A universal nuclease mutant and its application

Abstract

The present invention discloses a totipotent nuclease mutant and its application, relating to the field of biotechnology. The present invention provides a totipotent nuclease mutant comprising mutations at two positions, S22 and S115, compared to the wild-type totipotent nuclease shown in SEQ ID NO: 1. The totipotent nuclease mutant of the present invention still exhibits high specific activity in a high-salt environment and has broad application prospects in the preparation of medium-salt totipotent nuclease products with high specific activity and good stability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a omnipotent nuclease mutant and applications thereof. Background Art

[0002] Nucleases, a special class of enzymes, possess the ability to catalyze the hydrolysis of nuclease sequences, directly cleaving phosphodiester bonds. They lack nucleotide position specificity and can directly catalyze the hydrolysis of nucleotides at both the intermediate and terminal positions. Nucleases currently have a wide range of applications in the food, biological, and pharmaceutical sectors. The universal nuclease, a nonspecific endonuclease, is a highly active, nonspecific hydrolase derived from Serratia marcescens that can degrade most forms of nucleotides, including single-stranded, double-stranded, linear, and circular DNA and RNA molecules. The universal nuclease is a homodimer composed of two identical single peptide chains, with both subunits consisting of 246 amino acids. The universal nuclease requires divalent cations for activity and is active at pH 6-10, with an optimal reaction temperature of 35°C-44°C.

[0003] Currently, the primary commercially available omnipotent nuclease products are derived from Serratia marcescens, genetically engineered, and expressed in Escherichia coli (E. coli) using inclusion body solubilization and renaturation technology. This production method ensures efficient degradation and high enzyme purity, but scale-up is difficult and the renaturation process is complex, resulting in long production cycles, high costs, and unstable specific enzyme activity. Consequently, omnipotent nucleases face challenges such as long production cycles, high costs, and large batch-to-batch variability, severely restricting the stable supply of large-scale products. Furthermore, in practical applications, omnipotent nucleases require NaCl concentrations below 100mM to exhibit normal activity; higher salt concentrations reduce enzyme activity. Alternatively, salt-tolerant omnipotent nucleases have an optimal salt concentration of approximately 500mM. Activity decreases with decreasing salt concentrations, dropping by over 70% below 200mM. This necessitates sample preparation (e.g., dilution or liquid exchange) before use in 100-200mM salt conditions.

[0004] Chinese patent CN117384881A provides a totipotent nuclease and a method for preparing the same, comprising: (1) constructing a recombinant expression plasmid; (2) transforming a yeast strain and screening positive transformants; (3) fermenting the recombinant expression yeast strain; and (4) isolating and purifying the totipotent nuclease from the fermentation broth. By optimizing the nuclease gene sequence and the totipotent nuclease preparation method, this invention produces a highly pure and active totipotent nuclease that can meet the needs of the vaccine and other biological product industries.

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

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

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a totipotent nuclease mutant, which comprises mutations at the following two positions compared to the wild-type totipotent nuclease shown in SEQ ID NO: 1: S22 and S115.

[0009] SEQ ID NO: 1:

[0010] APISFSHAKNEAVKIYRDHPVSFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGE VNGDRSNFSFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN*

[0011] Preferably, the omnipotent nuclease mutant comprises the following mutations:

[0012] (1) S22R and S115R; or

[0013] (2) S22E and S115R; or

[0014] (3) S22S and S115R; or

[0015] (4) S22E and S115E; or

[0016] (5) S22K and S115R.

[0017] Further preferably, the totipotent nuclease mutant comprises mutations at the following three positions compared to the wild-type totipotent nuclease shown in SEQ ID NO: 1: S22, S115, and T101.

[0018] Specifically, the all-potent nuclease mutant comprises the following mutations: S22E, T101V, S115R; or S22E, T101V, S115E.

[0019] Preferably, the totipotent nuclease mutant comprises mutations at the following four positions compared to the wild-type totipotent nuclease shown in SEQ ID NO: 1: S22, S115, T101, and G199.

[0020] Specifically, the omnipotent nuclease mutant comprises the following mutations: S22E, T101V, S115R, and G199del.

[0021] Specifically, the amino acid sequence of the universal nuclease mutant is shown in any one of SEQ ID NO: 2 to SEQ ID NO: 7.

[0022] According to some embodiments of the present invention, the amino acid sequence of the totipotent nuclease mutant has at least 75% homology to the sequence shown in SEQ ID NO:1.

[0023] According to some embodiments of the present invention, the amino acid sequence of the totipotent nuclease mutant has at least 80% homology to the sequence shown in SEQ ID NO:1.

[0024] According to some embodiments of the present invention, the amino acid sequence of the totipotent nuclease mutant has at least 85% homology to the sequence shown in SEQ ID NO:1.

[0025] According to some embodiments of the present invention, the amino acid sequence of the totipotent nuclease mutant has at least 90% homology to the sequence shown in SEQ ID NO:1.

[0026] According to some embodiments of the present invention, the amino acid sequence of the totipotent nuclease mutant has at least 95% homology to the sequence shown in SEQ ID NO:1.

[0027] According to some embodiments of the present invention, the amino acid sequence of the totipotent nuclease mutant has at least 99% homology to the sequence shown in SEQ ID NO:1.

[0028] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned full-potent nuclease mutant.

[0029] In another aspect, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0030] Specifically, the expression vector is a plasmid, a phage particle or a viral vector.

[0031] Furthermore, the plasmid is selected from pPIC9K, pET28a, pET22b, pET32a, and pET50b;

[0032] Furthermore, the plasmid is pPIC9K.

[0033] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule or the aforementioned expression vector.

[0034] Specifically, the host cells include prokaryotic cells and eukaryotic cells.

[0035] Furthermore, the prokaryotic cells include Escherichia coli cells.

[0036] Furthermore, the Escherichia coli cells include but are not limited to BL21 (DE3) and BL21 (DE3) plys.

[0037] Furthermore, the eukaryotic cells include yeast cells.

[0038] In another aspect, the present invention provides a kit comprising the above-mentioned omnipotent nuclease mutant.

[0039] In another aspect, the present invention provides a method for preparing the above-mentioned omnipotent nuclease mutant, comprising the following steps:

[0040] (1) Transform the vector plasmid into the bacterial strain and culture it, then collect the bacterial cells for plasmid extraction;

[0041] (2) Linearization of recombinant plasmid;

[0042] (3) Electroporation of linearized plasmid;

[0043] (4) Transformation and expression of positive clones;

[0044] (5) Protein expression and concentration, followed by purification, yield the mutant of the universal nuclease.

[0045] According to some embodiments of the present invention, step (1) comprises:

[0046] S1. Transform the constructed recombinant plasmid MN-pPIC9K into the TOP10 strain and culture at 35-38°C overnight;

[0047] S2. Select a single colony containing the recombinant plasmid and place it in LB liquid medium, and culture it at 35-38°C overnight;

[0048] S3. Take the bacterial solution and inoculate it into LB liquid medium, and culture it at 35-38℃ overnight;

[0049] S4. Collect the bacteria and extract the plasmid.

[0050] According to some embodiments of the present invention, step (2) includes:

[0051] S1. Linearize the recombinant plasmid using endonuclease and digest it in a water bath at 35-38°C for 1-3 hours to form a Mut+ phenotype.

[0052] S2, add ethanol and NaAC to the enzyme digestion system, mix, centrifuge, discard the supernatant, resuspend the precipitate with 70% ethanol, centrifuge, and discard the supernatant;

[0053] S3. Dry the lower layer of precipitate and dissolve the precipitate.

[0054] Specifically, the endonuclease of S1 in step (2) is SacI endonuclease.

[0055] Specifically, the ethanol added in S2 of step (2) is anhydrous ethanol, and the amount of anhydrous ethanol added is 2 times the volume.

[0056] Specifically, the amount of NaAC added in S2 of step (2) is 1 / 10 volume.

[0057] Specifically, the centrifugal condition in S2 of step (2) is 1200 rpm and centrifugation for 10 min.

[0058] Specifically, the drying condition in S3 of step (2) is to place the mixture in an oven at 60° C. and let it stand for 10 minutes.

[0059] According to some embodiments of the present invention, step (3) includes:

[0060] pPIC9k recombinant plasmid was added to S1 and GS115 competent cells;

[0061] S2. Perform electric shock on the Pichia pastoris according to the preset parameters of the electroporator (voltage: 1.5 kV, capacitance: 25 uF, resistance: 200-400 ohms, click time: 10 ms);

[0062] S3. After the electric shock is completed, sorbitol is added to the electric rotating cup;

[0063] S4. Incubate the solution at 28-32°C for 1-2 h.

[0064] S5. Spread the transformed cells on YPD plates containing different concentrations of G418 for resistance selection (0.5 mg / ml, 1 mg / ml, 2 mg / ml), and culture inverted at 28-32°C for 2-4 days until colonies appear.

[0065] Specifically, the concentration of sorbitol in S3 of step (3) is 1 mol / L.

[0066] According to some embodiments of the present invention, step (4) comprises:

[0067] S1, inoculate positive strains and culture in BMGY liquid medium;

[0068] S2. Collect the bacteria by centrifugation and inoculate them into BMMY medium at an initial OD of 1.0;

[0069] S3. Add methanol and collect bacterial liquid samples.

[0070] Specifically, the culture conditions of S1 in step (4) are 28-32°C and 180-220 rpm for 20-24 hours.

[0071] Specifically, the culture conditions of S2 in step (4) are 28-32°C, 180-220 rpm, and 72-120 h.

[0072] Specifically, in step (4), 1% methanol is added to S3 every 24 hours.

[0073] According to some embodiments of the present invention, the expression and concentration of the protein in step (5) comprises:

[0074] S1. Take glycerol bacteria and activate them on YPD plate medium;

[0075] S2, inoculate 3 single colonies into the first-level YPG seed medium and culture overnight. After the OD600 reaches 6-10, transfer to the second-level YPG seed medium with an inoculum size of 0.5%. After overnight culture, the OD600 reaches 6-10 and then inoculate;

[0076] S3, inoculating the secondary seed culture medium into a fermenter containing BSM basal medium;

[0077] S4: Cultivate for 20-25 h until OD600 reaches 70-80 and the glycerol in the basal medium is depleted. Add glycerol feed medium at 45 ml / h and continue culturing.

[0078] When S5 and OD600 reach 150-200, stop adding glycerol and wait for the dissolved oxygen to rise rapidly. After 30 minutes, add methanol for induction and gradually increase the methanol addition rate to 28 ml / h. The dissolved oxygen is controlled to be greater than 20%.

[0079] S6. After induction for 72 h, stop the fermentation and collect the supernatant by centrifugation.

[0080] Specifically, the initial conditions of the fermentation tank in S3 of step (5) are 28°C, 300 rpm, pH=5.0, and ventilation volume 2.0 L / min.

[0081] According to some embodiments of the present invention, the purification in step (5) comprises:

[0082] S1. Adjust the conductivity of the fermentation supernatant to 20 mS / cm with NaH2PO4;

[0083] S2, load the supernatant protein solution at a flow rate of 5 mL / min;

[0084] S3. Wash the column with NaH2PO4 and NaCl until the effluent contains no protein;

[0085] S4, eluting with NaH2PO4 and NaCl, collecting the blue segments detected by G250;

[0086] S5. Wash the packing with 3 column volumes of deionized water and seal the column with 20% ethanol.

[0087] S6. The eluate was ultrafiltered through an ultrafiltration tube to 25 mM Tris-HCl pH 7.0, 5 mM MgCl2, 400 mM NaCl, 50% Glycerol, 0.05% Tween-20 and concentrated.

[0088] In another aspect, 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.

[0089] The beneficial effects of the present invention are:

[0090] This study has generated mutant sequences of the wild-type Zhongyan omnipotent nuclease derived from Vibrio cholerae by mutating them. These mutants are expected to improve specific yield and product stability. Furthermore, these mutant sequences can be secreted and purified in the Pichia pastoris expression system, facilitating industrial production. This important result is expected to provide Zhongyan omnipotent nuclease products with high specific activity and excellent stability, promoting the rapid development of related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 This is the structure diagram of the wild-type omnipotent nuclease.

[0092] Figure 2 Schematic diagram of the structure of the wild-type omnipotent nuclease at position S22.

[0093] Figure 3 Schematic diagram of the structure of the wild-type omnipotent nuclease at position S115.

[0094] Figure 4 The SDS-PAGE electrophoresis diagram of mutants 1-9. DETAILED DESCRIPTION

[0095] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0096] The amino acid sequence of the wild-type totipotent nuclease in the following examples is shown in SEQ ID NO: 1:

[0097] SEQ ID NO: 1:

[0098] APISFSHAKNEAVKIYRDHPVSFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGE VNGDRSNFSFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN*

[0099] The structure of wild-type omnipotent nuclease is shown in the figure Figure 1 As shown, the structural diagram of the wild-type universal nuclease at position S22 is shown in Figure 2 As shown, the structural diagram of the wild-type universal nuclease at position S115 is shown in Figure 3 shown.

[0100] The sequence of mutant 1 is shown in SEQ ID NO: 2 (S22E, S115R):

[0101] SEQ ID NO: 2:

[0102] APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGE VNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN*

[0103] The sequence of Mutant 2 is shown in SEQ ID NO:3: (S22R, S115R)

[0104] SEQ ID NO:3:

[0105] APISFSHAKNEAVKIYRDHPVRFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN*

[0106] The sequence of Mutant 3 is shown in SEQ ID NO:4 (S22S, S115R):

[0107] SEQ ID NO:4:

[0108] APISFSHAKNEAVKIYRDHPVSFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN*

[0109] The sequence of Mutant 4 is shown in SEQ ID NO:5 (S22K, S115R):

[0110] SEQ ID NO:5:

[0111] APISFSHAKNEAVKIYRDHPVKFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLTPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN*

[0112] The sequence of mutant 5 is shown in SEQ ID NO:6: (S22E, T101V, S115E)

[0113] SEQ ID NO:6:

[0114] APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLVPAIGE VNGDRSNFEFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQGNSNRFVREQCPN*

[0115] The sequence of mutant 6 is shown in SEQ ID NO:7: (S22E, T101V, S115R, G199del)

[0116] SEQ ID NO: 7:

[0117] APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQGKKGIPDLESCGYQVRKNENRASRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTSPEFNQMEADLHNLVPAIG EVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQNSNRFVREQCPN*

[0118] The sequence of mutant 7 is shown in SEQ ID NO:8: (S22E, G33E, S55E, S87E, T101V, S115R, G199del)

[0119] SEQ ID NO:8:

[0120] APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQEKKGIPDLESCGYQVRKNENRAERIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTEPEFNQMEADLHNLVPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQNSNRFVREQCPN*

[0121] The sequence of Mutant 8 is shown in SEQ ID NO:9: (S22E, G33E, S55R, S87R, T101V, S115R, G199del)

[0122] SEQ ID NO:9:

[0123] APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQEKKGIPDLESCGYQVRKNENRARRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTRPEFNQMEADLHNLVPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQNSNRFVREQCPN*

[0124] The sequence of Mutant 9 is shown in SEQ ID NO:10: (S22E, G33E, S55R, S87R, T101V, S115R, G199R)

[0125] SEQ ID NO:10:

[0126] APISFSHAKNEAVKIYRDHPVEFYCGCEIRWQEKKGIPDLESCGYQVRKNENRARRIEWEHVVPAWQFGHQLQCWQQGGRKNCTRTRPEFNQMEADLHNLVPAIGEVNGDRSNFRFSQWNGVDGATYGQCEMQVNFKERTAMPPERARGAIARTYLYMSEQYGLRLSKAQNQLMQAWNNQYPVSEWECVRDQKIEKVQRNSNRFVREQCPN*

[0127] Example 1

[0128] 1. Construction of wild-type and mutant strains of the Chinese salt omnipotent nuclease

[0129] 1.1 Large-scale extraction of Zhongyan omnipotent nuclease and mutant recombinant plasmids

[0130] (1) The constructed recombinant plasmid MN-pPIC9K (synthesized by GenScript) was transformed into the TOP10 strain, spread on LB plates (kanamycin resistance), and cultured at 37°C overnight;

[0131] (2) Select a single colony containing the recombinant plasmid and place it in 3 mL of LB liquid medium and culture it at 37°C overnight;

[0132] (3) Take 600 μL of bacterial solution for seed preservation, take 100 μL of bacterial solution and inoculate it into 200 mL of LB liquid medium (kanamycin resistance), and culture it at 37°C overnight;

[0133] (4) Collect the bacteria and use the OMEGA Plasmid Midi Kits (purchased from OMEGA, product number D6904) for plasmid extraction.

[0134] 1.2 Linearization and recovery of recombinant plasmid

[0135] (1) Linearize the MN-pPIC9K recombinant plasmid using SacI endonuclease and digest it in a water bath at 37°C for 2 h to form a Mut+ phenotype;

[0136] (2) Add 2 volumes of pre-chilled anhydrous ethanol and 1 / 10 volume of pH 5.2 NaAC to the enzyme digestion system and mix well. Let stand at -20°C for 20 minutes. Centrifuge at 1200 rpm for 10 minutes. Discard the supernatant after centrifugation. Centrifuge again for 1 minute. Remove any remaining supernatant with a 10-ul pipette. Resuspend the pellet in 500 ul of 70% ethanol and centrifuge for 10 minutes as above. Discard the supernatant. Centrifuge again for 1 minute. Remove any remaining ethanol with a 10-ul pipette.

[0137] (3) The lower precipitate was placed in an oven at 60°C for 10 min to allow the residual ethanol to evaporate;

[0138] (4) Dissolve the precipitate in 15-20 μL of sterile water per tube. (The plasmid should be transformed immediately after linearization and enzyme digestion. If not, it should be frozen at -20°C.)

[0139] 1.3 Electroporation of linearized plasmid

[0140] (1) Take 80 μL of GS115 competent cells and add 10 μL of linearized pPIC9k recombinant plasmid (about 5-10 μg).

[0141] (2) Place on ice for 5 minutes.

[0142] (3) Perform electric shock according to the preset parameters of the Pichia pastoris in the electroporation instrument (voltage: 1.5kV, capacitance: 25uF, resistance 200-400 ohms, electric shock time: 10 ms).

[0143] (4) Immediately after the electric shock, 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.

[0144] (5) Incubate at 30°C for 1-2 hours.

[0145] (6) Take 300 μl of transformed cells and spread them on YPD plates containing different concentrations of G418 (0.5 mg / ml, 1 mg / ml, 2 mg / ml) for selection of resistance. Incubate at 30°C for 2-4 days until colonies appear. (If the plasmid concentration is controlled, approximately 300-400 single colonies will grow from 300 μL of plating.)

[0146] 1.4 Screening of positive clones and PCR identification

[0147] According to the growth of strains on resistance plates with different concentrations, single clones with better growth on resistance plates were selected for PCR identification, with higher concentration resistance plates being preferred.

[0148] (1) Preparation of PCR reaction system

[0149] Table 1

[0150]

[0151] (2) Set up blank control (no plasmid template) and positive control (add 0.5 μL) plasmid for colony PCR;

[0152] Table 2

[0153]

[0154] (3) PCR products were detected by agarose gel electrophoresis.

[0155] 1.5 Transformation, expression, screening and identification of positive clones

[0156] (1) Select clones that are positive for colony PCR identification and preserve them;

[0157] (2) Inoculate the positive strain into 25 mL of BMGY liquid medium in a 250 mL baffled shake flask and culture at 30°C and 200 rpm for 24 h;

[0158] (3) Collect the cells by centrifugation and inoculate them into a 250 ml baffled shake flask containing 25 ml BMMY medium at an initial OD of 1.0. Culture at 28°C and 220 rpm for 72-120 h.

[0159] (4) Add 1% methanol every 24 hours. Take 1 ml of bacterial solution samples at different time points, place them in 1.5 ml EP tubes, and centrifuge to collect the supernatant and bacterial cells. Analyze the expression level of the target protein and the optimal harvest time of the bacterial solution. The time points are generally: 0, 24, 48, 72, 96, and 120 hours.

[0160] (5) For secretory expression, separate the supernatant of the sample; for intracellular expression, separate the bacterial pellet of the sample. SDS-PAGE, Western-Blot and activity assays can be used to detect and identify the expression of the recombinant protein.

[0161] 2. Expression and Purification

[0162] 2.1 Large-scale protein expression and concentration

[0163] (1) Take glycerol bacteria and activate the strain on YPD plate culture medium.

[0164] (2) Inoculate three single colonies into 50 ml of first-level YPG seed medium for overnight culture. After the OD600 reaches 6-10, transfer to 250 ml of second-level YPG seed medium with an inoculation volume of 0.5%. After overnight culture, the OD600 reaches 6-10. Microscopic examination shows no contamination by other bacteria and is used for inoculation.

[0165] (3) Prepare 2.5 L of BSM basal culture medium in a fermenter. After sterilization preparation is completed, inoculate the secondary seed culture medium into the fermenter. The initial conditions are 28 °C, 300 rpm, pH = 5.0, and ventilation volume 2.0 L / min.

[0166] (4) After culturing for 20-25 hours, when OD600 reaches 70-80 and the glycerol in the basal culture medium is exhausted, add glycerol feed medium at 45 ml / h and continue culturing.

[0167] (5) When OD600 reaches 150-200, stop adding glycerol and wait for the dissolved oxygen to rise rapidly. After 30 minutes, add methanol for induction and gradually increase the methanol addition rate to 28 ml / h. The dissolved oxygen is controlled to be greater than 20%.

[0168] (6) After 72 h of induction, stop the fermentation and collect the supernatant by centrifugation.

[0169] (7) After SDS-PAGE confirmation of normal expression, purification was performed.

[0170] 2.2 Purification of Zhongyan Universal Nuclease

[0171] (1) Adjust the conductivity of the fermentation supernatant to 20 mS / cm with 10 M NaH2PO4 (pH 6.0);

[0172] (2) Prepare a 5 mL pre-packed SP column;

[0173] (3) Load the supernatant protein solution at a flow rate of 5 mL / min;

[0174] (4) Wash the column with 20 M NaH2PO4, 200 mM NaCl (pH 6.0) until the effluent is protein-free (the G250 detection solution does not change color);

[0175] (5) Elute with 20 M NaH2PO4, 1 M NaCl (pH 6.0) and collect the blue-colored region detected by G250;

[0176] (6) Wash the packing with 3 column volumes of deionized water and seal the column with 20% ethanol;

[0177] (7) The eluate was ultrafiltered through a 10 kDa ultrafiltration tube to 25 mM Tris-HCl pH 7.0, 5 mM MgCl2, 400 mM NaCl, 50% Glycerol, 0.05% Tween-20 and concentrated to 1-2 mL. The concentrate was then subjected to SDS-PAGE analysis.

[0178] 3. Activity Assay of Zhongyan Universal Nuclease

[0179] 3.1 Enzyme activity determination method

[0180] 3.1.1 Reaction Buffer A

[0181] Solution formula: 25 mM Tris, 2.5 mM MgCl2, 150 mM NaCl, pH 7.6.

[0182] Preparation: Weigh 1.2414g Tris, 3.5064g NaCl, and 0.2033g MgCl2 on a microbalance. Dissolve in ultrapure water. Adjust pH to 7.6 with hydrochloric acid. Bring to 400ml with ultrapure water. Filter through a 0.22μM filter and store at 4°C.

[0183] 3.1.2 B calf thymus DNA

[0184] Solution concentration: 0.5 mg / ml calf thymus DNA.

[0185] Preparation steps: Dissolve 50 mg of calf thymus DNA powder in 10 ml of Buffer A to make a 5 mg / ml calf thymus DNA stock solution. For long-term storage, store at -20°C. Before activity testing, take 1 ml of the stock solution and add 9 ml of Buffer A to dilute it to 0.5 mg / ml calf thymus DNA. Pre-cool on ice until use. It can be stored temporarily at 4°C.

[0186] 3.1.3 C-terminated reaction solution

[0187] Solution concentration: 4% perchloric acid solution

[0188] Preparation steps: Dilute 11.26 ml of 70-72% perchloric acid with 188.74 ml of ultrapure water, mix thoroughly, and store in a brown bottle at 4°C in the dark. Pre-cool on ice before activity measurement.

[0189] 3.2 Reaction steps

[0190] 3.2.1 Dilution of test samples

[0191] Input amount: 1U (the input amount can be fine-tuned according to the slope change)

[0192] Dilution step: Use reaction buffer A to dilute the sample to be tested. Add 125 μl of enzyme solution to the reaction system. Based on the input amount of 1 U, the sample to be tested needs to be diluted to 0.008 U / μl.

[0193] 3.2.2 Reaction system

[0194] Table 3 Reaction system

[0195]

[0196] 3.2.3 Reaction conditions

[0197] Add calf thymus DNA to a 5ml centrifuge tube, and add the enzyme solution last. Complete the reaction system on ice, mix thoroughly by inverting, and immediately incubate in a 37°C water bath for 1 hour. While in the water bath, accurately time the reaction. At 15, 30, 45, and 60 minutes, remove 0.5ml of incubation solution from the centrifuge tube and transfer it to a 1.5ml centrifuge tube containing 0.5ml of ice-cold 4% perchloric acid. Vortex mix for 10 seconds and ice bath for 30 minutes. Keep the same operation for the four time periods. Then, centrifuge at 15,000 rpm and 4°C for 6 minutes, and collect 800μl of supernatant for detection.

[0198] 3.2.4 Activity determination

[0199] Select the cuvette mode and A260 wavelength on the enzyme reader and perform OD determination. Measure the blank control first and then the sample to be tested. Each tube of the sample to be tested needs to be read twice. The two OD 260nmIf the values ​​are consistent, continue to test the next tube of sample.

[0200] 3.3 Calculation of enzyme activity

[0201] Calculation formula:

[0202] Where: A = absorbance of the measured solution at time t;

[0203] V = total volume of incubation mixture;

[0204] t = incubation time of the measurement solution (min);

[0205] 30=defined 30min;

[0206] 2 = dilution factor for perchloric acid denaturation;

[0207] F = dilution factor of the sample;

[0208] V = volume of diluted sample added to the reaction system (ml).

[0209] Take time as the horizontal axis and remove the OD of the blank control. 260nm Use the vertical coordinate to fit the 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.

[0210] 4. Definition of enzyme activity

[0211] One unit of enzyme activity (U) is defined as the amount of enzyme that results in ΔA260 = 1.0 within 30 minutes in 25 mM Tris-HCl pH 7.6 (25°C), 2.5 mM MgCl2, 150 mM NaCl, and 50 μg / ml calf thymus DNA at 37°C.

[0212] 5. Determination of the Protein Concentration of the Medium Salt Universal Nuclease

[0213] 5.1 Detection Method

[0214] (1) Open the microplate reader, software, and select protein A280;

[0215] (2) Select 1 Abs at 1cm = 1mg / mL for sample type, select “background” for well type, test 4 replicates, and leave all other settings as default;

[0216] (3) Prepare dilution buffer (1 mL): 2× dilution buffer (100 mM Tris-HCl, pH 8.0, 500 mM NaCl, 4 mM CaCl2) and purified water in a volume ratio of 0.5 mL:0.5 mL and mix well.

[0217] (4) Dilution of the test sample: dilute the test sample with dilution buffer by 2 times, 4 times and 8 times in sequence, and mix the enzyme solution thoroughly;

[0218] (5) Take 2 μL of dilution buffer as a blank control, repeat 4 times, and click test;

[0219] (6) Sample testing: Take 2 μL of the original solution and 2-fold, 4-fold, and 8-fold dilutions, perform 4 repeated tests, and output the 4 test data of C0, C2, C4, and C8.

[0220] 5.2 Data Processing

[0221]

[0222] Where: C0, C2, C4, and C8 are the average values ​​of four replicate tests of the original solution, 2-fold, 4-fold, and 8-fold dilutions of the test sample, respectively; the extinction coefficient (0.1%) is the absorbance value of 1 mg / mL target protein at 280 nm (with an absorption cell thickness of 1 cm), which can be calculated based on the protein sequence.

[0223] 6. Protein Purity Analysis Method

[0224] 6.1 SDS-PAGE detection method

[0225] (1) Preparation of test samples: Dilute the test samples to 0.2 mg / mL and 0.02 mg / mL with the storage buffer and mix thoroughly;

[0226] (2) Take 10 μL of enzyme dilution solution, add an equal volume of 2× SDS PAGE loading buffer, mix thoroughly by pipetting, and then treat at 100°C for 10 min;

[0227] (3) Take 12% precast protein plus gel and install it on the electrophoresis instrument, add SDS-PAGE electrophoresis buffer, and add protein marker and sample to be tested;

[0228] (4) Constant voltage 150V, run the gel for 30-40 minutes;

[0229] (5) Take out the protein gel and stain and decolorize it using a staining and decolorizing instrument;

[0230] (6) Take out the protein gel and place it on a white plate, then put it into the gel imaging analysis system for photography.

[0231] 6.2 Data Processing

[0232] Purity analysis was performed using Quantity One, and gel images were processed using image processing software, retaining the complete gel image in tif format. The purity was determined to be ≥95%.

[0233] 7. Comparative Digestion Experiments of Wild-Type Medium Salt Universal Nuclease and Mutants

[0234] 7.1 Sample preparation

[0235] After AAV virus packaging, the culture medium supernatant was repeatedly frozen and thawed three times with liquid nitrogen to lyse the virus (pH 7-10 verified by pH test paper).

[0236] 7.2 Enzymatic digestion with universal nuclease

[0237] Table 4

[0238]

[0239] After digestion, centrifuge at 16,000 rpm for 10 min, collect the supernatant, and measure the HCD value of 293 cells.

[0240] 8. Results Analysis

[0241] 8.1 Protein Purification Results of Wild-Type Zhongyanuclease and Mutants

[0242] SDS-PAGE results showed that ( Figure 4 After expression and purification in Pichia pastoris, all nine mutants were successfully purified with minimal variability in purity. The amount of impurity bands in each target protein was less than 5% of the target protein, indicating that the protein purity of the wild-type mid-salt universal nuclease and the mutants was greater than 95%, allowing the samples to be used for specific activity determination.

[0243] 8.2 Specific activity results of wild-type mitochondria nuclease and mutants

[0244] Specific activity results showed that mutants 1-6 of the Zhongyan universal nuclease exhibited significantly increased specific activity, while mutants 7-9 showed no significant difference in specific activity compared to the wild type (Table 5). Furthermore, digestion assay results further demonstrated that, under the same activity input conditions, mutants 1-6 digested more DNA, demonstrating higher DNA digestion capacity (Table 6).

[0245] Table 5 Comparison of results between wild-type MSAN and mutants

[0246]

[0247] Table 6 Enzymatic hydrolysis results of wild-type Zhongyan universal nuclease and mutants

[0248]

[0249] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A universal nuclease mutant, characterized in that: The universal nuclease mutant is obtained by performing the following mutations based on the sequence shown in SEQ ID NO: 1: (1) S22E and S115R; or (2) S22R and S115R; or (3) S22K and S115R; or (4) S22E, T101V and S115E; or (5) S22E, T101V, S115R and G199del.

2. A nucleic acid molecule, characterized in that The invention comprises a nucleotide sequence encoding the full-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 constructed recombinant plasmid into the bacterial strain and culture it, then 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 and concentration, followed by purification, yield the mutant of the universal nuclease.

7. Use of the totipotent nuclease mutant 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.

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