Ppr high-salt-resistant omnipotent nuclease mutant and application thereof

Abstract

The application discloses a PPR high-salt-resistant omnipotent nuclease mutant and application thereof. The amino acid sequence of the PPR high-salt-resistant omnipotent nuclease mutant is shown in SEQ ID NO. 1. The application further discloses application of the PPR high-salt-resistant omnipotent nuclease mutant in removing nucleic acid. The PPR high-salt-resistant omnipotent nuclease mutant of the application replaces the alanine at the 165th position of the wild-type PPR high-salt-resistant omnipotent nuclease without a signal peptide with aspartic acid, can be overexpressed in a prokaryotic host and a large amount of protein can be obtained through purification. The PPR high-salt-resistant omnipotent nuclease mutant of the application maintains high activity under a wide range of salt concentration, temperature, pH and Mg 2+ range, and is compatible with most additives used in biological processes. In addition, the activity of the PPR high-salt-resistant omnipotent nuclease mutant is superior to other domestic competitive products, and can reach the level of the currently best-selling competitive product.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. More specifically, it relates to a PPR high-salt-tolerant totipotent nuclease mutant and its applications. Background Technology

[0002] Nucleic acid contamination, especially host genomic DNA contamination, is a key issue that needs to be addressed in almost all processes and the production of biopharmaceuticals. On the one hand, the presence of host DNA affects the purification of target products and downstream quality analysis; on the other hand, residual host DNA can trigger a severe immune response in the body and is one of the key parameters of biopharmaceuticals. Whether in laboratory-scale or production-scale sample preparation, such as protein purification, viral vector preparation, or sample processing in mNGS, removing nucleic acids can improve the process flow, making the selection of nucleases particularly important. PPR high-salt-tolerant totipotent nuclease is a type of nuclease derived from deep-sea barotropic luminescent bacteria (PPR). Photobacterium Profundum Non-specific, broad-spectrum endonucleases degrade long nucleic acids into short nucleotide chains of 2-5 bases in length, containing a 5'-phosphate group and a 3'-hydroxyl group, by cleaving phosphodiester bonds in the nucleic acid chain. These enzymes can efficiently degrade all types (single-stranded, double-stranded, linear, or circular) DNA and RNA, and typically exhibit optimal activity at high salt concentrations (500 mM NaCl or KCl). Processes conducted at higher salt concentrations can disrupt intermolecular electrostatic interactions, allowing nucleic acids to detach from proteins or viruses. Additionally, high salt concentrations can reduce protein aggregation caused by electrostatic interactions. Many biological experimental methods require high-salt conditions.

[0003] Therefore, there is a need to provide a novel high-salt tolerant totipotent nuclease that can be directly used in these high-salt conditions to reduce solution viscosity and remove nucleic acids. Summary of the Invention

[0004] One object of the present invention is to provide a PPR high-salt tolerant totipotent nuclease mutant, which is tolerant to a wide range of salt concentrations, temperatures, pH values, and Mg content. 2+ It maintains high activity across a wide concentration range and is compatible with most additives used in bioprocesses. Furthermore, its activity surpasses that of other domestic competitors, achieving the best performance level among currently available competitive products.

[0005] Another object of the present invention is to provide the application of the above-mentioned PPR high-salt pluripotent nuclease mutant.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The present invention first provides a PPR high-salt tolerant totipotent nuclease mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention relates to a PPR high-salt-tolerant totipotent nuclease mutant obtained by replacing alanine at position 165 (corresponding to position 183 of the full-length sequence of wild-type PPR high-salt-tolerant totipotent nuclease without a signal peptide) with aspartic acid. The sequence of the wild-type PPR high-salt-tolerant totipotent nuclease is obtained from the NCBI protein database (WP_011219706). The amino acid sequence of the wild-type PPR high-salt-tolerant totipotent nuclease without a signal peptide is shown in SEQ ID NO.3, and the nucleotide sequence after codon optimization is shown in SEQ ID NO.4.

[0008] The nucleotide sequence encoding the above-mentioned PPR high-salt pluripotent nuclease mutant is also within the scope of protection of this invention.

[0009] Furthermore, the nucleotide sequence encoding the above-mentioned PPR high-salt pluripotent nuclease mutant is shown in SEQ ID NO.2.

[0010] Recombinant plasmids containing the above nucleotide sequences are also within the scope of protection of this invention.

[0011] In a specific embodiment of the present invention, the recombinant plasmid is pET21a-PPRMut; pET21a-PPRMut is obtained by inserting the nucleotide sequence encoding a PPR high-salt pluripotent nuclease mutant as shown in SEQ ID NO.2 between the NdeI and XhoI restriction sites of the pET21a plasmid, while keeping the other sequences of pET-21a unchanged.

[0012] Recombinant cells containing the above-mentioned nucleotide sequences or recombinant plasmids are also within the scope of protection of this invention.

[0013] In a specific embodiment of the present invention, the host cell of the recombinant cell is a modified BL21 Escherichia coli.

[0014] In a preferred embodiment of the invention, the host cell is a BL21(DE3) competent cell.

[0015] The preparation method of the above-mentioned PPR high-salt pluripotent nuclease mutant in this invention includes the following steps: a1) Construct a recombinant plasmid pET21a-PPRMut containing the nucleotide sequence shown in SEQ ID NO.2; a2) The recombinant plasmid pET21a-PPRMut was transformed into host cells BL21(DE3) competent cells, and expression was induced to obtain bacterial cells; a3) The bacterial cells were broken, centrifuged, and the supernatant was obtained. After purification, the PPR high-salt tolerant totipotent nuclease mutant was obtained.

[0016] This invention further discloses the application of the above-mentioned PPR high-salt pluripotent nuclease mutant, the above-mentioned nucleotide sequence, or the above-mentioned recombinant plasmid or recombinant cell in the removal of nucleic acids or the preparation of nucleic acid-removed products.

[0017] In a specific embodiment of the present invention, the application is an application under high-salt conditions, wherein the NaCl concentration is 250-1000 mM.

[0018] In a preferred embodiment of the present invention, the NaCl concentration in the high-salt condition is 300-750 mM.

[0019] In a specific embodiment of the present invention, the pH range of the applied system is 6.5-9.5, and the Mg content is... 2+ The concentration range is 1-40 mM, and the temperature range is 0-50℃.

[0020] In a preferred embodiment of the present invention, the pH range of the system is 7.7-9.5, and the Mg content is... 2+ The concentration range is 1-40 mM, and the temperature range is 10-45℃.

[0021] In a specific embodiment of the present invention, the system further includes at least one additive selected from phosphate ions, ammonium sulfate, ammonium chloride, EDTA, Triton X-100, guanidine hydrochloride, and urea.

[0022] In a preferred embodiment of the present invention, the concentration ranges of the additives are phosphate ions 0-170 mM, ammonium sulfate 0-250 mM, ammonium chloride 0-300 mM, EDTA 0-5 mM, Triton X-100 0-15% (volume percentage), guanidine hydrochloride 0-250 mM, and urea 0-4 M.

[0023] In a more preferred embodiment of the present invention, the concentration range of the additives is 0-70 mM phosphate ions, 0-120 mM ammonium sulfate, 50-180 mM ammonium chloride, 0-2 mM EDTA, 0-3.5% Triton X-100, 0-100 mM guanidine hydrochloride, and 0-2 M urea.

[0024] In a specific embodiment of the present invention, the application scenario is nucleic acid removal in the biomedical field or molecular biology experiments.

[0025] In a preferred embodiment of the present invention, the application scenario is nucleic acid removal in protein purification, viral vector preparation, or mNGS sample processing.

[0026] The present invention further discloses a reagent or kit for removing nucleic acids, wherein the reagent or kit contains the above-mentioned PPR high-salt tolerant totipotent nuclease mutant.

[0027] The beneficial effects of this invention are as follows: This invention relates to a high-salt-tolerant totipotent nuclease mutant of PPR, which, compared to the wild-type PPR high-salt-tolerant totipotent nuclease, replaces alanine at position 165 with aspartic acid. This mutant can be overexpressed in prokaryotic hosts and purified in large quantities by affinity chromatography and ion exchange chromatography. The high-salt-tolerant totipotent nuclease mutant of this invention exhibits good performance across a wide range of salt concentrations, temperatures, pH levels, and Mg content. 2+ It maintains high activity within a wide range, and this PPR high-salt-tolerant totipotent nuclease mutant is compatible with most additives used in bioprocessing (phosphate ions, ammonium sulfate, ammonium chloride, EDTA, Triton X-100, guanidine hydrochloride, and urea). Furthermore, the activity of this PPR high-salt-tolerant totipotent nuclease mutant is superior to other domestically produced competing products, reaching the best performance level currently available in the market. Attached Figure Description

[0028] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0029] Figure 1 The SDS-PAGE results are for the PPR high-salt pluripotent nuclease mutant of this invention.

[0030] Figure 2 The images show the relative activity curves of the PPR high-salt tolerant totipotent nuclease mutant of this invention, the wild-type PPR high-salt tolerant totipotent nuclease, and other commercially available competitors at different NaCl concentrations.

[0031] Figure 3 The figures show the relative activity curves of the PPR high-salt tolerant totipotent nuclease mutant and the wild-type PPR high-salt tolerant totipotent nuclease at different temperature ranges.

[0032] Figure 4 The figures show the relative activity curves of the PPR high-salt pluripotent nuclease mutant and the wild-type PPR high-salt pluripotent nuclease at different pH ranges.

[0033] Figure 5 The present invention relates to a PPR high-salt-tolerant totipotent nuclease mutant and a wild-type PPR high-salt-tolerant totipotent nuclease in different Mg... 2+ Relative activity curves within a concentration range.

[0034] Figure 6 The images show the relative activity curves of the PPR high-salt tolerant totipotent nuclease mutant of this invention at different phosphate concentration ranges.

[0035] Figure 7 The relative activity curves of the PPR high-salt tolerant totipotent nuclease mutant of the present invention under different ammonium sulfate concentration ranges are shown.

[0036] Figure 8 The relative activity curves of the PPR high-salt tolerant totipotent nuclease mutant of the present invention under different ammonium chloride concentration ranges are shown.

[0037] Figure 9 The images show the relative activity curves of the PPR high-salt tolerant totipotent nuclease mutant of this invention at different EDTA concentration ranges.

[0038] Figure 10 The relative activity curves of the PPR high-salt tolerant totipotent nuclease mutant of the present invention at different Triton X-100 concentration ranges are shown.

[0039] Figure 11 The images show the relative activity curves of the PPR high-salt pluripotent nuclease mutant of this invention at different guanidine hydrochloride concentration ranges.

[0040] Figure 12 The relative activity curves of the PPR high-salt pluripotent nuclease mutant of the present invention under different urea concentration ranges are shown.

[0041] Figure 13 This is a comparison of the activities of the PPR high-salt tolerant totipotent nuclease mutant of the present invention with competing products; wherein, 1 is the PPR high-salt tolerant totipotent nuclease mutant, 2 is competing product Y, 3 is competing product J, 4 is competing product H, 5 is competing product N, M is a marker, and C is an enzyme-free control. Detailed Implementation

[0042] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention. Many other methods exist in the prior art; the following embodiments provide a specific solution.

[0043] Example 1: Obtaining a PPR high-salt-tolerant totipotent nuclease mutant By searching the NCBI protein database, the wild-type PPR high-salt-tolerant totipotent nuclease sequence without a signal peptide was obtained (SEQ ID NO.3, and its codon-optimized nucleotide sequence is shown in SEQ ID NO.4). Through multiple sequence comparison, protein structure analysis, and rational design, a PPR high-salt-tolerant totipotent nuclease mutant sequence with predicted improved enzyme performance was obtained. This sequence mutates alanine at position 165 (corresponding to position 183 of the full-length sequence) of the amino acid sequence shown in SEQ ID NO.3 of the wild-type PPR high-salt-tolerant totipotent nuclease without a signal peptide to aspartic acid. After heterologous protein expression and purification, the PPR high-salt-tolerant totipotent nuclease mutant was finally obtained (amino acid sequence shown in SEQ ID NO.1, i.e., the PPR high-salt-tolerant totipotent nuclease mutant without a signal peptide). The specific steps are as follows: I. Construction of Recombinant Cells The host-optimized nucleotide sequence of the PPR high-salt-tolerant totipotent nuclease mutant (shown in SEQ ID NO.2) was synthesized by Nanjing Genscript Biotech Co., Ltd., and this nucleotide sequence was inserted into an expression vector and transformed into E. coli. Details are as follows: The nucleotide sequence encoding the PPR high-salt-tolerant totipotent nuclease mutant (shown in SEQ ID NO.2) was inserted between the NdeI and XhoI restriction sites of the pET21a vector, while keeping the other sequences of pET21a unchanged, to obtain the corresponding recombinant plasmid pET21a-PPRMut. Subsequently, the recombinant plasmid pET21a-PPRMut was transformed into competent cells of Escherichia coli expression strain BL21(DE3), thereby constructing a recombinant Escherichia coli expression strain (i.e., recombinant bacteria) containing the recombinant plasmid of the PPR high-salt-tolerant totipotent nuclease mutant.

[0044] II. Expression and purification of PPR high-salt-tolerant totipotent nuclease mutant The recombinant bacteria were plated on ampicillin-resistant plates and incubated overnight at 37°C. The next day, single colonies were picked from the plates and inoculated into 10 mL of fresh LB medium, incubated overnight at 37°C and 220 rpm, and then inoculated into 1 L of LB medium and incubated at 37°C and 220 rpm until OD reached [value missing]. 600 The concentration was approximately 0.6-0.8, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 18℃ for 20-22 hours. The bacterial cells were collected by centrifugation, resuspended in lysis buffer, and the supernatant was collected after cell lysis. Affinity chromatography and ion exchange chromatography were then performed to obtain the purified product, which is the PPR high-salt tolerant totipotent nuclease mutant.

[0045] The PPR high-salt-tolerant totipotent nuclease mutant was dialyzed into Storage Buffer (25 mM Tris-HCl, 500 mM NaCl, 5 mM MgCl2, 50% Glycerol, pH 8.0). SDS-PAGE gel assay results are shown below. Figure 1 As shown in the figure, the protein sample obtained in this embodiment has a size of 26.4 kDa, which is consistent with the expected size of the PPR high-salt-tolerant totipotent nuclease mutant, indicating that the protein sample finally obtained in this embodiment is the PPR high-salt-tolerant totipotent nuclease mutant. Furthermore, sequencing confirmed its correctness, and its amino acid sequence is shown in SEQ ID NO.1.

[0046] Example 2: NaCl concentration tolerance test of PPR high-salt pluripotent nuclease mutant The PPR high-salt-tolerant totipotent nuclease mutant obtained in Example 1 was subjected to activity assays. One unit of activity of the PPR high-salt-tolerant totipotent nuclease mutant was defined as the production of OD100 after digesting 50 μg / ml of calf thymus DNA (Sigma, D-1501) in a reaction system of 37°C, 25 mM Tris-HCl, pH 8.0 (25°C), 500 mM NaCl, and 5 mM MgCl2 within 30 minutes. 260 The amount of enzyme required to change the absorbance at 1 nm is determined. Salt tolerance curves were performed on the PPR high-salt-tolerant totipotent nuclease mutant and other commercially available competing products after activity assays, i.e., the enzyme activity versus salt concentration curve. Specifically, the enzyme activity of the PPR high-salt-tolerant totipotent nuclease mutant under optimal conditions (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl2) was defined as 100% activity. First, a standard curve was plotted showing the cleavage of the fluorescently labeled substrate by the high-salt-tolerant totipotent nuclease under optimal conditions (i.e., the linear relationship between fluorescence value and enzyme amount under substrate excess). The fluorescence values ​​of the reaction system at different NaCl concentrations were detected, and the fluorescence values ​​were substituted into the standard curve to calculate the theoretical enzyme amount. The ratio of the actual enzyme amount to the theoretical enzyme amount is the relative activity. The specific procedures are as follows: I. Preparation of Materials and Reagents 1. Prepare the materials or reagents used in the experiment according to Table 1. Table 1. Preparation methods of materials or reagents used.

[0047] Note: The sequence of the fluorescently labeled DNA substrate is: 5'-FAM-TTCGAATTCGCGCCGGTTTTTCCGGCGCGAATTCGAA- Dabcyl-3'.

[0048] 2. Prepare 2× reaction buffer solution Prepare 2× reaction buffer solutions with four NaCl concentration gradients according to Table 2.

[0049] Table 24 2× reaction buffer solutions for NaCl concentration gradients

[0050] 3. Dissolve the fluorescently labeled DNA substrate Add an appropriate amount of RNase-free water to dissolve the fluorescently labeled DNA substrate to a concentration of 100 μM.

[0051] II. Testing the relative activity of PPR high-salt-tolerant totipotent nuclease mutants at different NaCl concentrations 1. Preparation of the reaction system 1) Dilute the PPR high-salt tolerant totipotent nuclease mutant The high-salt-tolerant nuclease mutant was diluted with a 1× dilution buffer to 0.008, 0.004, 0.002, 0.001, 0.0005, 0.00025, and 0.000125 U / μl, respectively, with each concentration having a volume of 50 μl. After dilution, the solutions were placed on ice for later use.

[0052] 2) Dilute the fluorescently labeled DNA substrate The fluorescently labeled DNA substrate was diluted to 5 μM with RNase-free water in a volume of 200 μl and then placed on ice for later use.

[0053] 3) Preparation of the reaction system The reaction systems were prepared according to Tables 3, 4, and 5, resulting in a total of 23 reaction systems. Note that the PPR high-salt-tolerant totipotent nuclease mutant was added as the final step after all other components in all systems had been added.

[0054] Table 3. Standard Curve Plotting of Reaction Systems

[0055] Note: Seven reaction systems with different enzyme concentrations, each with a volume of 20 μl.

[0056] Table 4 Reaction systems with different NaCl concentrations

[0057] Note: There are 12 reaction systems in total, each with 3 different NaCl concentration buffer systems and 3 different enzyme concentration gradients, with a volume of 20 μl.

[0058] Table 5. Control reaction system

[0059] Note: Four buffer systems with different NaCl concentrations, each with a volume of 20 μl.

[0060] 2. Test the relative activity at different NaCl concentrations. 1) Reaction Place the above reaction system in a PCR instrument and react at 37°C for 10 min. After the reaction is complete, place it on ice and immediately add 1 μl of 100 mM DTT (final concentration 5 mM) to terminate the reaction. Heat the PCR instrument at 55°C for 30 min to ensure complete enzyme inactivation. Place the reaction product on ice after the reaction is complete to detect fluorescence.

[0061] 2) Detection of fluorescence Add 180 μl of deionized water to the reaction product, vortex to mix, centrifuge briefly, and dispense into 80 μl / well. Set up 2 replicates for each reaction. After sample loading, use an ELISA reader to detect the fluorescence value.

[0062] 3) Draw a curve graph The relative activity curves of the PPR high-salt-tolerant totipotent nuclease mutant under different NaCl conditions were plotted based on the detected fluorescence values. The relative activities of four commercially available competing products—wild-type PPR high-salt-tolerant totipotent nuclease, domestic competitor Y (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 20159ES25), domestic competitor J (purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number M065-01A), imported competitor H (purchased from Arctic Zymes Technologies, catalog number 70910-202), and imported competitor N (purchased from NEB (New England Biolabs), catalog number M0764S)—at different NaCl concentrations were also plotted.

[0063] Test results are as follows Figure 2 As shown, this PPR high-salt tolerant totipotent nuclease mutant has a wide range of salt concentration tolerance, and its activity at different salt concentrations within 1M is higher than that of wild-type PPR high-salt tolerant totipotent nuclease, domestic competitor Y, and domestic competitor J, and is comparable to the activity of the best currently available imported competitor H and imported competitor N.

[0064] Example 3 Temperature tolerance range test of PPR high-salt pluripotent nuclease mutant The temperature tolerance of the PPR high-salt-tolerant totipotent nuclease mutant was determined, i.e., the enzyme activity versus temperature curve. The specific protocol was as follows: the enzyme activity of the PPR high-salt-tolerant totipotent nuclease mutant, after completing the activity determination, was defined as 100% activity under optimal conditions (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl2) at 37℃. Nucleic acid electrophoresis was used to detect the substrate digestion by the PPR high-salt-tolerant totipotent nuclease mutant at different temperatures. The relative enzyme activity at different temperatures was obtained by analyzing changes in substrate bands and grayscale values. The specific procedures are as follows: I. Preparation of Materials and Reagents Prepare the materials or reagents used in the experiment according to Table 6.

[0065] Preparation methods of materials or reagents used in Table 6

[0066] II. Testing the relative activity of PPR high-salt-tolerant totipotent nuclease mutants at different temperatures 1. Preparation of the reaction system 1) Dilute the PPR high-salt tolerant totipotent nuclease mutant The PPR high-salt pluripotent nuclease mutant was diluted to 0.05 U / μl using a 1× dilution buffer.

[0067] 2) Preparation of the reaction system The reaction systems were prepared according to Table 7, resulting in a total of 13 reaction systems (12 temperature gradients and a control, each with a volume of 20 μl). Note that the PPR high-salt-tolerant totipotent nuclease mutant was added as the last step after all other components of the systems had been added.

[0068] Table 7 Reaction System

[0069] Note: The control group, the PPR high-salt pluripotent nuclease mutant, was replaced with RNase-free water.

[0070] 2. Test the relative activity at different temperatures. 1) Reaction The above reaction system was placed in a metal bath at 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 37℃, 40℃, 45℃, and 50℃ for 10 min (or the PCR instrument was adjusted to the corresponding temperature, and the hot lid temperature was set to 50℃ to reduce sample evaporation). Immediately afterward, 1 μl of 100 mM DTT (final concentration 5 mM) was added to terminate the reaction. The metal bath was then heated at 55℃ for 30 min to ensure complete enzyme inactivation and obtain the reaction product.

[0071] 2) Detection by 1.5% agarose gel electrophoresis Add 4 μl of 6× DNA loading buffer to each of the above reaction products, and take 8 μl of each sample for agarose gel electrophoresis (electrophoresis conditions are shown in Table 8).

[0072] Table 8 Electrophoresis conditions

[0073] 3) Data Analysis The relative enzyme activity at different temperatures was obtained by analyzing the changes in substrate bands and grayscale values. The relative activities of wild-type PPR high-salt-tolerant totipotent nuclease at different temperatures were then tested using the same method, and curves were plotted.

[0074] Test results are as follows Figure 3 As shown, the PPR high-salt tolerant totipotent nuclease mutant has more than 80% activity in the temperature range of 10-45℃ and more than 60% activity in the temperature range of 0-50℃. Moreover, its activity at different temperatures is higher than that of the wild-type PPR high-salt tolerant totipotent nuclease.

[0075] Example 4: pH tolerance range test of PPR high-salt pluripotent nuclease mutant The pH tolerance of the PPR high-salt-tolerant totipotent nuclease mutant was determined, i.e., the enzyme activity versus pH curve. Specifically, the enzyme activity of the PPR high-salt-tolerant totipotent nuclease mutant under optimal conditions (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl2) was defined as 100% activity. Nucleic acid electrophoresis was used to detect the substrate digestion by the PPR high-salt-tolerant totipotent nuclease mutant under different pH conditions. The relative enzyme activity under different pH conditions was obtained by analyzing changes in substrate bands and grayscale values. The specific procedures are as follows: I. Preparation of Materials and Reagents Prepare the materials or reagents used in the experiment according to Table 9.

[0076] Table 9. Preparation methods of materials or reagents used.

[0077] II. Testing the relative activity of PPR high-salt-tolerant totipotent nuclease mutants under different pH conditions 1. Preparation of the reaction system 1) Dilute the PPR high-salt tolerant totipotent nuclease mutant The PPR high-salt pluripotent nuclease mutant was diluted to 0.05 U / μl using a 1× dilution buffer.

[0078] 2) Preparation of the reaction system The reaction systems were prepared according to Table 10, resulting in a total of 9 reaction systems (8 pH gradients and a control, each with a volume of 20 μl). Note that the PPR high-salt-tolerant totipotent nuclease mutant was added as the last step after all other components of the systems had been added.

[0079] Table 10 Reaction System

[0080] Note: The control group did not contain high-salt-tolerant nuclease and was replaced with RNase-free water.

[0081] 2. Test the relative activity under different pH conditions. 1) Reaction After reacting the above reaction system at 37℃ for 10 min (or setting the PCR instrument to 37℃ and the hot lid temperature to 50℃ to reduce sample evaporation), immediately add 1 μl of 100 mM DTT (final concentration 5 mM) to terminate the reaction. Heat in a metal bath at 55℃ for 30 min to ensure complete enzyme inactivation and obtain the reaction product.

[0082] 2) Detection by 1.5% agarose gel electrophoresis Add 4 μl of 6× DNA loading buffer to each of the above reaction products, and take 8 μl of each sample for agarose gel electrophoresis (electrophoresis conditions are shown in Table 8).

[0083] 3) Data Analysis The relative enzyme activity under different pH conditions was obtained by analyzing the changes in substrate bands and gray values.

[0084] The relative activities of wild-type PPR high-salt pluripotent nuclease at different pH values ​​were tested using the same method, and curves were plotted.

[0085] Test results are as follows Figure 4 The PPR high-salt-tolerant totipotent nuclease mutant exhibits over 80% activity in the pH range of 7.7-9.5 and over 20% activity in the pH range of 6.5-9.5. Furthermore, its activity at all pH levels is higher than that of the wild-type PPR high-salt-tolerant totipotent nuclease.

[0086] Example 5: PPR high-salt tolerant totipotent nuclease mutant with broad Mg content 2+ Concentration tolerance range Determination of the effect of PPR high-salt tolerant totipotent nuclease mutant on Mg 2+ Concentration tolerance, i.e., enzyme activity with Mg 2+Concentration change curves. Specifically, the enzyme activity of the PPR high-salt-tolerant totipotent nuclease mutant, after completing the activity determination process, was defined as 100% activity under optimal conditions (i.e., 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl2). Nucleic acid electrophoresis was used to detect different Mg... 2+ The digestion of substrates by PPR high-salt-tolerant totipotent nuclease mutants at different Mg concentrations was investigated by analyzing substrate band changes and grayscale value changes. 2+ Relative enzyme activity at different concentrations. The specific procedure is as follows: I. Preparation of Materials and Reagents Prepare the materials or reagents used in the experiment according to Table 11.

[0087] Preparation methods of materials or reagents used in Table 11

[0088] II. Testing PPR high-salt-tolerant totipotent nuclease mutants in different Mg... 2+ Relative activity at concentration 1. Preparation of the reaction system 1) Dilute the PPR high-salt tolerant totipotent nuclease mutant The PPR high-salt pluripotent nuclease mutant was diluted to 0.05 U / μl using a 1× dilution buffer.

[0089] 2) Preparation of the reaction system: The reaction systems were prepared according to Table 12, resulting in a total of 9 reaction systems (8 Mg ions). 2+ Concentration gradient and control (20 μl each). Note that the PPR high-salt-tolerant totipotent nuclease mutant should be added as the last step after all other components of the system have been added.

[0090] Table 12 Reaction System

[0091] Note: The control group did not contain high-salt-tolerant nuclease and was replaced with RNase-free water.

[0092] 2. Testing at different Mg 2+ Relative activity at concentration 1) Reaction After reacting the above reaction system at 37℃ for 10 min (or setting the PCR instrument to 37℃ and the hot lid temperature to 50℃ to reduce sample evaporation), immediately add 1 μl of 100 mM DTT (final concentration 5 mM) to terminate the reaction. Heat in a metal bath at 55℃ for 30 min to ensure complete enzyme inactivation and obtain the reaction product.

[0093] 2) Detection by 1.5% agarose gel electrophoresis Add 4 μl of 6× DNA loading buffer to each of the above reaction products, and take 8 μl of each sample for agarose gel electrophoresis (electrophoresis conditions are shown in Table 8).

[0094] 3) Data Analysis Analyzing the changes in substrate bands and grayscale values, we obtained different Mg... 2+ Relative enzyme activity at a given concentration.

[0095] Wild-type PPR high-salt tolerant totipotent nuclease was tested at different Mg levels using the same method. 2+ The relative activity at different concentrations was determined and plotted as a curve.

[0096] Test results are as follows Figure 5 PPR high-salt tolerant totipotent nuclease mutant in Mg 2+ It exhibits over 80% activity within a concentration range of 1-40 mM. Furthermore, different Mg... 2+ At all concentrations, the activity was higher than that of wild-type PPR high-salt pluripotent nuclease.

[0097] Example 6: Compatibility test of PPR high-salt pluripotent nuclease mutant with other additives The tolerance of the PPR high-salt-tolerant totipotent nuclease mutant to buffers and additives was determined, i.e., the enzyme activity was measured as a function of various buffer or additive concentrations. Specifically, the enzyme activity of the PPR high-salt-tolerant totipotent nuclease mutant under optimal conditions (25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl2) was defined as 100% activity. Nucleic acid electrophoresis was used to detect the substrate digestion by the PPR high-salt-tolerant totipotent nuclease mutant under different buffer and additive concentrations. The relative enzyme activity under different conditions was obtained by analyzing changes in substrate bands and grayscale values. Specific procedures are described in Examples 2-5. Only the 2× reaction buffer was replaced with buffers containing different concentrations of additives, as shown in Table 13, including different phosphate ion concentrations, different ammonium sulfate concentrations, different ammonium chloride concentrations, different EDTA concentrations, different Triton X-100 concentrations, different guanidine hydrochloride concentrations, and different urea concentrations.

[0098] Table 13 Components of 2× reaction buffer solution for various additives at different concentrations

[0099] The relative activity curves of the PPR high-salt-tolerant totipotent nuclease mutant under different phosphate, ammonium sulfate, ammonium chloride, EDTA, Triton X-100, guanidine hydrochloride, and urea concentration ranges are shown below. Figures 6-12 As shown in Table 14, the PPR high-salt tolerant toxoplasmic nuclease mutant has a wide tolerance to various buffers and additives. The reaction conditions with enzyme activity ≥80% are defined as the optimal conditions, and the reaction conditions with enzyme activity ≥20% are defined as the effective conditions.

[0100] Table 14 Tolerance range of various additives for PPR high-salt pluripotent nuclease mutants

[0101] Example 7: Activity comparison test of PPR high-salt tolerant totipotent nuclease mutant with competing products. The activities of the PPR high-salt-tolerant totipotent nuclease mutant and its competitors were compared. Specifically, nucleic acid electrophoresis was used to compare the cleavage activity of the PPR high-salt-tolerant totipotent nuclease mutant and its competitors on nucleic acid substrates under optimal conditions (i.e., 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl2). The specific procedures are as follows: I. Preparation of Materials and Reagents 1. Prepare the materials or reagents used in the experiment according to Table 15. Table 15. Preparation methods of materials or reagents used.

[0102] II. Testing the substrate cleavage activity of PPR high-salt tolerant totipotent nuclease mutant and competing products. 1. Preparation of the reaction system 1) Dilute the PPR high-salt tolerant totipotent nuclease mutant and competing products The PPR high-salt pluripotent nuclease mutant and four commercially available competing products—Y (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 20159ES25), J (purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number M065-01A), H (purchased from Arctic Zymes Technologies, catalog number 70910-202), and N (purchased from NEB (New England Biolabs), catalog number M0764S)—were serially diluted to 25, 2.5, 0.25, 0.025, 0.0025, and 0.00025 U / μl using a 1× dilution buffer.

[0103] 2) Preparation of the reaction system: Prepare the reaction systems according to Table 16. A total of 31 reaction systems were prepared (6 concentration gradients of each of the 5 high-salt-tolerant totipotent nucleases and a control, 20 μl in volume for each). Note that the high-salt-tolerant totipotent nucleases were added as the last step after all other components of the systems had been added.

[0104] Table 16 Reaction System

[0105] Note: The control group consisted of a high-salt-tolerant totimate nuclease mutant without PPR and competing products, and was replaced with RNase-free water.

[0106] 2. Test the cleavage activity on the substrate. 1) After reacting the above reaction system at 37℃ for 10 min (or setting the PCR instrument to 37℃ and the hot lid temperature to 50℃ to reduce sample evaporation), immediately add 1 μl of 100 mM DTT (final concentration 5 mM) to terminate the reaction. Heat in a metal bath at 55℃ for 30 min to ensure complete enzyme inactivation and obtain the reaction product.

[0107] 2) Detection by 1.5% agarose gel electrophoresis Add 4 μl of 6× DNA loading buffer to each of the above reaction products, and take 8 μl of each sample for agarose gel electrophoresis (electrophoresis conditions are shown in Table 8).

[0108] 3) Data Analysis Comparing the cleavage performance of the PPR high-salt-tolerant totipotent nuclease mutant with competing products under different enzyme dosages, as follows: Figure 13 As shown, the PPR high-salt tolerant totipotent nuclease mutant exhibits superior activity compared to domestic competitors Y and J, and imported competitor N, and is comparable to the best-performing imported competitor H currently on the market.

[0109] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A PPR high-salt-tolerant totipotent nuclease mutant, characterized in that, The amino acid sequence of the PPR high-salt pluripotent nuclease mutant is shown in SEQ ID NO.

1.

2. The nucleotide sequence encoding the PPR high-salt pluripotent nuclease mutant of claim 1.

3. The nucleotide sequence according to claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

4. A recombinant plasmid or recombinant cell comprising the nucleotide sequence of claim 2.

5. The use of the PPR high-salt tolerant totipotent nuclease mutant of claim 1, the nucleotide sequence of claim 2 or 3, or the recombinant plasmid or recombinant cell of claim 4 in the removal of nucleic acids or the preparation of nucleic acid-removed products.

6. The application according to claim 5, characterized in that, The application is performed under high-salt conditions, where the NaCl concentration is 250-1000 mM.

7. The application according to claim 6, characterized in that, The NaCl concentration is 300-750 mM.

8. The application according to claim 6, characterized in that, The pH range of the system used in this application is 6.5-9.5, and the Mg content is... 2+ The concentration range is 1-40 mM, and the temperature range is 0-50℃.

9. The application according to claim 8, characterized in that, The pH range of the system used in this application is 7.7-9.5, and the Mg content is... 2+ The concentration range is 1-40 mM, and the temperature range is 10-45℃.

10. The application according to claim 8, characterized in that, The system of the application also contains at least one additive selected from phosphate ions, ammonium sulfate, ammonium chloride, EDTA, Triton X-100, guanidine hydrochloride and urea.

11. The application according to claim 10, characterized in that, The concentration ranges of the additives are phosphate ions 0-170 mM, ammonium sulfate 0-250 mM, ammonium chloride 0-300 mM, EDTA 0-5 mM, Triton X-100 0-15%, guanidine hydrochloride 0-250 mM, and urea 0-4 M.

12. The application according to claim 11, characterized in that, The concentration ranges of the additives are as follows: phosphate ions 0-70 mM, ammonium sulfate 0-120 mM, ammonium chloride 50-180 mM, EDTA 0-2 mM, Triton X-100 0-3.5%, guanidine hydrochloride 0-100 mM, and urea 0-2 M.

13. The application according to claim 5, characterized in that, The applications are protein purification, viral vector preparation, or nucleic acid removal in mNGS sample processing.

14. A nucleic acid removal reagent or kit, characterized in that, The reagent or kit contains the PPR high-salt tolerant totipotent nuclease mutant of claim 1.

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