WO2024145824A1 - Dna polymerase mutant, preparation therefor and use thereof - Google Patents

Abstract

The present invention relates to a DNA polymerase mutant, the preparation therefor and the use thereof. The amino acid sequence of the DNA polymerase is as shown in SEQ ID NO: 1; and at least one of 18 amino acid residues at position 406, position 407, position 411, position 412, position 457, position 460, position 461, position 464, position 481, position 483, position 484, position 487, position 488, position 492, position 494, position 540, position 541 and position 667 of an amino acid sequence of SEQ ID NO: 1 is mutated to obtain a mutant protein having DNA polymerase activity. Compared with wild-type polymerase, the mutant 9°N DNA polymerase has higher polymerization activity. The mutant catalyzes the generation of more substrates within the same amount of time.

Description

A DNA polymerase mutant, its preparation and application Technical Field

The present invention relates to the fields of biotechnology, nucleic acid amplification and enzyme engineering, and in particular to a DNA polymerase mutant, preparation and application thereof.

Background technique

DNA polymerase is an important enzyme that plays an important role in cell DNA replication. It uses DNA as a replication template and replicates DNA from the 5' end to the 3' end. The main activity of DNA polymerase is to catalyze the synthesis of DNA (in the presence of templates, primers, dNTPs, etc.) and its complementary activities.

DNA polymerase is an important tool for ensuring nucleotide incorporation, SNPs detection or more extensive sequencing, such as sequencing by synthesis. However, in the process of sequencing by synthesis, the substrate is a non-natural nucleotide, and most natural polymerases have low ability to process or incorporate non-natural nucleotides and cannot be used in sequencing.

Thermoresistant DNA polymerases are mostly used in PCR technology. All thermoresistant DNA polymerases have 5'-3' polymerase activity, but not necessarily 3'-5' and 5'-3' exonuclease activity. 3'-5' exonuclease activity can eliminate mismatches and cut blunt ends; 5'-3' exonuclease activity can eliminate synthesis obstacles. Due to the above differences, thermoresistant DNA polymerases can be divided into three categories:

1. Common Thermostable DNA Polymerase

1. Taq DNA polymerase

It is isolated and extracted from a strain of aquatic Thermus yT1. It is the most active heat-resistant DNA polymerase found, reaching 200,000 units/mg. It has 5'-3' exonuclease activity, but not 3'-5' exonuclease activity, so it has no correction function for some single nucleotide mismatches during synthesis.

TaqDNA polymerase also has template-independent activity, which can add a single nucleotide tail to the 3' end of each strand of the PCR double-stranded product, so that the PCR product can have a 3' protruding single A nucleotide tail; on the other hand, when only dTTP exists, it can add a single T nucleotide tail to the 3' end of the blunt-ended plasmid, generating a 3' protruding single T nucleotide tail. Using this feature, the T-A cloning method of PCR products can be realized.

2. Tth DNA polymerase

Extracted from Thermus thermophilus HB8, the modified enzyme can effectively reverse transcribe RNA under high temperature and MnCl2 conditions; when Mg2+ is added, the polymerization activity of the enzyme is greatly increased, so that cDNA synthesis and amplification can be catalyzed by one enzyme.

2. High-fidelity DNA polymerase

1.pfu DNA polymerase

It is a high-fidelity, high-temperature resistant DNA polymerase refined from Pyrococcus furiosis. It does not have 5'-3' exonuclease activity, but has 3'-5' exonuclease activity, which can correct errors generated during PCR amplification and make the base mismatch rate of the product extremely low. The PCR product is blunt-ended and has no single A nucleotide protruding at the 3' end.

2. Vent DNA polymerase

The enzyme was isolated from Thermococcus Litoralis and does not have 5'-3' exonuclease activity, but has 3'-5' exonuclease activity, which can remove mismatched bases and has a proofreading function.

3. Thermostable DNA polymerases used in DNA sequencing

1. Promega sequencing-grade Taq DNA polymerase

It is based on the modification of TaqDNA polymerase to remove the 5'-3' exonuclease activity, ensure the high accuracy of sequencing, and produce sequencing bands with uniform intensity and clear background.

2. Bca Best DNA Polymerase

A DNA polymerase purified from Bacillus Caldotenax YT-G strain that lacks 5'-3' exonuclease activity. It has excellent elongation performance and can inhibit the formation of DNA secondary structure, resulting in uniform DNA sequencing bands.

3. Sac DNA polymerase

Isolated from acid-heat-bath fluidized bacterium, it has no 3'-5' exonuclease activity and can be used for DNA sequencing, but the ddNTP/dNTP ratio in the sequencing reaction must be high.

Currently, the core enzyme in mainstream second-generation sequencing is DNA polymerase, but there are many types of DNA polymerases, and natural DNA polymerases are difficult to apply to existing sequencing platforms. It takes a lot of resources to transform natural DNA polymerases to adapt to the requirements of enzymes in sequencing, such as improving reaction speed, stability, sequencing accuracy and other performance.

At present, a large number of research and developments are aimed at semi-rational design or computer simulation to modify the sites of polymerization activity of DNA polymerase, and to perform site-directed mutagenesis on DNA polymerase to improve its thermal stability and accelerate the reaction rate, thereby improving the overall quality of sequencing. The present invention aims to shorten the sequencing time by semi-rational design, performing site-directed mutagenesis on sites inside the wild-type 9°N DNA polymerase molecule to increase its reaction rate of incorporating non-natural nucleotides.

Summary of the invention

In order to improve the above technical problems, the present invention provides a recombinant heat-resistant 9°N DNA polymerase mutant, which improves the ability to incorporate non-natural nucleotides and thus enhances the overall catalytic efficiency of the enzyme in sequencing.

In a first aspect, the present invention provides a DNA polymerase mutant having DNA polymerase activity, which is any one of the following A1)-A3):

A1) DNA polymerase mutants obtained by replacing and/or deleting and/or adding amino acid residues to the amino acid sequence of 9°N DNA polymerase;

A2) A DNA polymerase mutant obtained by modifying the amino acid residues in the amino acid sequence of 9°N DNA polymerase;

A3) A fusion protein having DNA polymerase activity obtained by connecting a tag to the middle or/and N-terminus or/and C-terminus of A1) or A2).

In order to facilitate purification of the DNA polymerase mutant in A1) or A2), a tag as shown in Table 1 may be connected to the middle or/and N-terminus or/and C-terminus of the DNA polymerase mutant in A1) or A2).

Table 1 Tag sequences

Label	Residues	sequence
Poly-Arg	5-6	5×Arg, 6×Arg

Poly-His	2-10	6×His, 10×His
FLAG	8	DYKDDDDK
Strep-tag II	8	WSHQ
c-myc	10	EQKLISEEDL

In some embodiments, the aforementioned DNA polymerase mutants can be artificially synthesized, or their encoding genes can be synthesized first and then biologically expressed.

In some embodiments, the amino acid sequence of 9°N DNA polymerase is as shown in SEQ ID NO:1.

In some embodiments, the substitution and/or deletion and/or addition of amino acid residues in A1) is the substitution and/or deletion and/or addition of one or several amino acid residues;

And/or, the modification of the amino acid residues in A2) is modification of one or several amino acid residues.

In some embodiments, the DNA polymerase mutant in A1) has 75% or more identity with 9°N DNA polymerase. The above 75% or more identity can be 80%, 85%, 90% or more identity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

In some embodiments, the above-mentioned DNA polymerase mutant is a DNA polymerase mutant obtained by replacing and/or modifying at least one of the positions 406, 407, 411, 412, 457, 460, 461, 464, 481, 483, 484, 487, 488, 492, 494, 540, 541 and 667 of the 9°N DNA polymerase from the N-terminus.

In some embodiments, the fusion protein in A3) can be a protein obtained by connecting a his tag and/or a TEV enzyme recognition sequence to the middle or amino or carboxyl terminus of the DNA polymerase mutant in A1) or A2).

In some embodiments, the DNA polymerase mutant is any one of the following B1)-B20):

B1) A DNA polymerase mutant (ZYC5) obtained by replacing the serine residue at position 411 from the N-terminus of 9°N DNA polymerase with an alanine residue;

B2) A DNA polymerase mutant (ZYC6) obtained by replacing the serine residue at position 411 from the N-terminus of 9°N DNA polymerase with a leucine residue;

B3) A DNA polymerase mutant (ZYC8) obtained by replacing the leucine residue at position 457 from the N-terminus of 9°N DNA polymerase with a threonine residue;

B4) A DNA polymerase mutant (ZYC11) obtained by replacing the glutamine residue at position 461 from the N-terminus of 9°N DNA polymerase with an alanine residue;

B5) A DNA polymerase mutant (ZYC24) obtained by replacing the threonine residue at position 667 from the N-terminus of 9°N DNA polymerase with a glutamine residue;

B6) DNA polymerase mutants (ZYC1, ZYC2) obtained by replacing the arginine residue at position 406 from the N-terminus of 9°N DNA polymerase with alanine or leucine residue;

B7) DNA polymerase mutants (ZYC3, ZYC4) obtained by replacing the serine residue at position 407 from the N-terminus of 9°N DNA polymerase with an isoleucine or lysine residue;

B8) A DNA polymerase mutant (ZYC7) obtained by replacing the isoleucine residue at position 412 from the N-terminus of 9°N DNA polymerase with a leucine residue;

B9) A DNA polymerase mutant (ZYC9) obtained by replacing the leucine residue at position 457 from the N-terminus of 9°N DNA polymerase with an alanine residue;

B10) A DNA polymerase mutant (ZYC10) obtained by replacing the arginine residue at position 460 from the N-terminus of 9°N DNA polymerase with a glycine residue;

B11) A DNA polymerase mutant (ZYC12) obtained by replacing the 464th lysine residue from the N-terminus of 9°N DNA polymerase with a threonine residue;

B12) A DNA polymerase mutant (ZYC13) obtained by replacing the tyrosine residue at position 481 from the N-terminus of 9°N DNA polymerase with an alanine residue;

B13) DNA polymerase mutants (ZYC14, ZYC15) obtained by replacing the glutamine residue at position 483 from the N-terminus of 9°N DNA polymerase with a leucine or glutamic acid residue;

B14) DNA polymerase mutants (ZYC16, ZYC17) obtained by replacing the arginine residue at position 484 from the N-terminus of 9°N DNA polymerase with alanine or leucine residue;

B15) A DNA polymerase mutant (ZYC18) obtained by replacing the lysine residue at position 487 from the N-terminus of 9°N DNA polymerase with an arginine residue;

B16) A DNA polymerase mutant (ZYC19) obtained by replacing the isoleucine residue at position 488 from the N-terminus of 9°N DNA polymerase with an alanine residue;

B17) A DNA polymerase mutant (ZYC20) obtained by replacing the serine residue at position 492 from the N-terminus of 9°N DNA polymerase with an aspartic acid residue;

B18) A DNA polymerase mutant (ZYC21) obtained by replacing the tyrosine residue at position 494 from the N-terminus of 9°N DNA polymerase with an arginine or glutamic acid residue;

B19) A DNA polymerase mutant (ZYC22) obtained by replacing the aspartic acid residue at position 540 from the N-terminus of 9°N DNA polymerase with a serine residue;

B20) A DNA polymerase mutant (ZYC23) was obtained by replacing the threonine residue at position 541 of 9°N DNA polymerase from the N-terminus with an alanine residue.

In some embodiments, the DNA polymerase mutant is any one of the following B1)-B5):

B1) A DNA polymerase mutant (ZYC5) obtained by replacing the serine residue at position 411 from the N-terminus of 9°N DNA polymerase with an alanine residue;

B2) A DNA polymerase mutant (ZYC6) obtained by replacing the serine residue at position 411 from the N-terminus of 9°N DNA polymerase with a leucine residue;

B3) A DNA polymerase mutant (ZYC8) obtained by replacing the leucine residue at position 457 from the N-terminus of 9°N DNA polymerase with a threonine residue;

B4) A DNA polymerase mutant (ZYC11) obtained by replacing the glutamine residue at position 461 from the N-terminus of 9°N DNA polymerase with an alanine residue;

B5) A DNA polymerase mutant (ZYC24) was obtained by replacing the threonine residue at position 667 from the N-terminus of 9°N DNA polymerase with a glutamine residue.

In some embodiments, the above-mentioned protein can use nucleotides or nucleotide analogs as substrates. The nucleotide analogs are substances obtained by modifying nucleotides. The nucleotide analogs can specifically be substances obtained by modifying nucleotides with fluorescent groups.

In a second aspect, the present invention provides a biological material related to the above-mentioned DNA polymerase mutant, wherein the biological material is any one of the following C1) to C5):

C1) a nucleic acid molecule encoding the above-mentioned DNA polymerase mutant;

C2) an expression cassette containing the nucleic acid molecule described in C1);

C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3);

C5) A transgenic cell line containing the nucleic acid molecule described in C1), or a transgenic cell line containing the expression cassette described in C2).

In some embodiments, the nucleic acid molecule in C1) is the following 1), 2) or 3):

1) A cDNA molecule or a DNA molecule encoding the above-mentioned DNA polymerase mutant obtained by replacing at least one nucleotide in the sequence of the gene encoding 9°N DNA polymerase;

2) a cDNA molecule or a genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the above-mentioned DNA polymerase mutant;

3) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined in 1) under stringent conditions and encodes the above-mentioned DNA polymerase mutant.

The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

Those skilled in the art can easily mutate the nucleotide sequence encoding the above-mentioned DNA polymerase mutant of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the DNA polymerase mutant of the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above-mentioned DNA polymerase mutant and have the above-mentioned DNA polymerase mutant function.

The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the amino acid sequence of the coding DNA polymerase mutant of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

In the above biological materials, the stringent conditions are hybridization and washing at 68°C in a 2×SSC, 0.1% SDS solution for 5 min each time, and hybridization and washing at 68°C in a 0.5×SSC, 0.1% SDS solution for 15 min each time; or hybridization and washing at 65°C in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution.

In the above biological material, the expression cassette containing the nucleic acid molecule encoding the above DNA polymerase mutant (the above DNA polymerase mutant gene expression cassette) mentioned in C2) refers to a DNA capable of expressing the above DNA polymerase mutant in a host cell, and the DNA may include not only a promoter for initiating transcription of the above DNA polymerase mutant gene, but also a terminator for terminating transcription of the above DNA polymerase mutant gene. Furthermore, the expression cassette may also include an enhancer sequence.

Existing expression vectors can be used to construct a recombinant vector containing the above-mentioned DNA polymerase mutant gene expression cassette.

In the above biological material, the vector may be a plasmid, a cosmid, a phage or a virus vector. Specifically, the plasmid may be pET-22(b).

The recombinant vector may be a recombinant vector obtained by inserting the nucleic acid molecule encoding the above-mentioned DNA polymerase mutant into the multiple cloning site of the vector.

In the above biological material, the microorganism can be yeast, bacteria, algae or fungi. Among them, the bacteria can be Escherichia coli, such as Escherichia coli BL21 (DE3).

In an embodiment of the present invention, the recombinant microorganism is specifically a vector obtained by introducing the recombinant vector into Escherichia coli BL21 (DE3).

In the above biological materials, the transgenic cell line may or may not include propagation materials.

In a third aspect, the present invention provides a method for preparing the above-mentioned DNA polymerase mutant, comprising introducing the coding gene of the above-mentioned DNA polymerase mutant into a biological cell to express the coding gene of the above-mentioned DNA polymerase mutant, thereby obtaining the above-mentioned DNA polymerase mutant.

In the above method, the introduction of the gene encoding the DNA polymerase mutant into the biological cell may be the introduction of a recombinant expression vector containing the gene encoding the DNA polymerase mutant into the biological cell to obtain a recombinant cell.

The encoding gene of the above-mentioned DNA polymerase mutant can specifically be the nucleic acid molecule described in C1) above.

The recombinant expression vector can be obtained by introducing the coding gene of the above DNA polymerase mutant into an expression vector. The expression vector can be a plasmid, a cosmid, a phage or a viral vector. The plasmid can specifically be pET-22(b).

In the above method, the biological cell can be a microorganism, an animal cell or a plant cell. The microorganism can be specifically Escherichia coli, such as Escherichia coli BL21 (DE3).

In the above method, the expression of the gene encoding the DNA polymerase mutant may specifically be achieved by culturing the recombinant cell to obtain a culture, and expressing the gene encoding the DNA polymerase mutant in the recombinant cell.

The method may further include purifying the DNA polymerase mutant from the culture. Purification of the DNA polymerase mutant from the culture may be carried out by affinity chromatography and ion exchange chromatography.

In a fourth aspect, the present invention provides any of the following applications:

E1) Use of the above DNA polymerase mutant as a DNA polymerase;

E2) Use of the above biological materials in the preparation of DNA polymerase;

E3) Use of the above DNA polymerase mutant in DNA polymerization reaction;

E4) Use of the above DNA polymerase mutant in preparing polymerase chain reaction products;

E5) Application of the above biological materials in polymerase chain reaction;

E6) Use of the above biological materials in the preparation of polymerase chain reaction products;

E7) Application of the above method in preparing DNA polymerization reaction products;

E8) Application of the above DNA polymerase mutants in sequencing;

E9) Application of the above biological materials in sequencing;

E10) Use of the above DNA polymerase mutant in preparing sequencing products;

E11) Use of the above biological materials in preparing sequencing products;

E12) Application of the above method in preparing sequencing products.

In the present invention, the DNA polymerase can use nucleotides or nucleotide analogs as substrates. The nucleotide analogs are substances obtained by modifying nucleotides. The nucleotide analogs can specifically be substances obtained by modifying nucleotides with fluorescent groups.

The coding gene of the above-mentioned DNA polymerase mutant can be obtained by mutating one or several nucleotides in the DNA sequence encoding 9°N DNA polymerase, and/or connecting the coding sequence of the tag shown in Table 1 in the middle and/or 5′ end and/or 3′ end of its sequence.

By analyzing and predicting the structure of the polymerization functional region in the SEQ NO.1 sequence, as shown in Figure 1, the site information of mutants that may improve the polymerization activity of the 9°N DNA polymerase mutant was screened.

The present invention also provides a kit for implementing a non-natural nucleotide incorporation reaction, wherein the modified nucleotide is modified at the 3' sugar hydroxyl group so that the substituent is larger in size than the naturally occurring 3' hydroxyl group, and the modified nucleotide at the 5' phosphate group of the template so that the substituent is larger in size than the naturally occurring 5' phosphate group, and the performance of the enzyme is described by detecting the speed at which the separated protein incorporates the non-natural nucleotide by FRET signal at a specified time.

The present invention is based on the tertiary structure of the DNA polymerase of the SEQ NO:1 sequence, and designs a series of site mutations outside the molecule, inside the molecule, near the substrate, and near the DNA binding site. The resulting protein mutants are used in the above-mentioned detection. The rate of incorporation of non-natural nucleotides by some mutant proteins is higher than that of the DNA polymerase shown in the SEQ NO:1 sequence. Preferred examples are that the 411th serine mutates to alanine, the 411th serine mutates to leucine, the 457th leucine mutates to threonine, the 461st glutamine mutates to alanine, and the 676th threonine mutates to glutamine.

Mutant: refers to a gene that has at least one base (nucleotide) change, deletion or insertion relative to the natural or wild-type gene. The mutation (change, deletion and/or insertion of one or more nucleotides) may be in the coding region of the gene or in the intron, 3'UTR, 5'UTR or promoter region. As a non-limiting example, a mutant gene may be a gene that has an insertion in the promoter region that can increase or decrease gene expression; it may be a gene with a deletion that results in the production of a non-functional protein, a truncated protein, a dominant negative protein or no protein; or, it may be a gene with one or more point mutations that results in a change in the amino acid of the encoded protein or results in abnormal splicing of the gene transcript.

Wild-type: refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence found in an organism that has not been intentionally modified by human manipulation.

Beneficial Effects

The mutant 9°N DNA polymerase has higher polymerization activity than the wild-type polymerase. The mutant catalyzes the generation of more substrates in the same amount of time.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic diagram of the 9°N DNA polymerase structure and mutation site. Site-directed mutagenesis is performed based on the structural diagram.

Figure 2: SDS-PAGE electrophoresis of 9°N DNA polymerase after purification, the purity can reach more than 90%.

Figure 3: FRET detection results of ZYC4, showing that the initial reaction rate V0 (the amount of template added substrate per unit time) is 1.37 times that of the wild type.

Figure 4: FRET detection results of ZYC5, showing that the initial reaction rate V0 (the amount of substrate added to the template per unit time) is 1.33 times that of the wild type.

Figure 5: FRET detection results of ZYC11, showing that the initial reaction rate V0 (the amount of template added substrate per unit time) is 1.35 times that of the wild type.

Detailed ways

The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

According to an embodiment of the present invention, the isolated protein is SEQ NO:1 and a mutant having one of the following: R406A/L, S407I/K, S411A/L, I412L, L457T/A, R460G, Q461A, K464T, Y481A, Q483L/E, R484A/L, K487R, I488A, S492D, Y494R, D540S, T541A, T667Q.

Example 1: Preparation of wild-type 9°N DNA polymerase SEQ NO: 1 (9N-WT) and mutant 9°N DNA polymerase proteins

(1) Construction of polymerase SEQ NO:1 (9N-WT) expression strain and induced expression of recombinant protein

The plasmid containing the 9N-WT gene fragment subcloned into the pET-22(b) vector was transformed into Transetta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd.) Escherichia coli to obtain recombinant protein engineering bacteria, which were inoculated into LB medium containing ampicillin and cultured at 37°C and 200rpm for 3 to 4 hours for activation. The activated bacterial solution was added to a new LB medium containing ampicillin at a ratio of 1:100, and cultured at 37°C with shaking until OD600nm reached 0.8-1.1. After cooling in an ice water bath, IPTG was added at a final concentration of 0.5mM and cultured overnight at 25°C with shaking. The induced bacterial solution was centrifuged at 8000rpm for 10 minutes to collect the bacteria.

(2) Protein purification

The buffer used for protein purification is as follows:

Lysis buffer: 50 mM MOPS, 500 mM NaCl, 5% Glycerol, pH 7.6;

A buffer: 50mM MOPS, 500mM NaCl, 20mM Imidazole, 5% Glycerol, pH 7.6;

C buffer: 50 mM MOPS, 50 mM NaCl, 5% Glycerol, pH 7.0;

D Buffer: 50mM MOPS, 50mM NaCl, 500mM Imidazole, 5% Glycerol, pH 7.0;

E Buffer: 50 mM MOPS, 5% Glycerol, pH 7.0;

F Buffer: 50mM MOPS, 1M Nacl, 5% Glycerol, pH 7.0;

Dialysis buffer: 20 mM Tris-HCl, 200 mM KCl, 0.2 mM EDTA, pH 7.4.

Lysis buffer was added at a ratio of 1:10 for bacterial weight (g)/buffer volume (ml) to resuspend the bacteria; PMSF was added to a final concentration of 1 mM. The sample was added to a high-pressure homogenizer (ATS), the pressure was raised to 700-800 MPa, and lysis was performed at 4°C for 2-3 cycles; the lysed bacterial solution was ultracentrifuged at 4°C and 18,000 rpm for 40 min; the supernatant obtained after centrifugation was heated in a 75°C water bath for 30 min, and stirred regularly during the period to heat and mix; the crude enzyme solution obtained above was centrifuged at 16,000 rpm and 4°C for 30 min, and vacuum filtered with a 0.45 μm filter membrane (Merck Millipore). The obtained sample was used for subsequent experiments.

Affinity column purification was performed using a purifier (Biorad NGC Quest 100). The filtrate was loaded onto a pre-equilibrated Histrap HP column (Cat. No. 17-5248-02, cytiva), and the column was equilibrated with the Lysis buffer for 10 CV, with a retention time of 2.5 min; after all samples were loaded onto the column, the column was rinsed with Lysis buffer until the baseline of UV absorption was balanced; the column was rinsed with B buffer for 20 CV, and then with C buffer for 10 CV, and finally the target protein was eluted with D buffer until the components with a peak A280nm greater than 400 mAU were collected for the next step of purification.

The sample eluted from the affinity column purification was diluted 5 times with E Buffer and then loaded onto a Hitrap Q HP column (Cat. No. 17-1154-01, cytiva) pre-equilibrated with E Buffer + 2% F Buffer, and the flow-through was collected;

Use E Buffer + 2% F Buffer to pre-equilibrate Hitrap SP HP (17-1152-01) column, load the above flow-through sample onto the column, rinse the column with E Buffer + 2% F Buffer for 5CV, and then use 2-50% QB Buffer for gradient elution. Start collecting when A280nm is greater than 100mAU, and discard when it is lower than 100mAU. Collect 5ml in each tube;

The protein eluted from the SP column was placed in a 10 kDa dialysis bag (Cat. No. 132576, Spectrum) and dialyzed into Dialysis buffer overnight. The dialyzed sample was first measured for concentration using an ELISA reader (CLARIOstar Plus, BMG), and then Triton X-100 and glycerol were added to make the final concentrations 0.1% and 50%, respectively, and stored at -80°C.

(3) Identification of protein purity

At the same time, the protein purity was determined according to the SDS-PAGE electrophoresis gel image (Figure 2). The purity of the purified wild-type 9N-WT and ZYC1-ZYC24 proteins was more than 90%, and the size of the target protein was about 92 kd.

(4) Preparation of recombinant vectors of 9°N DNA polymerase point mutants (ZYC1-ZYC24)

Using the plasmid of 9N-WT gene fragment SEQ NO:2 as a template, single point mutation primers were designed for PCR amplification, and the amplified fragments were connected and transformed into Transetta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd.) Escherichia coli to obtain mutant recombinant protein engineering bacteria. The method for preparing 9°N DNA polymerase point mutants can also be prepared according to the wild-type preparation method.

Table 1 shows the mutation position and mutation information of 9°N DNA polymerase single point mutant (compared with SEQ NO: 1).

Table 19 Mutation positions and mutation information of single-point mutants of DNA polymerase

Example 2: Activity detection of recombinant 9°N DNA polymerase and its mutants

(1) F/R-cy5 template synthesis

Primer synthesis

Primers were synthesized by Shanghai Bioengineering Co., Ltd. The sequences are as follows:

Primer 1 (5’-3’) CCGAGTGTCGGGACGGTGACCCAAGCTGCACCAG

Primer 2 (5’-3’) AGCCCAGTCTGGTGCAGCTTGGGTCACCGTCCCG

Primer 1 and Primer 2 are reverse complementary sequences, wherein cy5 is connected to the 5' end of Primer 1 and Primer 2, and after annealing, they are complementary and matched to form a template and primer mixture for enzyme activity determination.

(2) Preparation conditions of F/R-cy5

The reaction was carried out in a PCR instrument. The reaction system and reaction procedure were as follows, wherein 5X Anneal buffer was purchased from Solebao (Beijing).

reaction system:

Reaction procedure: 95℃5min

60℃10min

After the reaction, the mass concentration of the double-stranded protein was measured using Qubit, and the molecular weight was converted into its molar concentration. The protein activity detection reaction system is shown in Table 2.

Table 2 Protein activity detection reaction system

F/R-Cy5	0.1uM
dATP-Cy3-N3	0.4uM
10*Reactionbuffer	5ul
dC/dG/dT Mixture	0.4uM
Enzyme	20ul
H2O	Add water to 50ul

10*Reaction buffer composition: 200mM Tris, 100mM (NH ₄ ) ₂ SO ₄ , 100mM KCL, 5mM MgSO ₄ , pH=8.8, dC/dG/dT purchased from Shanghai Biotech Co., Ltd., is a substrate after mixing equal volumes of dCTP (100mM), dGTP (100mM), and dTTP (100mM).

The above reaction was carried out in a microplate reader (CLARIOstarPlus BMG) at 42° for 40 min, and the FRET Cy5 (excitation 530 nm/emission 676 nm) signal was detected. The activity of 150 pmol of non-natural base dATP-Cy3-N3 added per unit time was defined as 1 U.

The polymerization activity of the preferred exemplified 9°N DNA polymerase mutant is higher than that of the wild type, and the results are shown in Table 3.

Table 39 Polymerization activity of 9°N DNA polymerase mutants

9°N DNA polymerase name	Activity (U)	Amino acids in 9°N DNA polymerase mutants
9N-WT	1
ZGar	1.36	A
ZGar	1.61	L

ZGar	1.19	T
ZYC24	1.13	Q

The amino acid sequence of the ZYC5 mutant is that the serine at position 411 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to alanine;

The amino acid sequence of the ZYC6 mutant is that the serine at position 411 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to leucine;

The amino acid sequence of the ZYC8 mutant is that the leucine at position 457 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to threonine;

The amino acid sequence of the ZYC24 mutant is that the threonine at position 667 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to glutamine;

The speed of the preferred 9°N DNA polymerase mutant polymerizing the substrate (dATP-cy3-N3) in the initial time period of the reaction is represented by V _0. The stronger the FRET Cy5 (excitation 530nm/emission 676nm) signal, the faster the initial reaction speed, indicating that the dATP-cy3-N3 added in the same initial time is faster. The initial speed is calculated using the first 4 minutes of the reaction, and the speed of adding dATP-cy3-N3 by the wild-type 9°N DNA polymerase is 1. The reaction speed of the 9°N DNA polymerase mutant is represented by its multiple relationship, see Table 4 and Figures 3-5.

Table 4 Reaction rates of 9°N DNA polymerase mutants

9°N DNA polymerase name		V 0
9N-WT	1
ZGar	1.37
ZGar	1.33
ZYC11	1.35

The amino acid sequence of the ZYC5 mutant is that the serine at position 411 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to alanine;

The amino acid sequence of the ZYC6 mutant is that the serine at position 411 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to leucine;

The amino acid sequence of the ZYC11 mutant is that the glutamine at position 461 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to alanine;

The results showed that compared with the wild-type 9°N DNA polymerase, the enzymatic activity and initial reaction rate of mutant proteins such as ZYC5, ZYC6, ZYC8, ZYC11, and ZYC24 were better than those of the wild-type. In the sequencing reaction, they can shorten the reaction time and improve the catalytic efficiency.

The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (8)

1. A DNA polymerase mutant having DNA polymerase activity, which is any one of the following A1)-A3):

   A1) DNA polymerase mutants obtained by replacing and/or deleting and/or adding amino acid residues to the amino acid sequence of 9°N DNA polymerase;

   A2) A DNA polymerase mutant obtained by modifying the amino acid residues in the amino acid sequence of 9°N DNA polymerase;

   A3) A fusion protein having DNA polymerase activity obtained by removing a sequence on the vector or connecting a tag sequence in the middle or/and N-terminus or/and C-terminus of A1) or A2);

   The amino acid sequence of 9°N DNA polymerase is shown in SEQ ID NO:1.
2. The DNA polymerase mutant according to claim 1, characterized in that

   The substitution and/or deletion and/or addition of amino acid residues in A1) is substitution and/or deletion and/or addition of one or several amino acid residues;

   and/or, the modification of the amino acid residues in A2) is modification of one or several amino acid residues;

   and/or, the DNA polymerase mutant in A1) has 75% or more identity with 9°N DNA polymerase; the above-mentioned 75% or more identity may be 80%, 85%, 90% or more identity, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
3. The DNA polymerase mutant according to claim 1 or 2 is characterized in that the DNA polymerase mutant is a DNA polymerase mutant obtained by replacing and/or modifying at least one of the 406th, 407th, 411th, 412th, 457th, 460th, 461st, 464th, 481st, 483rd, 484th, 487th, 488th, 492nd, 494th, 540th, 541st and 667th positions of the 9°N DNA polymerase from the N-terminus.
4. The DNA polymerase mutant according to any one of claims 1 to 3, characterized in that the DNA polymerase mutant is any one of the following B1) to B20):

   B1) A DNA polymerase mutant (ZYC5) obtained by replacing the serine residue at position 411 from the N-terminus of 9°N DNA polymerase with an alanine residue;

   B2) A DNA polymerase mutant (ZYC6) obtained by replacing the serine residue at position 411 from the N-terminus of 9°N DNA polymerase with a leucine residue;

   B3) A DNA polymerase mutant (ZYC8) obtained by replacing the leucine residue at position 457 from the N-terminus of 9°N DNA polymerase with a threonine residue;

   B4) A DNA polymerase mutant (ZYC11) obtained by replacing the glutamine residue at position 461 from the N-terminus of 9°N DNA polymerase with an alanine residue;

   B5) A DNA polymerase mutant (ZYC24) obtained by replacing the threonine residue at position 667 from the N-terminus of 9°N DNA polymerase with a glutamine residue;

   B6) DNA polymerase mutants (ZYC1, ZYC2) obtained by replacing the arginine residue at position 406 from the N-terminus of 9°N DNA polymerase with alanine or leucine residue;

   B7) DNA polymerase mutants (ZYC3, ZYC4) obtained by replacing the serine residue at position 407 from the N-terminus of 9°N DNA polymerase with an isoleucine or lysine residue;

   B8) A DNA polymerase mutant (ZYC7) obtained by replacing the isoleucine residue at position 412 from the N-terminus of 9°N DNA polymerase with a leucine residue;

   B9) A DNA polymerase mutant (ZYC9) obtained by replacing the leucine residue at position 457 from the N-terminus of 9°N DNA polymerase with an alanine residue;

   B10) A DNA polymerase mutant (ZYC10) obtained by replacing the arginine residue at position 460 from the N-terminus of 9°N DNA polymerase with a glycine residue;

   B11) A DNA polymerase mutant (ZYC12) obtained by replacing the 464th lysine residue from the N-terminus of 9°N DNA polymerase with a threonine residue;

   B12) A DNA polymerase mutant (ZYC13) obtained by replacing the tyrosine residue at position 481 from the N-terminus of 9°N DNA polymerase with an alanine residue;

   B13) DNA polymerase mutants (ZYC14, ZYC15) obtained by replacing the glutamine residue at position 483 from the N-terminus of 9°N DNA polymerase with a leucine or glutamic acid residue;

   B14) DNA polymerase mutants (ZYC16, ZYC17) obtained by replacing the arginine residue at position 484 from the N-terminus of 9°N DNA polymerase with an alanine or leucine residue;

   B15) A DNA polymerase mutant (ZYC18) obtained by replacing the lysine residue at position 487 from the N-terminus of 9°N DNA polymerase with an arginine residue;

   B16) A DNA polymerase mutant (ZYC19) obtained by replacing the isoleucine residue at position 488 from the N-terminus of 9°N DNA polymerase with an alanine residue;

   B17) A DNA polymerase mutant (ZYC20) obtained by replacing the serine residue at position 492 from the N-terminus of 9°N DNA polymerase with an aspartic acid residue;

   B18) A DNA polymerase mutant (ZYC21) obtained by replacing the tyrosine residue at position 494 from the N-terminus of 9°N DNA polymerase with an arginine or glutamic acid residue;

   B19) A DNA polymerase mutant (ZYC22) obtained by replacing the aspartic acid residue at position 540 from the N-terminus of 9°N DNA polymerase with a serine residue;

   B20) A DNA polymerase mutant (ZYC23) obtained by replacing the 541st threonine residue from the N-terminus of 9°N DNA polymerase with an alanine residue;

   Preferably, it is any one of B1) to B5):

   B1) A DNA polymerase mutant (ZYC5) obtained by replacing the serine residue at position 411 from the N-terminus of 9°N DNA polymerase with an alanine residue;

   B2) A DNA polymerase mutant (ZYC6) obtained by replacing the serine residue at position 411 from the N-terminus of 9°N DNA polymerase with a leucine residue;

   B3) A DNA polymerase mutant (ZYC8) obtained by replacing the leucine residue at position 457 from the N-terminus of 9°N DNA polymerase with a threonine residue;

   B4) A DNA polymerase mutant (ZYC11) obtained by replacing the glutamine residue at position 461 from the N-terminus of 9°N DNA polymerase with an alanine residue;

   B5) A DNA polymerase mutant (ZYC24) was obtained by replacing the threonine residue at position 667 from the N-terminus of 9°N DNA polymerase with a glutamine residue.
5. A biological material related to the DNA polymerase mutant according to any one of claims 1 to 4, characterized in that the biological material is any one of the following C1) to C5):

   C1) A nucleic acid molecule encoding the DNA polymerase mutant according to any one of claims 1 to 4;

   C2) an expression cassette containing the nucleic acid molecule described in C1);

   C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

   C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3);

   C5) A transgenic cell line containing the nucleic acid molecule described in C1), or a transgenic cell line containing the expression cassette described in C2).
6. The biomaterial according to claim 5, characterized in that the nucleic acid molecule in C1) is the following 1), 2) or 3):

   1) A cDNA molecule or a DNA molecule encoding the DNA polymerase mutant according to any one of claims 1 to 4 obtained by replacing at least one nucleotide in the sequence of the gene encoding 9°N DNA polymerase;

   2) a cDNA molecule or a genomic DNA molecule having 75% or more identity with the nucleotide sequence defined in 1), and encoding the DNA polymerase mutant described in any one of claims 1 to 4;

   3) A cDNA molecule or a genomic DNA molecule which hybridizes with the nucleotide sequence defined in 1) under stringent conditions and encodes the DNA polymerase mutant according to any one of claims 1 to 4.
7. A method for preparing the DNA polymerase mutant described in any one of claims 1 to 4, comprising introducing the coding gene of the DNA polymerase mutant described in any one of claims 1 to 4 into a biological cell so that the coding gene of the DNA polymerase mutant described in any one of claims 1 to 4 is expressed to obtain the DNA polymerase mutant described in any one of claims 1 to 4.
8. Any of the following applications:

   E1) Use of the DNA polymerase mutant according to any one of claims 1 to 4 as a DNA polymerase;

   E2) Use of the biological material according to claim 5 or 6 in the preparation of DNA polymerase;

   E3) Use of the DNA polymerase mutant according to any one of claims 1 to 4 in a DNA polymerization reaction;

   E4) Use of the DNA polymerase mutant according to any one of claims 1 to 4 in the preparation of polymerase chain reaction products;

   E5) Use of the biological material according to claim 5 or 6 in polymerase chain reaction;

   E6) Use of the biological material according to claim 5 or 6 in preparing a polymerase chain reaction product;

   E7) Use of the method according to claim 7 in preparing a DNA polymerization reaction product;

   E8) Use of the DNA polymerase mutant according to any one of claims 1 to 4 in sequencing;

   E9) Use of the biological material according to claim 5 or 6 in sequencing;

   E10) Use of the DNA polymerase mutant according to any one of claims 1 to 4 in preparing a sequencing product;

   E11) Use of the biological material according to claim 5 or 6 in preparing a sequencing product;

   E12) Use of the method according to claim 7 in preparing sequencing products.

Images