

primer ligation R (SEQ ID No. 6):

5’-ATAAGCGGCCGCTTACGCGAACGCGAAGTCCGACT-3’。

The PCR reaction system (total reaction solution: 50. Mu.L) consisted of a Primer ligation F1. Mu.L at a concentration of 10. Mu.M, a Primer ligation R1. Mu.L at a concentration of 10. Mu.M, 10 XTaq Buffer 5. Mu.L, mgCl ₂. Mu. L, dNTP (Shanghai) 1. Mu. L, DNA template 2.5. Mu. L, taq enzyme (Shanghai) 0.5. Mu. L, ddH ₂ O36. Mu.L at a concentration of 25. Mu.M.

The PCR reaction system was repeatedly conducted at 94℃for 30 seconds, at 58℃for 30 seconds, at 72℃for 1 minute, and then at 4℃after heating at 72℃for 10 minutes, after heating at 94℃for 3 minutes, using a thermal cycler (manufactured by BIO-RAD).

(2) The PCR product was purified using a PCR purification kit (Shanghai Biotechnology).

(3) The purified PCR product was digested with restriction enzymes Xho I (manufactured by NEB) and Not I (manufactured by NEB), and reacted with pET-28a (+) digested with restriction enzymes Xho I (manufactured by NEB) and Not I (manufactured by NEB) by T4 ligase for 30 minutes at 4 ℃.

(4) Coli TOP10 strain was transformed with the reaction solution of (3), selection was performed on LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose, 1% agar, 50. Mu.g/mL kanamycin sulfate (manufactured by Michelin), pH=7.4), colonies grown after overnight culture at 37℃were picked, plasmids were extracted after expansion culture, and the correct plasmids were identified as pET-28a (+) -T7RNAP by PCR reaction (see FIG. 2).

2. Construction of G640W mutant T7RNAP expression vector

Site-directed mutagenesis was performed on the T7RNAP gene of the pET-28a (+) -T7RNAP plasmid prepared in step one by the following procedure, so that the G mutation at 1918 of the nucleotide sequence (SEQ ID NO. 4) of the wild-type T7RNAP gene was made T, to obtain G640W mutant T7RNAP.

(1) The PCR reaction of site-directed mutagenesis was performed using pET-28a (+) -T7RNAP as a template plasmid with the following composition of reagents and reaction conditions.

The PCR reaction primers were as follows:

Primer mutation F (SEQ ID NO. 7):

5’-CTTACTGGTCCAAAGAGTTCGGCTTCC-3’;

primer mutation R (SEQ ID NO. 8):

5’-CTTTGGACCAGTAAGCCAGCGTCATGACTG-3’。

the PCR reaction system (total reaction amount: 50. Mu.L) consisted of Primer mutation F1. Mu.L at a concentration of 10. Mu.M, primer mutation R1. Mu.L at a concentration of 10. Mu.M, 10 XTaq Buffer 5. Mu.L, mgCl ₂. Mu. L, dNTP (Shanghai) 1. Mu. L, DNA template 2.5. Mu. L, taq enzyme (Shanghai) 0.5. Mu. L, ddH ₂ O36. Mu.L at a concentration of 25. Mu.M.

The PCR reaction conditions were that the sample was repeatedly heated at 94℃for 3 minutes using a thermal cycler (manufactured by BIO-RAD), then heated at 94℃for 35 times for 30 seconds, and then stored at 4℃after being repeatedly heated at 55℃for 30 seconds, and then heated at 72℃for 2 minutes for 41 seconds.

After the PCR reaction is finished, 10 units of restriction enzyme DpnI (Shanghai) is added, the mixture is digested for 1 hour at 37 ℃, the mixture is transformed into escherichia coli TOP10 according to a conventional method, plasmid sequencing is extracted by using an extraction and purification plasmid kit (Shanghai), the sequencing result is correct, namely pET-28a (+) -G640W-T7RNAP plasmid (figure 3), the nucleotide sequence of G640W mutant T7RNAP is shown as SEQ ID NO.2, the coded amino acid sequence is shown as SEQ ID NO.1, namely 640 glycine of the amino acid sequence of wild T7RNAP (SEQ ID NO. 3) is replaced by tryptophan.

3. Construction of G640W mutant T7RNAP engineering bacteria

(1) PET-28a (+) -G640W-T7RNAP plasmid was transformed into E.coli BL21 according to the conventional method and selected on LB agar medium (1% peptone, 0.5% yeast extract, 1% NaCl, 0.5% glucose, 1% agar, 50. Mu.g/mL kanamycin sulfate (microphone), pH=7.4), colonies grown after overnight culture at 37℃were picked, and the correct G640W mutant T7RNAP engineering bacteria were identified by colony PCR.

(2) Inoculating the G640W mutant T7RNAP engineering bacteria obtained in the step (1) in 100mL of LB liquid medium, and culturing for 18-20h in a 500mL conical flask at 37 ℃ and 220rpm in a shaking way.

(3) And (3) preserving the G640W mutant T7RNAP engineering bacteria glycerol bacteria obtained in the step (2) according to a conventional method, and preserving at-80 ℃.

EXAMPLE 2 purification of G640W mutant T7RNAP expression

1. Inducible expression of G640W mutant T7RNAP

(1) A500 mL Erlenmeyer flask was filled with 100mL of LB liquid medium, and 400. Mu.L of the glycerol stock of the G640W mutant T7RNAP engineering bacterium obtained in example 1 was inoculated thereto, and cultured at 37℃and 220rpm with shaking overnight.

(2) The bacterial liquid cultured in (1) was inoculated in 500mL of LB liquid medium in a 2000mL Erlenmeyer flask at 37℃and 220rpm to perform the culture.

(3) After the cultivation of (2) was performed for about 2.5 hours (OD ₆₀₀ value of about 0.4 to 0.6), 500. Mu.L of 0.1M IPTG (isopropyl-. Beta. -thiogalactoside) was added to each flask, and the cultivation was continued under shaking at 25℃and 180rpm for 18 to 20 hours.

(4) After the completion of the culture, the cells were collected by centrifugation at 8000rpm at 4℃for 15 minutes. In the case where cell disruption is not immediately performed, the cells were stored at-20 ℃.

2. Isolation and purification of G640W mutant T7RNAP

(1) The recovered cells were added with an appropriate amount of cell lysate (50 mM Tris-HCL,50mM NaCl,5mM EDTA,5% (v/v) glycerol, 12.5. Mu.g/mL lysozyme (BBI), 0.008% sodium deoxycholate (Michael system), 5mM DTT (Beyotime system), 0.3mM benzamidine (Inoki system), 50. Mu.g/mL PMSF (BBI system), 20. Mu.g/mL bacitracin (BBI system), 8mM ammonium sulfate (Arbitartan Ding Zhi), pH=7.9, and sonicated with a sonicator (New Zhi system).

The ultrasonic conditions are that a beaker filled with a thallus suspension to be cracked is placed in an ice-water mixture at 0 ℃, an ultrasonic probe is inserted into a proper position of the thallus, the ultrasonic power is 400W, the ultrasonic power is 2s, the ultrasonic treatment is stopped for 4s, the ultrasonic treatment is carried out for 20min, and the liquid temperature is always kept below 10 ℃ in the ultrasonic process.

After completion of sonication, the supernatant was centrifuged at 10000rpm for 10min at 4℃and diluted with TED-A (50mM PB,0.5M NaCl,3mM imidazole, pH=7.5) and filtered through a 0.45 μm aqueous filter membrane, and the 4℃was temporarily stored overnight for affinity purification using nickel chelating resins.

(2) Purification of the target protein was performed by affinity purification and ion exchange purification using a histidine hexamer tag added to the G640W mutant T7RNA polymerase, using the following method.

(2-1) A column containing 30mL of IDA packing (manufactured by Seisan blue dawn) was fixedly connected to a low pressure chromatography system (Bio-Lab 100 manufactured by Jiangsu Hanbang), and washed with Nucleasefreewater, 2mL/min,10CV. Equilibrated to baseline with TED-a (50mM PB,0.5M NaCl,3mM imidazole, ph=7.5) at a flow rate of 3mL/min,8-10CV.

And (3) loading the loading stock solution obtained in the step (1) at a flow rate of 0.5-1.0mL/min. After loading, the sample was rinsed with TED-A at 2mL/min and 12CV to a plateau at baseline (UV, cd). The relevant impurity was eluted again with 3% TED-B (50mM PB,0.5M NaCl,1M imidazole, ph=7.5), 10CV. Then eluted with 10% TED-B (50mM PB,0.5M NaCl,1M imidazole, ph=7.5) to the onset of uv=280 nm for the low pressure chromatography system, collected by separate tubes, 0.5CV per tube.

20UL of sample was taken per tube, and 5 XSDS loading buffer was added thereto at 100℃for 10min. SDS-PAGE was performed at 180V for 30min. The purity of the sample was observed after staining with SDS-PAGE rapid staining decolorizer (GenScript) and the results are shown in FIG. 4.

(2-2) TED column chromatography was selected based on the protein purity shown by SDS-PAGE in (2-1), pooled and dialyzed 2 times at 4℃with 10 volumes of storage buffer (50 mM Tris-HCL,5mM EDTA,0.1% (v/v) Triton X-100,50% (v/v) glycerol, pH=8) for about 6 hours each time.

(2-3) A column containing 30mL of DEAE-650M packing (manufactured by TOYOPEARL) was fixedly connected to a low pressure chromatography system (manufactured by Jiangsu Han Pont), and washed with Nuclease FREE WATER, 2mL/min,10CV. DEAE-a (50 mM Tris-HCl,20mM NaCl,0.1% Triton X-100,5% glycerol, ph=7.9) equilibrated to baseline (UV, cd) plateau at a flow rate of 1-2mL/min, about 10CV.

Diluting the dialysate obtained in (2-2) with DEAE-A for 5 times volume, and loading into sample, 0.5-1.0mL/min. DEAE-A (50 mM Tris-HCl,20mM NaCl,0.1% Triton X-100,5% glycerol, pH=7.9), 1-2mL/min,20CV. The relevant impurities were washed with 10% DEAE-B (50 mM Tris-HCl,1M NaCl,0.1% Triton X-100,5% glycerol, pH=7.9), 1-2mL/min,10CV. Then eluted with 20% DEAE-B (50 mM Tris-HCl,1M NaCl,0.1% Triton X-100,5% glycerol, pH=7.9), 1-2mL/min, and collected in tubes at the beginning of UV=280 nm, 0.5CV per tube.

Mu.L of each tube was sampled, and 5 XSDS loading buffer was added thereto at 100℃for 10 minutes. SDS-PAGE was performed at 180V for 30min. The purity of the sample was observed after staining with SDS-PAGE rapid staining decolorizer (GenScript) and the results are shown in FIG. 5.

(2-4) Was dialyzed with 10 volumes of storage buffer (50 mM Tris-HCL,5mM EDTA,0.1% Triton X-100,50% glycerol, pH=8) at 4℃for about 8 hours each time. Detection example 1 detection of RNase and DNase contamination

(1) Protein concentration measurement was performed by spectrophotometry, and the protein concentration of the G640W mutant T7RNA polymerase in the dialysate obtained in example 2 (2-4) was measured to be 0.43mg/mL using a modified Bradford protein concentration measurement kit (Shanghai).

(2) Nickase residual assay 4. Mu.L of the sample to be tested and 1. Mu.L of pBR322 DNA (NEB) at a concentration of 0.5. Mu.g/. Mu.L were added to a 10. Mu.L reaction system, incubated at 37℃for 4h, and then subjected to agarose gel electrophoresis. As a result, as shown in FIG. 6, the agarose gel electrophoresis of the DNA band did not change.

(3) Endonuclease residue detection 4. Mu.L of the sample to be detected and 1. Mu.L of lambda DNA (NEB) at a concentration of 0.35. Mu.g/. Mu.L were added to 10. Mu.L of the reaction system, incubated at 37℃for 4 hours, and then subjected to agarose gel electrophoresis detection. As a result, as shown in FIG. 7, the agarose gel electrophoresis of the DNA band did not change.

(4) Exonucleas residual detection 4. Mu.L of the sample to be tested and 1. Mu.L of lambda DNA-HINDIIIDIGEST (NEB) with a concentration of 0.5. Mu.g/. Mu.L were added to 10. Mu.L of the reaction system, incubated at 37℃for 4h, and then subjected to agarose gel electrophoresis detection. As a result, as shown in FIG. 8, the agarose gel electrophoresis of the DNA band did not change.

(5) RNase residual detection 4. Mu.L of the sample to be detected and 1. Mu.L of MS2 RNA (Roche) at a concentration of 1. Mu.g/. Mu.L were added to a 10. Mu.L reaction system, incubated at 37℃for 4 hours, and then subjected to agarose gel electrophoresis detection. As a result, as shown in FIG. 9, the agarose gel electrophoresis band was unchanged.

The detection results of the residual RNase and DNase enzymes of the G640W mutant T7RNA polymerase sample obtained by the separation and purification of the example 2 are shown in figures 6-9, and the sample obtained by the preparation method has no pollution of the relevant RNase and DNase enzymes.

Detection example 2 Activity measurement

The activity was measured by measuring the amount of RNA produced in an in vitro transcription reaction, and a DNA having a T7 promoter sequence specifically recognizing T7RNA polymerase was used as the template DNA. Here, a linear double-stranded DNA (SEQ ID NO. 9) containing a T7 promoter sequence was used as a template and obtained by PCR amplification.

1. The in vitro transcription reaction is carried out by using SEQ ID NO.9 as a transcription template and adopting the following reagent composition and reaction conditions.

The reaction system (total reaction solution: 20. Mu.L) consisted of nucleic FREE WATER 4.6.6. Mu.L, 10X reaction Buffer 2. Mu.L, RNase Inhibitor 1. Mu.L, inorganic pyrophosphatase (100U/. Mu.L, next generation) 0.4. Mu. L, dNTP (100 mM ATP, CTP, GTP, UTP, shanghai manufacturing) each 2. Mu. L, DNA template (SEQ ID NO. 9) 2. Mu. L, T7 RNAPase 2. Mu.L.

The experimental group adopts the G640W mutant T7RNA polymerase of the example 2, the control group is wild type T7RNA polymerase, and the enzyme concentration is 0.3-0.5mg/mL, and the repeated 2 times.

Reaction conditions 37 ℃ incubation reaction for 2 hours.

The product was sampled and subjected to agarose gel electrophoresis as shown in fig. 10.

The amount of RNA produced was quantitatively measured using a commercially available (XR) RNA quantification kit (Thermofisher. RTM.) for Qubit 4 and Qubit 4 (Thermofisher. RTM.) and the data are shown in Table 1.

TABLE 1 amount of RNA produced by in vitro transcription reaction

As can be seen from FIG. 10 and Table 1, the in vitro transcriptional catalytic activity of the T7RNAP-G640W mutant is significantly higher than that of the wild type T7RNAP compared to the transcriptional activity of the wild type T7RNAP.

2. The corresponding reagents were added in a super clean bench according to the reaction system in step 1, wherein T7RNAP (experimental group using the G640W mutant T7RNA polymerase of example 2, control group using commercially available T7RNA polymerase, repeated 2 times), and after mixing and centrifugation, the mixture was placed in a PCR apparatus, incubated at 37℃for 2 hours, and 1. Mu.L of DNase I was added to each sample for 15 minutes. The product was sampled and subjected to agarose gel electrophoresis as shown in fig. 11. The RNA quantification results are shown in Table 2.

TABLE 2 Activity test in vitro transcription reaction System and amount of RNA produced

According to the data in the table, the G640W mutant T7 RNA polymerase obtained by the invention has good transcription catalytic activity. Compared with the traditional in vitro transcription reaction, the method has high synthesis amount, and compared with the level of the related kit (the synthesis amount of each reaction (20 mu L) is 150-180 mu g of RNA), the yield of RNA is improved by 30-50%.

Test example 3 thermal stability test

The thermal stability test was performed by measuring the amount of RNA generated in an in vitro transcription reaction at 46 ℃. The reaction system was incubated at 46℃for 2 hours with test example 2.

The corresponding reagents were added to the reaction system of step 1 of detection example 2 in an ultra clean bench, wherein T7RNAP (experimental group using the mutant T7RNA polymerase of G640W of example 2, control group using wild type T7RNA polymerase, commercially available T7RNA polymerase, repeated 2 times), and after mixing and centrifugation, the mixture was placed in a PCR apparatus, incubated at 46℃for 2 hours, and 1. Mu.L of DNase I was added to each sample and incubated for 15 minutes. The product was sampled and subjected to agarose gel electrophoresis as shown in fig. 12. The RNA quantification results are shown in Table 3.

TABLE 3 thermal stability test in vitro transcription reaction System and amount of RNA produced

	RNA synthesis amount (μg)
		T7RNAP-G640W	168.07
T7RNAP-G640W	169.34
		Wild type T7RNAP	65.36
Wild type T7RNAP	67.27
		Commercial product 1	126.06
Commercial product 1	123.12
		Commercial product 2	108.05
Commercial product 2	110.56

From the data in the above table, the thermal stability of the T7RNAP-G640W mutant was significantly higher than that of the wild-type T7RNAP. The G640W mutant T7RNA polymerase obtained by the invention still has higher in-vitro transcription catalytic activity under the temperature condition of 46 ℃, which is higher than the related T7RNAP on the market.

Claims

1. A T7 RNA polymerase mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 1.

2. A coding gene for the mutant T7RNA polymerase of claim 1, wherein the nucleotide sequence of the coding gene is shown in SEQ ID No. 2.

3. A recombinant vector comprising the coding gene of claim 2.

4. A recombinant genetically engineered bacterium comprising the recombinant vector of claim 3.

5. A method for preparing a T7RNA polymerase mutant without DNase and RNase pollution, which is characterized by comprising the following steps:

The composition of the cell lysate comprises 20-50mM Tris-HCl,10-50mM NaCl,3-10mM EDTA, 3-10% glycerol, 10-20 mug/mL lysozyme, 0.005-0.010% sodium deoxycholate, 3-8mM dithiothreitol, 0.1-0.5mM benzamidine, 20-80 mug/mL phenylmethylsulfonyl fluoride, 10-40 mug/mL bacitracin, 5-15mM ammonium sulfate, and pH=7.5-8.0;

The affinity chromatography impurity-washing liquid comprises 20-60mM phosphate buffer solution, 0.2-0.5MNaCl,30mM imidazole, and pH=7.5-8.0;

The affinity chromatography eluent comprises 20-60mM phosphate buffer, 0.2-0.5MNaCl,100mM imidazole, and pH=7.5-8.0;

the composition of the stock solution comprises 30-50mM Tris-HCl,4-8mM EDTA,0.1% -0.2% Triton X-100,45-55% glycerol, and pH=7.5-8.0;

The ion exchange chromatography impurity washing liquid comprises 20-80mM Tris-HCl,100mM NaCl,0.1% -0.3% Triton X-100,3% -8% glycerol, and pH=7.5-8.0;

6. The method according to claim 5, wherein in the step (2), the supernatant of the cell disruption is diluted with an affinity chromatography buffer A and then filtered to obtain an affinity chromatography sample solution;

the affinity chromatography buffer A comprises 20-60mM phosphate buffer, 0.2-0.5M NaCl,3-10mM imidazole and pH=7.5-8.0.

7. The method according to claim 6, wherein in the step (3), before loading, the column is washed with water without nuclease, and then the column is equilibrated with the affinity chromatography buffer A, after loading, the impurities are eluted with the affinity chromatography buffer A, and then the elution is collected when the elution is started by using the affinity chromatography eluent at a wavelength of 280nm ultraviolet detection.

8. The method according to claim 5, wherein in the step (4), before loading, the column is washed with water without nuclease, and then is equilibrated with the ion exchange chromatography buffer A, after loading, the ion exchange chromatography buffer A is used for equilibration, then the impurity is eluted with the ion exchange chromatography eluting solution, and then the eluting solution is collected when the eluting solution is eluted until the eluting solution starts to peak at the ultraviolet detection wavelength of 280 nm;

The ion exchange chromatography buffer A comprises 20-80mM Tris-HCl,10-50mM NaCl,0.1% -0.3% Triton X-100,3% -8% glycerol, and pH=7.5-8.0.

9. The method according to claim 5, wherein the original plasmid of the recombinant expression vector is pET-28a (+), and the fermentation culture comprises the steps of amplifying the activated recombinant genetically engineered bacterium to OD ₆₀₀ =0.4-0.6, adding IPTG with a final concentration of 0.1mM, and shaking culture at 25 ℃ and 180rpm for 18-20 hours.

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