All-round nuclease mutant and application thereof

Abstract

The invention discloses an all-potent nuclease mutant and application thereof, and relates to the technical field of biology. The invention provides an all-potent nuclease mutant. Compared with wild type all-potent nuclease as shown in SEQ ID NO: 1, the all-potent nuclease mutant comprises mutations at the following two positions: S22 and S115. The all-purpose nuclease mutant disclosed by the invention still has relatively high specific activity in a high-salt environment, and has a wide application prospect in preparation of medium-salt all-purpose nuclease products with high specific activity and good stability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to a mutant of a universal nuclease and its application. Background Art

[0002] As a special type of enzyme, nucleases have the property of catalyzing the hydrolysis of nuclease sequences, can directly cleave phosphodiester bonds, and have no nucleotide position specificity, and can directly carry out the catalytic hydrolysis reaction of nucleotides whether at the middle position or the terminal position of nucleotides. At present, nucleases have extensive application values in the fields of food, biology, medicine, etc. The universal nuclease, as a non-specific endonuclease, is a highly active non-specific hydrolase derived from Serratia marcescens, and can degrade most forms of nucleotides, and can hydrolyze both single-stranded, double-stranded, linear, and circular DNA and RNA molecules. The universal nuclease is a homodimer composed of two identical single peptide chains, and both subunits are composed of 246 amino acids. The universal nuclease requires divalent cations to exert its activity, has activity at pH 6-10, and the optimal reaction temperature is 35°C - 44°C.

[0003] At present, the universal nuclease products on the market are mainly derived from Serratia marcescens, expressed in Escherichia coli (E. coli) after genetic engineering transformation, and the inclusion body dissolution and renaturation technology is adopted. This production method ensures the high degradation ability and high purity of the enzyme, but it is difficult to scale up, the renaturation treatment process is complex, resulting in a long production cycle, high cost, and unstable specific activity of the obtained enzyme. Therefore, the universal nuclease faces problems such as a long production cycle, high cost, and large batch-to-batch differences, which seriously restricts the stable supply of large quantities of products. In addition, in practical applications, the universal nuclease needs to have a NaCl concentration lower than 100 mM to exert normal activity, and the higher the salt concentration, the lower the enzyme activity. Another type is the salt-tolerant universal nuclease, with an optimal salt concentration of about 500 mM. As the salt concentration decreases, the activity also decreases. When it is lower than 200 mM, the activity decreases by more than 70%. This makes it necessary to process some samples (such as dilution, liquid replacement) before use under the salt condition of 100 - 200 mM.

[0004] Chinese Patent CN117384881A provides a universal nuclease and its preparation method, including (1) constructing a recombinant expression plasmid; (2) transforming yeast strains and screening positive transformants; (3) fermenting yeast recombinant expression strains; (4) separating and purifying the universal nuclease in the fermentation broth. This invention obtained a universal nuclease with high purity and strong activity by optimizing the nuclease gene sequence and the preparation method of the universal nuclease, which can meet the needs of the biological products industry such as vaccines.

[0005] Currently, there is a lack of a medium-salt universal nuclease with optimal enzyme activity under 100-200 mM salt conditions on the market. Summary of the Invention

[0006] The object of the present invention is to provide a universal nuclease mutant and its application.

[0007] To achieve the above object of the invention, the technical solution of the present invention is as follows: On the one hand, the present invention provides a universal nuclease mutant, and the universal nuclease mutant contains mutations at the following two positions compared to the wild-type universal nuclease shown in SEQ ID NO:1: S22 and S115.

[0008] SEQ ID NO:1: APISFSHAKNEAVKIYRDHPVSFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRSNFSFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN* Preferably, the universal nuclease mutant contains the following mutations: (1) S22R and S115R; or; (2) S22E and S115R; or; (3) S22S and S115R; or; (4) S22E and S115E; or; (5) S22K and S115R.

[0009] More preferably, the universal nuclease mutant contains mutations at the following three positions compared to the wild-type universal nuclease shown in SEQ ID NO:1: S22, S115, T101.

[0010] Specifically, the universal nuclease mutant contains the following mutations: S22E, T101V, S115R; or S22E, T101V, S115E.

[0011] Preferably, the universal nuclease mutant contains mutations at the following four positions compared to the wild-type universal nuclease shown in SEQ ID NO:1: S22, S115, T101, G199.

[0012] Specifically, the omnipotent nuclease mutant contains the following mutations: S22E, T101V, S115R, G199del.

[0013] Specifically, the amino acid sequence of the omnipotent nuclease mutant is shown as any one of SEQ ID NO:2-SEQ ID NO:7.

[0014] According to some embodiments of the present invention, the amino acid sequence of the omnipotent nuclease mutant has at least 75% homology with the sequence shown in SEQ ID NO:1.

[0015] According to some embodiments of the present invention, the amino acid sequence of the omnipotent nuclease mutant has at least 80% homology with the sequence shown in SEQ ID NO:1.

[0016] According to some embodiments of the present invention, the amino acid sequence of the omnipotent nuclease mutant has at least 85% homology with the sequence shown in SEQ ID NO:1.

[0017] According to some embodiments of the present invention, the amino acid sequence of the omnipotent nuclease mutant has at least 90% homology with the sequence shown in SEQ ID NO:1.

[0018] According to some embodiments of the present invention, the amino acid sequence of the omnipotent nuclease mutant has at least 95% homology with the sequence shown in SEQ ID NO:1.

[0019] According to some embodiments of the present invention, the amino acid sequence of the omnipotent nuclease mutant has at least 99% homology with the sequence shown in SEQ ID NO:1.

[0020] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned omnipotent nuclease mutant.

[0021] In another aspect, the present invention provides an expression vector, and the expression vector comprises the above-mentioned nucleic acid molecule.

[0022] Specifically, the expression vector is a plasmid, a phage particle or a viral vector.

[0023] Further, the plasmid is selected from pPIC9K, pET28a, pET22b, pET32a, pET50b; Still further, the plasmid is pPIC9K.

[0024] In another aspect, the present invention provides a host cell, and the host cell comprises the above-mentioned nucleic acid molecule or the above-mentioned expression vector.

[0025] Specifically, the host cells include prokaryotic cells and eukaryotic cells.

[0026] Further, the prokaryotic cells include Escherichia coli cells.

[0027] Still further, the Escherichia coli cells include, but are not limited to, BL21(DE3), BL21(DE3)plys.

[0028] Further, the eukaryotic cells include yeast cells.

[0029] In another aspect, the present invention provides a kit, which includes the above-mentioned omnipotent nuclease mutant.

[0030] In another aspect, the present invention provides a method for preparing the above-mentioned omnipotent nuclease mutant, including the following steps: (1) Transform the vector plasmid into a strain for culture, collect the bacterial cells and extract the plasmid. (2) Linearize the recombinant plasmid. (3) Electrotransform the linearized plasmid. (4) Transform and express the positive clone. (5) Express and concentrate the protein, and obtain the mutant of the omnipotent nuclease after purification.

[0031] According to some embodiments of the present invention, step (1) includes: S1. Transform the constructed recombinant plasmid MN-pPIC9K into TOP10 strain and culture overnight at 35-38°C. S2. Pick a single colony containing the recombinant plasmid into LB liquid medium and culture overnight at 35-38°C. S3. Inoculate the bacterial liquid into LB liquid medium and culture overnight at 35-38°C. S4. Collect the bacterial cells and extract the plasmid.

[0032] According to some embodiments of the present invention, step (2) includes: S1. Use an endonuclease to linearize the recombinant plasmid, incubate in a water bath at 35-38°C for 1-3 h to form a Mut+ phenotype. S2. Add ethanol and NaAC to the digestion system, mix well, centrifuge, discard the supernatant, resuspend the precipitate with 70% ethanol, centrifuge, and discard the supernatant. S3. Dry the lower precipitate and dissolve the precipitate.

[0033] Specifically, the endonuclease in S1 of step (2) is SacI endonuclease.

[0034] Specifically, the ethanol added in S2 of step (2) is absolute ethanol, and the added amount of absolute ethanol is 2 volumes.

[0035] Specifically, the addition amount of NaAC in S2 of step (2) is 1 / 10 volume.

[0036] Specifically, the centrifugation conditions in S2 of step (2) are 1200 rpm for 10 min.

[0037] Specifically, the drying condition in S3 of step (2) is to place it in an oven at 60 °C and let it stand for 10 min.

[0038] According to some embodiments of the present invention, step (3) includes: S1. Add the pPIC9k recombinant plasmid to the GS115 competent cells; S2. Perform electrotransformation according to the preset Pichia pastoris parameters of the electrotransformer used (voltage: 1.5 kV, capacitance: 25 uF, resistance 200 - 400 ohms, electrotransformation time: 10 ms); S3. After electrotransformation, add sorbitol to the electroporation cuvette; S4. Incubate the solution at 28 - 32 °C for 1 - 2 h; S5. Spread the transformed cells on YPD plates containing different concentrations of G418 screening resistance (0.5 mg / ml, 1 mg / ml, 2 mg / ml), and incubate them upside down at 28 - 32 °C for 2 - 4 d until colonies appear.

[0039] Specifically, the concentration of sorbitol in S3 of step (3) is 1 mol / L.

[0040] According to some embodiments of the present invention, step (4) includes: S1. Inoculate the positive strain in BMGY liquid medium for culture; S2. Centrifuge to collect the bacterial cells, and inoculate the bacterial cells into BMMY medium for culture according to the initial OD = 1.0; S3. Supplement methanol and collect the bacterial liquid samples.

[0041] Specifically, the culture conditions in S1 of step (4) are to culture at 28 - 32 °C, 180 - 220 rpm for 20 - 24 h.

[0042] Specifically, the culture conditions in S2 of step (4) are to culture at 28 - 32 °C, 180 - 220 rpm for 72 - 120 h.

[0043] Specifically, 1% methanol is supplemented every 24 h in S3 of step (4).

[0044] According to some embodiments of the present invention, the protein expression and concentration in step (5) include: S1. Take the glycerol stock and activate the strain on YPD plate medium; S2. Inoculate 3 single colonies into the first-stage YPG seed medium and culture overnight. After the OD600 reaches 6 - 10, transfer them to the second-stage YPG seed medium with an inoculation amount of 0.5%. After overnight culture, when the OD600 reaches 6 - 10, proceed with the inoculation; S3. Inoculate the second-stage seed medium into a fermenter containing the BSM basal medium; S4. Culture for 20 - 25 h until the OD600 reaches 70 - 80 and the glycerol in the basal medium is exhausted. Then, supplement the glycerol feeding medium at a rate of 45 ml / h and continue the culture; S5. When the OD600 reaches 150 - 200, stop supplementing glycerol. Wait for the dissolved oxygen to rise rapidly. After 30 min, supplement methanol for induction and gradually increase the methanol feeding rate to 28 ml / h, with the dissolved oxygen controlled to be greater than 20%; S6. Induce for 72 h, stop fermentation, and collect the supernatant by centrifugation.

[0045] Specifically, the initial conditions of the fermenter in S3 of step (5) are 28°C, 300 rpm, pH = 5.0, and the ventilation rate is 2.0 L / min.

[0046] According to some embodiments of the present invention, the purification described in step (5) includes: S1. Adjust the conductivity of the fermentation supernatant to 20 mS / cm with NaH2PO4; S2. Load the supernatant protein solution at a flow rate of 5 mL / min; S3. Wash the column with NaH2PO4 and NaCl until no protein is detected in the effluent; S4. Elute with NaH2PO4 and NaCl, and collect the section where the detection by G250 shows blue; S5. Wash the packing material with 3 column volumes of deionized water and seal the column with 20% ethanol; S6. Ultrafilter and change the buffer of the eluate to 25 mM Tris-HCl pH 7.0, 5 mM MgCl2, 400 mM NaCl, 50% Glycerol, 0.05% Tween-20 and concentrate it.

[0047] On the other hand, the present invention provides the use of the above-mentioned omnipotent nuclease or nucleic acid molecule or expression vector or host cell or kit in the preparation of biological products.

[0048] The beneficial effects of the present invention are: The present invention has mutated a wild-type moderate-salt omnipotent nuclease from Vibrio cholerae to obtain a moderate-salt omnipotent nuclease mutant sequence with high specific activity. The mutant with high specific activity is expected to improve the unit yield and product stability. In addition, these mutant sequences can be secreted and expressed and purified in the Pichia pastoris expression system, providing the possibility for industrial production. This important result is expected to provide moderate-salt omnipotent nuclease products with high specific activity and good stability, and promote the rapid development of related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a structural diagram of the wild-type omnipotent nuclease.

[0050] Figure 2 It is a schematic structural diagram at the S22 position of the wild-type omnipotent nuclease.

[0051] Figure 3 It is a schematic structural diagram at the S115 position of the wild-type omnipotent nuclease.

[0052] Figure 4 It is an SDS-PAGE electrophoresis diagram of mutants 1-9. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further clarified below in conjunction with specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention, not all of them. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operation methods used are all conventional operation methods, the equipment used is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0054] The amino acid sequence of the wild-type omnipotent nuclease in the following embodiments is shown in SEQ ID NO:1: SEQ ID NO:1: APISFSHAKNEAVKIYRDHPVSFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRSNFSFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN* The structural diagram of the wild-type omnipotent nuclease is as Figure 1As shown, the schematic diagram of the structure at position S22 of wild-type omnipotent nuclease is as Figure 2 shown, and the schematic diagram of the structure at position S115 of wild-type omnipotent nuclease is as Figure 3 shown.

[0055] The sequence of Mutant 1 is shown in SEQ ID NO:2 (S22E, S115R): SEQ ID NO:2: APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN* The sequence of Mutant 2 is shown in SEQ ID NO:3: (S22R, S115R) SEQ ID NO:3: APISFSHAKNEAVKIYRDHPVRFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN* The sequence of Mutant 3 is shown in SEQ ID NO:4 (S22S, S115R): SEQ ID NO:4: APISFSHAKNEAVKIYRDHPVSFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN* The sequence of Mutant 4 is shown in SEQ ID NO:5 (S22K, S115R): SEQ ID NO:5: APISFSHAKNEAVKIYRDHPVKFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN* The sequence of Mutant 5 is shown in SEQ ID NO:6: (S22E, T101V, S115E) SEQ ID NO:6: APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLVPAIGEVNGDRSNFEFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN* The sequence of Mutant 6 is shown in SEQ ID NO:7: (S22E, T101V, S115R, G199del) SEQ ID NO:7: APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLVPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQNSNRFVREQCPN* The sequence of Mutant 7 is shown in SEQ ID NO:8: (S22E, G33E, S55E, S87E, T101V, S115R, G199del) SEQ ID NO:8: APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQEKKGIPDLESCGYQVRKNENRAERIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTEPEFNQMEADLHNLVPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQNSNRFVREQCPN* The sequence of Mutant 8 is shown in SEQ ID NO:9: (S22E, G33E, S55R, S87R, T101V, S115R, G199del) SEQ ID NO:9: APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQEKKGIPDLESCGYQVRKNENRARRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTRPEFNQMEADLHNLVPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQNSNRFVREQCPN* The sequence of Mutant 9 is shown in SEQ ID NO:10: (S22E, G33E, S55R, S87R, T101V, S115R, G199R) SEQ ID NO:10: APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQEKKGIPDLESCGYQVRKNENRARRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTRPEFNQMEADLHNLVPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQRNSNRFVREQCPN* Example 1 1. Method for constructing strains of wild-type mesophilic omnipotent nuclease and mutants 1.1 Large-scale extraction of recombinant plasmids of mesophilic omnipotent nuclease and mutants (1) Transform the constructed recombinant plasmid MN-pPIC9K (synthesized and prepared by GenScript) into TOP10 strain, coat it on an LB plate (kanamycin resistant), and culture it overnight at 37°C; (2) Pick a single colony containing the recombinant plasmid into 3 mL of LB liquid medium and culture it overnight at 37°C; (3) Take 600 μL of the bacterial solution for preservation, take 100 μL of the bacterial solution and inoculate it into 200 mL of LB liquid medium (kanamycin resistant), and culture it overnight at 37°C; (4) Collect the bacteria and use the OMEGA Plasmid Midi Kits kit (purchased from OMEGA, catalog number D6904) to extract the plasmid.

[0056] 1.2 Linearization and recovery of the recombinant plasmid (1) Use SacI endonuclease to linearize the MN-pPIC9K recombinant plasmid, incubate it in a water bath at 37°C for 2 h to form the Mut+ phenotype; (2) Add 2 volumes of pre-cooled absolute ethanol and 1 / 10 volume of NaAC with pH 5.2 to the enzyme digestion system, mix well; let it stand at -20°C for 20 min; centrifuge at 1200 rpm for 10 min, discard the supernatant after centrifugation, centrifuge again for 1 min, and use a 10 μL pipette to aspirate a small amount of the remaining supernatant. Resuspend the precipitate with 500 μL of 70% ethanol, centrifuge as above for 10 min, discard the supernatant, centrifuge again for 1 min, and use a 10 μL pipette to aspirate a small amount of the remaining ethanol.

[0057] (3) Let the lower precipitate stand in an oven at 60°C for 10 min to volatilize the remaining ethanol; (4) Dissolve the precipitate in 15 - 20 μL of sterile water per tube. (After linearization and enzyme digestion recovery of the plasmid, it should be transformed immediately. If not, it should be stored at -20°C.)

[0058] 1.3 Electroporation of the linearized plasmid (1) Take 80 μL of GS115 competent cells and add 10 μL of the linearized pPIC9k recombinant plasmid (about 5 - 10 μg).

[0059] (2) Place it on ice for 5 min.

[0060] (3) Perform electroporation according to the preset Pichia pastoris parameters of the electroporator used (voltage: 1.5 kV, capacitance: 25 μF, resistance 200 - 400 ohms, electroporation time: 10 ms).

[0061] (4) Immediately after electroporation, add 1 ml of pre-cooled 1 mol / L sorbitol to the electroporation cup, and then transfer all the solution in the electroporation cup to a 50 ml sterile centrifuge tube.

[0062] (5) Incubate statically at 30 °C for 1 - 2 h.

[0063] (6) Take 300 µl of the transformed cells and spread them on YPD plates containing different concentrations of G418 screening resistance (0.5 mg / ml, 1 mg / ml, 2 mg / ml), and incubate them upside down at 30 °C for 2 - 4 d until colonies appear. (Controlling the plasmid concentration and spreading 300 μL can grow approximately 300 - 400 single colonies).

[0064] 1.4 Screening and Identification of Positive Clones by PCR According to the growth of strains on resistance plates with different concentrations, pick monoclonal colonies with better growth on the resistance plates for PCR identification, and preferably choose the resistance plates with higher concentrations first.

[0065] (1) Preparation of PCR reaction system Table 1

[0066] (2) Set up blank controls (without plasmid template) and positive controls (adding 0.5 μL plasmid) for colony PCR; Table 2

[0067] (3) Detect the PCR products by agarose gel electrophoresis.

[0068] 1.5 Screening and Identification of Positive Clone Transformation and Expression (1) Select and preserve the clones identified as positive by colony PCR; (2) Inoculate the positive strain into a 250 ml baffled flask containing 25 mL of BMGY liquid medium and culture it at 30 °C and 200 rpm for 24 h; (3) Centrifuge to collect the bacterial cells, inoculate the bacterial cells into a 250 ml baffled flask containing 25 ml of BMMY medium at an initial OD = 1.0, and culture it at 28 °C and 220 rpm for 72 - 120 h.

[0069] (4) Add 1% methanol every 24 h. Take samples of the bacterial liquid at different time points, with a sampling volume of 1 ml, place them in 1.5 ml EP tubes, centrifuge to collect the supernatant and the bacterial cells respectively, and analyze the expression level of the target protein and the optimal harvest time of the bacterial liquid. The time points are generally taken as: 0, 24, 48, 72, 96, and 120 h.

[0070] (5) For secreted expression, separate the supernatant of the sample; for intracellular expression, separate the bacterial cell pellet of the sample, and the expression of the recombinant protein can be detected and identified by SDS - PAGE, Western - Blot, and activity experiments.

[0071] 2. Expression and purification 2.1 High-level protein expression and concentration (1)Take glycerol bacteria for strain activation on YPD plate medium.

[0072] (2)Inoculate 3 single colonies into 50 ml of first-stage YPG seed medium and culture overnight. After the OD600 reaches 6 - 10, transfer it to 250 ml of second-stage YPG seed medium with an inoculation amount of 0.5%. After overnight culture, when the OD600 reaches 6 - 10 and there is no contamination of miscellaneous bacteria under microscopic examination, it is used for inoculation.

[0073] (3)Prepare 2.5 L of BSM basal medium in a fermenter. After the sterilization is completed, inoculate the second-stage seed medium into the fermenter. The initial conditions are 28 °C, 300 rpm, pH = 5.0, and the aeration rate is 2.0 L / min.

[0074] (4)Cultivate for 20 - 25 h until the OD600 reaches 70 - 80 and the glycerol in the basal medium is exhausted. Then, supplement the glycerol feeding medium at a rate of 45 ml / h and continue the cultivation.

[0075] (5)When the OD600 reaches 150 - 200, stop supplementing glycerol. Wait for the dissolved oxygen to rise rapidly. After 30 min, supplement methanol for induction and gradually increase the methanol feeding rate to 28 ml / h, with the dissolved oxygen controlled to be greater than 20%.

[0076] (6)Induce for 72 h, stop fermentation, and collect the supernatant by centrifugation.

[0077] (7)After identifying normal expression by SDS-PAGE, proceed with purification.

[0078] 2.2 Purification of medium-salt universal nuclease (1)Adjust the conductivity of the fermentation supernatant to 20 mS / cm with 10 M NaH2PO4 (pH 6.0); (2)Prepare 5 mL of pre-packed SP column; (3)Load the supernatant protein solution at a flow rate of 5 mL / min; (4)Wash the column with 20 M NaH2PO4, 200 mM NaCl (pH 6.0) until the effluent contains no protein (the G250 detection solution does not change color); (5)Elute with 20 M NaH2PO4, 1 M NaCl (pH 6.0), and collect the section where the G250 detection shows blue; (6)Wash the packing with 3 column volumes of deionized water and seal the column with 20% ethanol; (7)The eluate was ultrafiltered and buffer-exchanged to 25 mM Tris-HCl pH 7.0, 5 mM MgCl2, 400 mM NaCl, 50% Glycerol, 0.05% Tween-20 through a 10 kDa ultrafiltration tube and concentrated to 1-2 mL. The concentrated solution was taken for SDS-PAGE detection.

[0079] 3. Enzyme Activity Identification of Medium-Salt Omnipotent Nuclease 3.1 Enzyme Activity Assay Method 3.1.1 A Reaction Buffer Solution formula: 25 mM Tris, 2.5 mM MgCl2, 150 mM NaCl, pH 7.6.

[0080] Preparation steps: Weigh 1.2414 g of Tris, 3.5064 g of NaCl and 0.2033 g of MgCl2 with a ten-thousandth balance, dissolve them in ultrapure water, adjust the pH to 7.6 with hydrochloric acid, and make up the volume to 400 ml with ultrapure water. Filter with a 0.22 μM filter membrane and store at 4°C.

[0081] 3.1.2 B Calf Thymus DNA Solution concentration: 0.5 mg / ml calf thymus DNA.

[0082] Preparation steps: Take 50 mg of calf thymus DNA powder and dissolve it in 10 ml of Buffer A to make a 5 mg / ml calf thymus DNA stock solution. For long-term storage, store it at -20°C. Before activity measurement, take 1 ml of the stock solution and dilute it with 9 ml of Buffer A to 0.5 mg / ml calf thymus DNA, pre-cool on ice and keep it at 4°C for short-term storage.

[0083] 3.1.3 C Reaction Termination Solution Solution concentration: 4% perchloric acid solution Preparation steps: Dilute 11.26 ml of 70 - 72% perchloric acid with 188.74 ml of ultrapure water, mix well and store in a brown bottle at 4°C in the dark. Before activity measurement, pre-cool on ice.

[0084] 3.2 Reaction Steps 3.2.1 Dilution of the Sample to be Measured Input amount: 1 U (the input amount can be fine-tuned according to the slope change) Dilution steps: Dilute the sample to be measured with A reaction buffer. 125 μl of enzyme solution needs to be added to the reaction system. Calculated according to the input amount of 1 U, the sample to be measured needs to be diluted to 0.008 U / μl.

[0085] 3.2.2 Reaction System Table 3 Reaction System

[0086] 3.2.3 Reaction conditions Add calf thymus DNA into a 5 ml centrifuge tube, and add the enzyme solution at the end. The above reaction system is loaded on ice, and after mixing by inverting up and down, it is immediately incubated in a 37 °C water bath for 1 hour. Accurately time when it is placed in the water bath. At the 15th, 30th, 45th, and 60th minutes of the reaction, take 0.5 ml of the incubation solution from the centrifuge tube and transfer it to a 1.5 ml centrifuge tube containing 0.5 ml of 4% perchloric acid pre-cooled on ice in advance. Vortex for 10 s and ice-bath for 30 minutes. The operations in the four time periods are kept consistent. Then centrifuge at 15000 r / min at 4 °C for 6 min, and take 800 μl of the supernatant for detection.

[0087] 3.2.4 Activity determination Select the cuvette mode on the microplate reader, measure the OD at the wavelength of A260. First measure the blank control and then measure the sample to be tested. Each tube of the sample to be tested needs to be read twice, and the two OD 260nm values are consistent, and then continue to detect the next tube of the sample to be tested.

[0088] 3.3 Calculation of enzyme activity Calculation formula:

[0089] In the formula: A = absorbance of the measured solution at time t; V = total volume of the incubation mixture; t = incubation time of the measured solution (min); 30 = defined 30 min; 2 = dilution factor for perchloric acid denaturation; F = dilution factor of the sample; V = volume of the diluted sample added to the reaction system (ml).

[0090] With time as the abscissa and the OD after removing the blank control 260nm as the ordinate, fit a straight line, select "cubic curve regression" to fit the standard curve, and calculate the enzyme activity according to the slope of the sample to be tested.

[0091] 4. Definition of enzyme activity Under the reaction conditions of 37 °C, in 25 mM Tris-HCl pH 7.6 (25 °C), 2.5 mM MgCl2, 150 mM NaCl and 50 μg / ml calf thymus DNA, the amount of enzyme that causes ΔA260 = 1.0 within 30 minutes is defined as one unit of enzyme activity (U).

[0092] 5. Determination of the protein concentration of medium-salt omnipotent nuclease 5.1 Detection Method (1)Turn on the microplate reader and the software in sequence, and select Protein A280; (2)Select the sample type as 1 Abs at 1cm = 1mg / mL, select the well type as "background", detect 4 replicates, and keep other settings default; (3)Prepare the dilution buffer (1 mL): Mix 0.5 mL of 2× dilution buffer (buffer) (100 mM Tris-HCl, pH 8.0, 500 mM NaCl, 4 mM CaCl2) and 0.5 mL of purified water evenly; (4)Dilute the sample to be tested: Dilute the sample to be tested 2-fold, 4-fold, and 8-fold successively with the dilution buffer, and mix the enzyme solution well; (5)Take 2 μL of the dilution buffer as the blank control, with 4 replicates, and click to detect; (6)Sample detection: Take 2 μL of the stock solution and the 2-fold, 4-fold, and 8-fold diluted solutions, each with 4 replicates for detection, and output the 4 test data of C0, C2, C4, and C8.

[0093] 5.2 Data Processing

[0094] Among them: C0, C2, C4, and C8 are the average values of 4 replicates of the stock solution, 2-fold, 4-fold, and 8-fold diluted solutions of the sample to be tested respectively; the extinction coefficient (0.1%) is the absorbance value of the target protein at 280 nm (the thickness of the absorption cell is 1 cm) at a concentration of 1 mg / mL, which can be calculated according to the protein sequence.

[0095] 6. Protein Purity Analysis Method 6.1 SDS-PAGE Detection Method (1)Preparation of the sample to be tested: Dilute the sample to be tested to 0.2 mg / mL and 0.02 mg / mL with the stock buffer and mix well; (2)Take 10 μL of the enzyme dilution, add an equal volume of 2× SDS PAGE loading buffer, mix well by pipetting, and treat at 100 °C for 10 min; (3)Install the 12% precast protein plus gel on the electrophoresis apparatus, add SDS-PAGE electrophoresis buffer, protein marker, and the sample to be tested; (4)Run the gel at a constant voltage of 150 V for 30 - 40 min; (5)Take out the protein gel and stain and decolorize it with a staining and decolorizing instrument; (6) Take out the protein gel and place it on a whiteboard, then put it into a gel imaging analysis system for photographing.

[0096] 6.2 Data processing Use Quantity One for purity analysis and an image processing software for gel image processing. Keep the complete gel image in tif format. The identified purity is ≥95%.

[0097] 7. Digestive comparison experiment between wild-type medium-salt omnipotent nuclease and mutants 7.1 Sample preparation The supernatant of the culture medium after AAV packaging of the virus is repeatedly frozen and thawed 3 times with liquid nitrogen to lyse the virus (verified by pH test paper with pH 7 - 10).

[0098] 7.2 Omnipotent nuclease enzymatic digestion Table 4

[0099] After the digestion is completed, centrifuge at 16,000 rpm for 10 min, take the supernatant, and detect the HCD value of 293 cells.

[0100] 8. Result analysis 8.1 Protein purification results of wild-type medium-salt omnipotent nuclease and mutants The SDS-PAGE results show that Figure 4 ), after expression and purification by Pichia pastoris, all 9 mutants can be successfully purified, and the purity differences are small. The amount of impurity bands in the target protein is less than 5% of the amount of the target protein. Therefore, the protein purities of both wild-type medium-salt omnipotent nuclease and mutants are greater than 95%, and the samples can be used for the determination of specific activity data.

[0101] 8.2 Specific activity results of wild-type medium-salt omnipotent nuclease and mutants The specific activity results show that mutants 1 - 6 of medium-salt omnipotent nuclease show a significant increase in specific activity. However, there is no obvious difference in the specific activity data between mutants 7 - 9 and the wild type (Table 5). At the same time, the digestion experiment results further show that under the condition of the same active input amount, mutants 1 - 6 digest more DNA and show higher DNA digestion ability (Table 6).

[0102] Table 5 Comparison of results between wild-type MSAN and mutants

[0103] Table 6 Enzymatic digestion results of wild-type medium-salt omnipotent nuclease and mutants

[0104] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A universal nuclease mutant, characterized in that: The universal nuclease mutant comprises the following mutations: (1) S22E and S115R; or (2) S22R and S115R; or (3) S22S and S115R; or (4) S22K and S115R; or (5) S22E, T101V and S115E; or (6) S22E, T101V, S115R and G199del.

2. A nucleic acid molecule, characterized in that It comprises a nucleotide sequence encoding the all-potent nuclease mutant according to claim 1.

3. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule according to claim 2.

4. A host cell, characterized in that The host cell comprises the nucleic acid molecule according to claim 2 or the expression vector according to claim 3.

5. A kit, characterized in that: The kit comprises the all-potent nuclease mutant according to claim 1.

6. The method for preparing the universal nuclease mutant according to claim 1, characterized in that: The following steps are involved: (1) Transform the vector plasmid into the bacterial strain, culture it, and collect the bacterial cells for plasmid extraction; (2) Linearization of recombinant plasmid; (3) Electroporation of linearized plasmid; (4) Transformation and expression of positive clones; (5) Protein expression, concentration, and purification are performed to obtain mutants of the universal nuclease.

7. Use of the all-potent nuclease according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3, the host cell according to claim 4, or the kit according to claim 5 in the preparation of biological products.

Images