A medium-temperature nuclease mutant protein and its preparation method and application

Abstract

The present invention discloses a mesophilic endonuclease mutant protein, its preparation method, and application. Through extensive and in-depth research and extensive screening, the present invention unexpectedly obtained, for the first time, a mutant protein with significantly improved KmAgo enzyme activity. The mutant protein is based on the mesophilic nuclease Argonaute (KmAgo, wild-type sequence shown in SEQ ID NO. 1) from Kurthia massiliensis, or its active fragments, variants, and derivatives. Compared with the wild-type, the mutant protein of the present invention has significantly improved enzymatic activity by up to 7 times; it also maintains excellent thermal stability; and has potential applications in nucleic acid detection technology and the development of gene editing tools.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and relates to a medium-temperature nuclease mutant protein and a preparation method and application thereof. Background Art

[0002] Argonaute (Ago) proteins, as key components of the RNA-induced silencing complex (RISC), play a crucial role in the cleavage and recruitment of small RNA molecules. They are a family of proteins that has garnered considerable attention following the discovery of RNA interference (RNAi). Argonaute proteins are widely found in both eukaryotes and prokaryotes, and their functions differ in these two species. In eukaryotes, Ago proteins primarily participate in the RNAi process and play a crucial role in the formation of the RISC complex. In prokaryotes, Ago proteins primarily serve a host defense function, participating in bacterial defense mechanisms against foreign gene invasion and in host replication and damage repair.

[0003] Prokaryotic Argonaute proteins (pAgos) are more functionally and structurally diverse than eAgos, but their physiological functions have long remained elusive. Early studies focused on pAgos derived from thermophilic organisms. With the exception of MpAgo, which prefers to cleave single-stranded DNA (ssDNA) and RNA targets using RNA guides (gDNA) with 5'-terminal hydroxylation (5'OH), all other thermophilic pAgos prefer to cleave ssDNA and / or RNA targets using gDNA with 5'-terminal phosphorylation (5'P). Thermophilic pAgos exhibit only low levels of gDNA-guided cleavage activity under mesophilic conditions, limiting the development of thermophilic pAgo-based applications. Recent research has focused on pAgos derived from mesophilic organisms, hoping to identify pAgos that can effectively cleave target DNA and / or RNA under mesophilic conditions. In 2020, Ma Lixin's team discovered the mesophilic nuclease Argonaute (KmAgo) protein from kurthia massiliensis, which has the ability to effectively cut target DNA / RNA, but its wild-type cutting ability is weak and its activity is poor.

[0004] Similar to the currently commonly used CRISPR-Cas9 and CRISPR-Cas12a / 13a, pAgos has been proposed as a next-generation genome editing tool. However, the low endonuclease activity of the KmAgo protein at mesophilic temperatures significantly limits its application in gene editing and nucleic acid detection.

[0005] Therefore, it is urgent to develop a method to obtain KmAgo mutants with high catalytic activity. Modification of KmAgo catalytic activity will further reveal its structure-function relationship. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a mesophilic endonuclease mutant protein and its preparation method and application, which has improved enzyme activity and thermal stability.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A mesophilic endonuclease mutant protein KmAgo or an active fragment, variant form, or derivative protein thereof, wherein the mesophilic endonuclease is derived from the mesophilic prokaryotic organism Kurthia massiliensis, and the mutant protein KmAgo has a core amino acid mutation at one or more sites selected from the following:

[0009] (1)W599S;

[0010] (2)H602E;

[0011] (3) E12L;

[0012] (4)E621I;

[0013] (5)Y130D;

[0014] (6)E614G;

[0015] (7) C68L;

[0016] (8) C480L;

[0017] (9)R291P;

[0018] (10)A654I;

[0019] (11)S715V;

[0020] (12)C201D;

[0021] (13)S162D;

[0022] (14)W445I;

[0023] (15)N541A,

[0024] The numbering of the mutation sites is based on the sequence shown in SEQ ID NO. 1. The KmAgo mutant protein has the same or substantially the same sequence as the sequence shown in SEQ ID NO. 1 except for one or more amino acid mutations.

[0025] As one of the preferred technical solutions, the mutant protein KmAgo has a core amino acid mutation at one or more sites selected from the following:

[0026] (1)W599S;

[0027] (2)H602E;

[0028] (3) E12L;

[0029] (4)E621I;

[0030] (5)Y130D;

[0031] (6)E614G;

[0032] (7) C68L;

[0033] (8) C480L;

[0034] (9)R291P;

[0035] (10)A654I;

[0036] The numbering of the mutation sites is based on the sequence shown in SEQ ID NO.1.

[0037] As one of the preferred technical solutions, the medium temperature is 10-80°C, more preferably 32-44°C, and even more preferably 37°C.

[0038] As one of the preferred technical solutions, the ratio of the enzyme activity Q1 of the KmAgo mutant protein to the enzyme activity Q0 of the wild type: Q1 / Q0 is ≥3, preferably ≥4, more preferably ≥5, and most preferably ≥7.

[0039] As one of the preferred technical solutions, the mutant protein KmAgo has core amino acid mutations selected from the following sites:

[0040] (1)W599S;

[0041] (2)H602E;

[0042] (3) E12L;

[0043] (4)E621I;

[0044] (5)Y130D;

[0045] (6) E12L and W445I and E614G and Y130D and S715V and E621I;

[0046] (7) W599S and E12L and W445I and E614G and Y130D and H602E and S715V and E621I and A654I and S162D and C201D and R291P;

[0047] (8)W599S and E12L and W445I and E614G and Y130D and H602E and N541A and S715V and

[0048] E621I and A654I and S162D and C201D and R291P;

[0049] (9) W599S and E12L and W445I and E614G and Y130D and S715V and E621I and C201D;

[0050] (10) W599S and E12L and W445I and E614G and Y130D and H602E and S715V and E621I and A654I and S162D and C201D;

[0051] (11) W599S and W445I and E614G and Y130D and S715V and E621I and A654I and S162D;

[0052] (12)W599S and E12L and W445I and E614G and Y130D and N541A and S715V and E621I and

[0053] A654I and S162D and C201D and R291P;

[0054] The numbering of the mutation sites is based on SEQ ID NO.1.

[0055] As one of the further preferred technical solutions, the amino acid sequence of the mutant protein KmAgo is selected from:

[0056] (1) the amino acid sequence shown in SEQ ID NO. 1, with the W at position 599 mutated to S (W599S);

[0057] (2) the amino acid sequence shown in SEQ ID NO. 1, with H at position 602 mutated to E (H602E);

[0058] (3) the amino acid sequence shown in SEQ ID NO. 1, with E at position 12 mutated to L (E12L);

[0059] (4) the amino acid sequence shown in SEQ ID NO. 1, wherein E at position 621 is mutated to I (E621I);

[0060] (5) the amino acid sequence shown in SEQ ID NO. 1, wherein Y at position 130 is mutated to D (Y130D);

[0061] (6) (S4) The amino acid sequence shown in SEQ ID NO. 1, wherein E at position 12 is mutated to L (E12L), W at position 445 is mutated to I (W445I), E at position 614 is mutated to G (E614G), Y at position 130 is mutated to D (Y130D), S at position 715 is mutated to V (S715V), and

[0062] E mutated to I (E621I);

[0063] (7) (S12) The amino acid sequence shown in SEQ ID NO. 1, wherein W at position 599 is mutated to S (W599S), E at position 12 is mutated to L (E12L), W at position 445 is mutated to I (W445I), and

[0064] The E at position 14 was mutated to G (E614G), and the Y at position 130 was mutated to D (Y130D), and the

[0065] The S at position 61 was mutated to V (S715V), and the E at position 621 was mutated to I (E621I), and the H at position 602 was mutated to E (H602E), and the A at position 654 was mutated to I (A654I), and the S at position 162 was mutated to D (S162D), and the C at position 201 was mutated to D (C201D), and the R at position 291 was mutated to P

[0066] (R291P);

[0067] (8) (S15) The amino acid sequence shown in SEQ ID NO. 1, wherein W at position 599 is mutated to S (W599S), E at position 12 is mutated to L (E12L), W at position 445 is mutated to I (W445I), and

[0068] The E at position 14 was mutated to G (E614G), and the Y at position 130 was mutated to D (Y130D), and the

[0069] The S at position 61 was mutated to V (S715V), and the E at position 621 was mutated to I (E621I), and the H at position 602 was mutated to E (H602E), and the A at position 654 was mutated to I (A654I), and the S at position 162 was mutated to D (S162D), and the C at position 201 was mutated to D (C201D), and the R at position 291 was mutated to P

[0070] (R291P), and the N at position 541 was mutated to A (N541A);

[0071] (9)(S10) The amino acid sequence shown in SEQ ID NO. 1, wherein W at position 599 is mutated to S (W599S), E at position 12 is mutated to L (E12L), W at position 445 is mutated to I (W445I), and

[0072] The E at position 14 was mutated to G (E614G), and the Y at position 130 was mutated to D (Y130D), and the

[0073] The S at position 621 was mutated to V (S715V), the E at position 621 was mutated to I (E621I), and the C at position 201 was mutated to D (C201D);

[0074] (10) (S11) The amino acid sequence shown in SEQ ID NO. 1, wherein W at position 599 is mutated to S (W599S), E at position 12 is mutated to L (E12L), W at position 445 is mutated to I (W445I), and

[0075] The E at position 14 was mutated to G (E614G), and the Y at position 130 was mutated to D (Y130D), and the

[0076] The S at position 61 was mutated to V (S715V), and the E at position 621 was mutated to I (E621I), and the H at position 602 was mutated to E (H602E), and the A at position 654 was mutated to I (A654I), and the S at position 162 was mutated to D (S162D), and the C at position 201 was mutated to D (C201D);

[0077] (11) (S8) the amino acid sequence shown in SEQ ID NO. 1, wherein W at position 599 is mutated to S (W599S), W at position 445 is mutated to I (W445I), E at position 614 is mutated to G (E614G), Y at position 130 is mutated to D (Y130D), S at position 715 is mutated to V (S715V), E at position 621 is mutated to I (E621I), A at position 654 is mutated to I (A654I), and S at position 162 is mutated to D (S162D);

[0078] (12)(S20) The amino acid sequence shown in SEQ ID NO. 1, wherein W at position 599 is mutated to S (W599S), E at position 12 is mutated to L (E12L), W at position 445 is mutated to I (W445I), and

[0079] E at position 14 mutated to G (E614G), and Y at position 130 mutated to D (Y130D), and S at position 715 mutated to V (S715V), and E at position 621 mutated to I (E621I), and A at position 654 mutated to I (A654I), and S at position 162 mutated to D (S162D), and C at position 201 mutated to D (C201D), and R at position 291 mutated to P (R291P), and N at position 541 mutated to A

[0080] (N541A).

[0081] As one of the further preferred technical solutions, the mutant protein KmAgo has a core amino acid mutation selected from the following sites:

[0082] (S4) E12L and W445I and E614G and Y130D and S715V and E621I;

[0083] (S12) W599S and E12L and W445I and E614G and Y130D and H602E and S715V and E621I and A654I and S162D and C201D and R291P;

[0084] (S15) W599S and E12L and W445I and E614G and Y130D and H602E and N541A and S715V and E621I and A654I and S162D and C201D and R291P;

[0085] (S10) W599S and E12L and W445I and E614G and Y130D and S715V and E621I and C201D;

[0086] (S11) W599S and E12L and W445I and E614G and Y130D and H602E and S715V and E621I and A654I and S162D and C201D;

[0087] (S8) W599S and W445I and E614G and Y130D and S715V and E621I and A654I and S162D;

[0088] (S20) W599S and E12L and W445I and E614G and Y130D and N541A and S715V and E621I and A654I and S162D and C201D and R291P;

[0089] The numbering of the mutation sites is based on SEQ ID NO.1.

[0090] As one of the further preferred technical solutions, the ratio of the enzyme activity Q1 of the KmAgo mutant protein to the enzyme activity Q0 of the wild type: the Q1 / Q0 ratio is 3.0-5.0, preferably 4.1-5.0.

[0091] As one of the preferred technical solutions, the active fragments, variant forms, and derivative proteins include:

[0092] The amino acid sequence formed by the aforementioned single-site mutation in the sequence shown in SEQ ID NO. 1 further has one or more (e.g., generally 1-30, preferably 1-10, more preferably 1-6, even more preferably 1-3, and most preferably 1) amino acid residues deleted, inserted and / or substituted, and still has a directed single-stranded DNA cleavage activity (cA1), which is significantly higher than the corresponding activity (cA0) of the wild-type KmAgo enzyme shown in SEQ ID NO. 1, wherein the significantly higher value is (cA1-cA0) / cA0 ≥ 10%-800%, for example ≥ 15%, ≥ 20%, ≥ 40%, ≥ 50%, ≥ 100%, ≥ 200%, or ≥ 600% or more;

[0093] Protein modifications (usually without altering the primary structure) include: chemical derivatization of proteins in vivo or in vitro, such as acetylation or carboxylation; glycosylation; sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine); proteins modified to increase their resistance to proteolysis or optimize their solubility;

[0094] Fusion proteins or conjugates formed by the aforementioned mutant proteins or active fragments thereof and other proteins or markers;

[0095] Contains one or more other mutations to further enhance the enzymatic activity of the KmAgo mutant protein.

[0096] 2. An isolated polynucleotide encoding the aforementioned mutant protein KmAgo or its active fragment, variant form, or derivative protein.

[0097] 3. A vector comprising the isolated polynucleotide.

[0098] As one of the preferred technical solutions, the vector is a recombinant expression vector, which refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0099] As one of the preferred technical solutions, the polynucleotide can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the nucleotide sequence encoding the KmAgo mutant protein in the embodiments of the present invention or a degenerate variant.

[0100] 4. A host cell containing the aforementioned vector, or a host cell containing the aforementioned isolated polynucleotide in its nucleic acid.

[0101] As one of the preferred technical solutions, the host cells include cells derived from the following microorganisms:

[0102] Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipites, Candida shehatae, Candida tropicalis, and Escherichia coli.

[0103] The mutant protein of the present invention can be produced by conventional recombinant transformation methods in the art, and the mutant protein can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, high-pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods. The host cell of the present invention can express the KmAgo protein mutant protein.

[0104] As one of the further preferred technical solutions, the host cell is cultured under conditions suitable for expression, thereby expressing the KmAgo mutant protein; and

[0105] The KmAgo mutant protein is isolated.

[0106] The obtained mutant protein can be optionally purified to obtain a purer mutant protein product.

[0107] As one of the further preferred technical solutions, the conditions suitable for expression include conventional techniques in the art, and the purification techniques include nickel column purification, ion exchange chromatography, and the like.

[0108] 5. A method for preparing the aforementioned mesophilic nuclease mutant protein KmAgo or its active fragment, variant form, or derivative protein, comprising culturing the aforementioned host cell to express the mutant protein or its active fragment, variant form, or derivative protein.

[0109] 6. Application of the aforementioned mesophilic endonuclease mutant protein KmAgo or its active fragments, variant forms, and derivative proteins in nucleic acid detection and gene editing.

[0110] 7. An in vitro DNA cleavage system based on the aforementioned mutant protein KmAgo or its active fragments, variant forms, or derivatives, comprising:

[0111] (A) a guide DNA (gDNA) that binds to a predetermined target site; and

[0112] (B) programmable nuclease Argonaute (KmAgo), wherein the programmable nuclease is the aforementioned KmAgo mutant protein or its active fragment, variant form, or derivative protein; and

[0113] (C) Target DNA, which has a nucleotide sequence that is largely complementary to the guide DNA.

[0114] As one of the preferred technical solutions, the length of the guide DNA is 21 to 45 nt, more preferably 15 to 21 nt, and even more preferably 16 to 18 nt.

[0115] As one of the preferred technical solutions, the guide DNA and the target nucleic acid have reverse complementary fragments.

[0116] As one of the preferred technical solutions, the target DNA is a target nucleic acid carrying a reporter molecule, and the reporter molecule is a nucleic acid molecule carrying a fluorescent group and a quenching group respectively.

[0117] As one of the further preferred technical solutions, the fluorescent group includes: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof, and FAM is further preferred.

[0118] As one of the further preferred technical solutions, the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof, and is further preferably BHQ.

[0119] 8. A method for in vitro DNA cleavage based on the above system, the specific steps are as follows:

[0120] S1. Formation of KmAgo-guide complex based on KmAgo and gDNA;

[0121] S2. The KmAgo-guide complex is brought into contact with the target DNA, and the KmAgo-guide complex cuts the target DNA at a specific site.

[0122] As one of the preferred technical solutions, the nuclease activity of the KmAgo and KmAgo-guide complex requires the presence of at least one selected from Mn 2+ , Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ ,Co 2+ , Zn 2+ and Ni 2+ Or any combination of divalent metal ions therein.

[0123] As one of the further preferred technical solutions, the cations of KmAgo and KmAgo-guide complex are Mn 2 +

[0124] As one of the further preferred technical solutions, the concentration of the divalent metal ions is 10 μM to 3 mM, more preferably 10 μM to 200 mM, and even more preferably 20 μM.

[0125] The beneficial effects of the present invention are:

[0126] Through extensive and intensive research and extensive screening, the present invention unexpectedly obtained, for the first time, mutant proteins with significantly improved KmAgo enzyme activity. These proteins are based on a mutant protein of the mesophilic nuclease Argonaute from Kurthia massiliensis (KmAgo, wild-type sequence shown in SEQ ID NO. 1), or an active fragment, variant form, or derivative thereof. The KmAgo mutant protein derivative is identical or substantially identical to the sequence shown in SEQ ID NO. 1, except for one or more amino acid mutations.

[0127] The present invention uses machine learning to transform target scoring, supplemented by the crystal structure of the enzyme or the structural co-evolution analysis of homology modeling to select target sites, and obtains mutants with improved enzyme catalytic activity by screening. In the present invention, a total of 15 mutation hotspots were finally selected, including sites W599, H602, E621, Y130, E12, E614, C68, C480, R291, A654, S715, C201, S162, W445 and N541. Further, the inventors screened these candidate sites and used fluorescence pure enzyme screening. The screening results showed that 10 single-point mutants, W599S, H602E, E621I, Y130D, E12L, E614G, C68L, C480L, R291P and A654I, can improve the enzyme catalytic activity of KmAgo. Furthermore, based on the activity data of mutants with improved enzyme catalytic activity screened out by mutation, the inventors obtained multi-point combination mutants with further improved activity through machine learning: S4, S12, S15, S10, S11, S8, and S20.

[0128] The KmAgo mutant protein of the present invention exhibits nuclease activity at temperatures between 10 and 80°C, preferably at a mesophilic temperature of 32 to 44°C, and more preferably at 37°C. The KmAgo mutant protein of the present invention exhibits significantly enhanced enzymatic activity compared to the wild-type protein and exhibits improved high-temperature thermal stability.

[0129] KmAgo mutant proteins significantly enhance the activity of directed cleavage of single-stranded DNA targets, with increases of up to 300% to 700%, thus providing assistance for pathogen detection, genotyping, and disease progression monitoring. The method of the present invention enables site-specific modification of extracellular genetic material and can therefore be effectively applied in biotechnology fields such as nucleic acid detection and gene editing. This enhances the protein's potential for application in nucleic acid detection technology and also lays the foundation for the development of new genetic manipulation tools.

[0130] The main advantages of the present invention are as follows:

[0131] (1) The mutants screened by the present invention have significantly improved enzyme catalytic activity compared to the wild type, with the maximum being up to 7 times;

[0132] (2) The thermal stability of the mutant of the present invention is not adversely affected by the mutation and maintains very good thermal stability;

[0133] (3) The mutants of the present invention have application potential in nucleic acid detection technology and the development of gene editing tool technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration.

[0135] Figure 1 Figure 2 is the SDS-PAGE electrophoresis diagram of the wild-type KmAgo and mutants, where A is the electrophoresis diagram of the wild-type KmAgo, B is the electrophoresis diagram of the single-site mutant, and C is the electrophoresis diagram of the multi-site mutant. M: Marker; Lane 1: Flow-through; Lanes 2-9: Purified samples;

[0136] Figure 2 The gel images show target shearing nucleic acid of wild-type KmAgo and mutants;

[0137] Figure 3 The substrate cleavage efficiency and time curves of wild-type KmAgo and mutants;

[0138] Figure 4 Comparison of the cleavage activity of wild-type KmAgo and mutants. DETAILED DESCRIPTION

[0139] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0140] Example 1 Construction and synthesis of wild-type KmAgo plasmid vector and selection of mutation sites

[0141] The wild-type KmAgo nucleotide sequence was searched in the NCBI database and sent to Sangon Biotech for synthesis. After codon optimization, the pET28a(+) vector was selected, and the NdeI and XhoI restriction sites were selected. The synthesized plasmid product was transformed into competent Escherichia coli Ecoli-BL21(DE3) cells. Plates were spread (containing 50 μg / ml kanamycin) and incubated inverted overnight at 37°C. The next day, a single colony was picked for sequencing to verify the correctness of the plasmid sequence. After correct alignment, the plasmid was extracted. Plasmid extraction procedures were followed according to the instructions of the Axygen Plasmid Extraction Kit. Plasmid concentration was determined using a Nano-300.

[0142] The wild-type (WT) amino acid sequence of the Argonaute protein (KmAgo) from the mesophilic prokaryotic bacterium Kurthia massiliensis involved in the present invention is shown in SEQ ID NO.1.

[0143] The existing unsupervised model was used to score the KmAgo modified targets with the help of machine learning, and the target sites were selected by structural co-evolution analysis supplemented by the crystal structure of the enzyme or homology modeling. Amino acid residues that met both requirements were selected as target sites (see Table 1).

[0144] Table 1 Target sites and their distances from the catalytic center

[0145]

[0146] Example 2 Expression of single-site mutant proteins and determination of cleavage activity

[0147] The pET28a-KmAgo plasmid was transformed into Escherichia coli BL21(DE3). A single colony was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured in a shaker at 37°C and 220 rpm. When the OD600 of the cells reached 0.6-0.8, 0.5 mM IPTG was added and the cells were shaken overnight at 18°C. The cells were harvested by centrifugation at 4000 rpm for 15-20 minutes, washed with Buffer A (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM imidazole), resuspended in Buffer A, and disrupted by sonication. The supernatant was collected by centrifugation at 13,300 rpm for 45 minutes. The supernatant was filtered and purified with Ni-NTA. Wash the column with 10mM, 20mM and 250mM imidazole for 1 column volume each, collect the eluted fractions containing the highly purified target protein, and replace the buffer with Buffer B (20mMTris-HCl pH 7.5, 500mM NaCl) by ultrafiltration. Collect the purified protein and identify its purity using SDS-polyacrylamide gel. Figure 1 The protein was divided into small portions, quick-frozen in liquid nitrogen, and stored at -80°C.

[0148] Design a 16nt gDNA sequence and a 30nt target DNA sequence and send them to the company for synthesis. The gDNA and target DNA sequences are shown in Table 2.

[0149] Table 2. Sequences of gDNA and target DNA

[0150]

[0151] Prepare reaction buffer (containing 15mM Tris-HCl pH 8.8, 250mM NaCl), add MnCl2 to a final concentration of 20μM, 100nM KmAgo, 1μM synthetic gDNA and 1μM complementary single-stranded DNA target nucleic acid to the reaction buffer, react at 490nm excitation wavelength and 550nm emission wavelength at 37℃ for 120min, measure the fluorescence value at the end point, and perform three replicates for each experiment. Figure 3 shown.

[0152] Prepare reaction buffer (containing 15mM Tris-HCl pH 8.8, 250mM NaCl), add 20μM MnCl2, 100nM KmAgo, 1μM synthetic gDNA and 1μM complementary single-stranded DNA target nucleic acid to the reaction buffer, incubate at 37°C for 30min, take 6-10μL sample, add loading buffer (containing 95% (deionized) formamide, 0.5mmol / L EDTA, 0.025% bromophenol blue, and 0.025% xylene cyanol) in a 1:1 ratio (volume ratio), and perform electrophoresis detection under 16% nucleic acid Urea-PAGE, as shown in Figure 2. Figure 2 shown.

[0153] result

[0154] The enzyme activity of some mutant proteins with single-point mutations remains basically unchanged or slightly decreases, such as S715V, C201D, S162D, W445I, and N541A.

[0155] The enzyme activity improvement data are shown in Table 3. After screening, 10 single-point mutants with improved enzyme activity were obtained, including W599S, H602E, E621I, Y130D, E12L, E614G, C68L, C480L, R291P and A654I.

[0156] Table 3. Enzyme activity improvement

[0157]

[0158] The five preferred single point mutants are shown in Table 4.

[0159] Table 4. Five preferred single-point mutants

[0160]

[0161]

[0162] Subsequently, a supervised model based on machine learning was used to perform multi-site combination of effective sites, and the multi-site combination mutants with further improved activity were obtained: (S4) E12L and W445I and E614G and Y130D and S715V and E621I; (S12) W599S and E12L and W445I and E614G and Y130D and H602E and S715V and E621I and A654I and S162D and C201D and R291P; (S15) W599 and E12L and W445I and E614G and Y130D and H602E and N541A and S715V and E621I and A654I and S162D and C201D and R291P; 10) W599S and E12L and W445I and E614G and Y130D and S715V and E621I and C201D; (S11) W599 and E12L and W445I and E614G and Y130D and H602E and S715V and E621I and A654I and S162D and C201D; (S8) W599S and W445I and E614G and Y130D and S715V and E621I and A654I and S162D and (S20)W599S and E12L and W445I and E614G and Y130D and N541A and S715V and E621I and A654I and S162D and C201D and R291P.

[0163] The enzyme activity data were then measured using the above enzyme activity detection method, and the enzyme activity improvement data are shown in Table 5.

[0164] Table 5. Enzyme activity improvement

[0165]

[0166] Figure 4 A bar graph shows the enhanced activity of KmAgo single-site and multi-site mutant proteins. The results show that the mutant proteins can increase activity by up to sevenfold compared to the wild-type protein. They can also be used for in vitro genetic testing, demonstrating promising application prospects.

[0167] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A mesophilic endonuclease mutant protein Km Ago is characterized by The mesophilic endonuclease is derived from a mesophilic prokaryotic organism kurthia massiliensis , the mutant protein Km Ago is derived from the wild-type sequence shown in SEQ ID NO. 1 by mutation of the following sites: (1) W599S; or (2) W599S and E12L and W445I and E614G and Y130D and H602E and S715V and E621I and A654I and S162D and C201D and R291P; or (3) W599S and E12L and W445I and E614G and Y130D and H602E and N541A and S715V and E621I and A654I and S162D and C201D and R291P; or (4) W599S and E12L and W445I and E614G and Y130D and S715V and E621I and C201D; or (5) W599S and E12L and W445I and E614G and Y130D and H602E and S715V and E621I and A654I and S162D and C201D; or (6) W599S and W445I and E614G and Y130D and S715V and E621I and A654I and S162D; or (7) W599S and E12L and W445I and E614G and Y130D and N541A and S715V and E621I and A654I and S162D and C201D and R291P.

2. An isolated polynucleotide, characterized in that It encodes the mutant protein according to claim 1 Km Ago.

3. A carrier, characterized in that It contains the isolated polynucleotide according to claim 2.

4. A host cell, characterized in that It contains the vector according to claim 3.

5. A mesophilic endonuclease mutant protein according to claim 1 Km The preparation method of Ago is characterized by: Cultivate the host cell according to claim 4 to express the mutant protein according to claim 1.

6. A mesophilic endonuclease mutant protein according to claim 1 Km Application of Ago in nucleic acid detection and gene editing.

7. A mutant protein according to claim 1 Km The in vitro DNA cleavage system of Ago is characterized by: include: (A) a guide DNA that binds to a predetermined target site; and (B) Programmable endonuclease Argonaute Km Ago, wherein the programmable endonuclease is the one described in claim 1 Km Ago mutant proteins; and (C) A target DNA having a nucleotide sequence that is largely complementary to the guide DNA.

8. A method for in vitro DNA cleavage based on the system of claim 7, characterized in that: The specific steps are as follows: S1. Based on Km Ago and gDNA formation Km Ago guide complex; S2. Order Km The Ago guide complex contacts the target DNA. Km The Ago guide complex cleaves the target DNA at a specific site.

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