Pfu DNA polymerase mutant and application thereof in PCR

Abstract

The invention belongs to the technical field of PCR (Polymerase Chain Reaction), relates to a Pfu DNA (Deoxyribose Nucleic Acid) polymerase mutant and application of the Pfu DNA polymerase mutant in PCR, and particularly relates to a method for substituting an H680 and / or A684 amino acid site of an amino acid sequence of a wild type Pfu DNA polymerase or a wild type Pfu DNA polymerase derivative. Compared with the unmutated mutant, the substituted mutant has a lower Km value and higher affinity with a substrate, so that the catalytic activity of the Pfu DNA polymerase is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of PCR, and specifically relates to Pfu DNA polymerase mutants and their applications in PCR. Background Art

[0002] The PCR technology is the core of molecular biology and has powerful functions. It can efficiently and rapidly perform enzymatic amplification of specific DNA or RNA sequences from different sources in vitro. The Pfu polymerase was discovered in the thermophilic archaeon Pyrococcus. This enzyme has both 5'-3' polymerase activity and 3'-5' exonuclease activity. When replicating DNA in vivo, it can correct misincorporated bases during the polymerization reaction, with extremely high fidelity. However, with the development of new interdisciplinary fields such as synthetic biology, researchers have an increasingly high pursuit of polymerases with different properties. For example, there is a need to improve the affinity for substrates, possess the ability to amplify long fragments, be compatible with high-GC templates, and be able to directly extract DNA from crude extracts. Therefore, it is urgent to develop different Pfu polymerase mutants to meet the growing demands in laboratories and industrial production. Summary of the Invention

[0003] The object of the present invention is to address the above problems existing in the prior art and provide Pfu DNA polymerase mutants with strong affinity for substrates and high catalytic activity, as well as their applications in PCR.

[0004] To achieve the above object, the technical solution of the present invention is as follows:

[0005] The present invention provides a Pfu DNA polymerase mutant, wherein the Pfu DNA polymerase mutant has at least one amino acid site substitution selected from the following occurring in the amino acid sequence of wild-type Pfu DNA polymerase or a wild-type Pfu DNA polymerase derivative: H680, A684.

[0006] Specifically, the substitution at H680 is K, and the substitution at A684 is Q.

[0007] Specifically, the amino acid sequences of the wild-type Pfu DNA polymerase and its H680K mutant, A684Q mutant, and H680K+A684Q mutant are shown in SEQ ID NO.1-4 respectively;

[0008] The amino acid sequences of the wild-type Pfu DNA polymerase derivative and its H680K mutant, A684Q mutant, and H680K+A684Q mutant are shown in SEQ ID NO.5-8 respectively.

[0009] The present invention also provides a nucleotide sequence encoding the aforementioned Pfu DNA polymerase mutant. The nucleotide sequences encoding the wild-type Pfu DNA polymerase and its H680K mutant, A684Q mutant, and H680K+A684Q mutant are shown in SEQ ID NOs. 9-12 respectively;

[0010] The nucleotide sequences encoding the wild-type Pfu DNA polymerase derivative and its H680K mutant, A684Q mutant, and H680K+A684Q mutant are shown in SEQ ID NOs. 13-16 respectively.

[0011] The present invention also provides a recombinant vector containing the aforementioned nucleotide sequence.

[0012] The present invention also provides a recombinant cell containing the aforementioned nucleotide sequence or the aforementioned recombinant vector.

[0013] The present invention also provides an application of the aforementioned Pfu DNA polymerase mutant, the aforementioned nucleotide sequence, the aforementioned recombinant vector, and the aforementioned recombinant cell in the field of PCR.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The H680K mutant and A684Q mutant of the present invention have lower Km values compared with the unmutated Pfu DNA polymerase. The lower the Km value, the higher the affinity with the substrate, thereby enhancing the catalytic activity of the Pfu DNA polymerase. In addition, compared with the unmutated Pfu DNA polymerase, the H680K mutant mutates the 680th histidine of the Pfu DNA polymerase to lysine, enhancing the electrostatic interaction with the DNA template by enhancing the surface electrostatic potential of the enzyme, thereby improving the amplification specificity; the A684Q mutant mutates the 684th alanine of the Pfu DNA polymerase to glutamine. The side chain of glutamine can extend deeper into the double helix groove of the DNA template, thereby enhancing the affinity of the Pfu DNA polymerase for the DNA template and reducing the salt tolerance, thereby enhancing its compatibility with high-GC DNA templates. Description of the Drawings

[0016] Figure 1 The PCR amplification result obtained by using the H680K mutant of the wild-type Pfu DNA polymerase derivative as the DNA template for PCR amplification of upland cotton gDNA.

[0017] Figure 2 The structural comparison diagram of the wild-type Pfu DNA polymerase derivative and its H680K mutant.

[0018] Figure 3 The PCR amplification results obtained by using the A684Q mutant of the wild-type Pfu DNA polymerase derivative as a DNA template with pet16b-derived plasmid DNA under potassium salts at a concentration of 10 mM to 100 mM for PCR amplification.

[0019] Figure 4 The PCR amplification results obtained by using the A684Q mutant of the wild-type Pfu DNA polymerase derivative as a DNA template with Nipponbare rice gDNA under potassium salts at concentrations of 50 mM and 100 mM for PCR amplification.

[0020] Figure 5 The structural comparison diagram of the wild-type Pfu DNA polymerase derivative and its A684Q mutant. Detailed implementation manners

[0021] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0022] The present invention provides a Pfu DNA polymerase mutant, which is a substitution at at least one amino acid site selected from the following in the amino acid sequence of the wild-type Pfu DNA polymerase or the wild-type Pfu DNA polymerase derivative: H680, A684.

[0023] The substitution at H680 is K, and the substitution at A684 is Q.

[0024] The amino acid sequences of the wild-type Pfu DNA polymerase and its H680K mutant, A684Q mutant, and H680K + A684Q mutant are shown in SEQ ID NO.1-4 respectively;

[0025] A double-stranded DNA binding protein sso7d is fused to the C-terminus of the wild-type Pfu DNA polymerase to obtain a wild-type Pfu DNA polymerase derivative. The amino acid sequences of the wild-type Pfu DNA polymerase derivative and its H680K mutant, A684Q mutant, and H680K + A684Q mutant are shown in SEQ ID NO.5-8 respectively.

[0026] A nucleotide sequence encoding the aforementioned Pfu DNA polymerase mutant, and the nucleotide sequences encoding the wild-type Pfu DNA polymerase and its H680K mutant, A684Q mutant, and H680K + A684Q mutant are shown in SEQ ID NO.9-12 respectively;

[0027] The nucleotide sequences encoding wild-type Pfu DNA polymerase derivatives and their H680K mutants, A684Q mutants, and H680K+A684Q mutants are shown in SEQ ID NO.13-16, respectively.

[0028] A recombinant vector containing the aforementioned nucleotide sequence.

[0029] A recombinant cell containing the aforementioned nucleotide sequence or the aforementioned recombinant vector.

[0030] Use of the aforementioned Pfu DNA polymerase mutants, the aforementioned nucleotide sequences, the aforementioned recombinant vectors, and the aforementioned recombinant cells in the field of PCR.

[0031] Hereinafter, the H680K mutant, A684Q mutant, and H680K+A684Q mutant of the wild-type Pfu DNA polymerase derivative will be taken as examples for explanation. The main materials and instruments used in the present invention are shown in Table 1:

[0032] Table 1 Main materials and instruments

[0033]

[0034] Example 1 Enzymatic kinetic analysis of wild-type Pfu DNA polymerase derivative mutants

[0035] Enzymatic kinetic analysis method: Pretreat the hairpin DNA by heating at 98°C for 5 min and annealing on ice for 30 min; use 0.1-2 μM concentration of hairpin DNA as the substrate template, mix it with dNTP, 1 nM wild-type Pfu DNA polymerase derivative or its mutant, and add MgCl 2 to initiate DNA synthesis at 72°C; the reaction buffer contains 25 mM Tris-HCl (pH 8.8), 10 mM KCl, 2.5 mM MgCl 2 , 200 μM dNTP, 0.05% Triton X-100, 1X EvaGreen; plot the fluorescence curve, which is obtained by subtracting the fluorescence value of each reaction background, calculate the first derivative of the fluorescence curve, and obtain the initial rate of each reaction at different template amounts. Use GraphPad Prism 8.0 software to fit the Michaelis-Menten kinetic equation to obtain k cat value and K mValue. Three biological replicates were set for each group. The base sequence of the hairpin DNA was: 5’-CCAGCATTATGAAAGTGACACGTGCACCATTGGTGCACGTG-3’. The results of the enzymatic kinetic analysis of the wild-type Pfu DNA polymerase derivative and its A684Q, H680K, and A684Q+H680K mutants are shown in Table 2.

[0036] Table 2 Results of the enzymatic kinetic analysis of the wild-type Pfu DNA polymerase derivative and its mutants

[0037]

[0038] As can be seen from Table 2, the Km value of the wild-type Pfu DNA polymerase derivative was 0.45±0.07, while the Km values of its A684Q and H680K mutants were 0.34±0.03 and 0.31±0.06, respectively. It can be seen that the A684Q, H680K, and A684Q+H680K mutants all had lower Km values. The lower the Km value, the stronger the binding ability to the substrate. Among them, the A684Q+H680K mutant had the lowest Km value, indicating that the A684Q+H680K mutant had the strongest binding ability to the substrate.

[0039] Example 2 Analysis of the amplification performance of the wild-type Pfu DNA polymerase derivative mutants

[0040] The A684Q and H680K mutants of the wild-type Pfu DNA polymerase derivative were used to perform PCR amplification on different DNA templates respectively. The PCR amplification system included: dNTP 200 μmol / L, wild-type Pfu DNA polymerase derivative mutant 40 U / mL, hsp20 200 pg / uL, Tris 120 mmol / L, KCl 10 mmol / L, (NH 4 ) 2 SO 4 6 mmol / L, MgCl 22 mmol / L, 0.1% w / v Triton X-100, 0.001% w / v BSA; the pH value is 8.3 - 9.2. PCR amplification method: Add the DNA template, forward primer, and reverse primer to the aforementioned PCR amplification system and perform the reaction using a PCR instrument. The PCR amplification program is shown in Table 3. The DNA templates used for the A684Q mutant are the pet16b-derived plasmid DNA with a target length of 10 kbp and the Nipponbare rice gDNA with a target length of 417 kbp. Among them, the Nipponbare rice gDNA is a high-GC template, and the PCR amplification is carried out under different concentrations of potassium salts from 10 mM to 100 mM; the DNA template used for the H680K mutant is the upland cotton gDNA with a target length of 1.3 kbp. The corresponding forward primers and reverse primers are shown in Table 4.

[0041] Table 3 PCR Amplification Program

[0042]

[0043] Table 4 Forward Primers and Reverse Primers

[0044]

[0045]

[0046] The PCR amplification results obtained by using the H680K mutant with upland cotton gDNA as the DNA template for PCR amplification are as Figure 1 shown. In the figure, M represents the corresponding DNA template, WT represents the wild-type Pfu DNA polymerase derivative, and HK represents the H680K mutant of the wild-type Pfu DNA polymerase derivative. It can be seen that the H680K mutant can significantly improve the specificity of polymerase amplification. Use the Alphafold3 software to perform a structural analysis on the wild-type Pfu DNA polymerase derivative and its H680K mutant. The analysis results are as Figure 2 shown. In the figure, H680 represents the wild-type Pfu DNA polymerase derivative, and K680 represents the H680K mutant. It can be seen that the H680K mutant increases the surface charge of the wild-type Pfu DNA polymerase derivative.

[0047] The PCR amplification results obtained by using the A684Q mutant with pet16b-derived plasmid DNA as the DNA template for PCR amplification are as Figure 3As shown in the figure, M in the figure represents the corresponding DNA template, and AQ represents the A684Q mutant of Pfu DNA polymerase. It can be seen that the salt tolerance of the A684Q mutant is 50 mM, while the salt tolerance of the wild-type Pfu DNA polymerase derivative is generally 100 mM, and the salt tolerance of the A684Q mutant is lower. The PCR amplification results obtained by using the A684Q mutant with Nipponbare rice gDNA as the DNA template are as Figure 4 shown. In the figure, M represents the corresponding DNA template, WT represents the wild-type Pfu DNA polymerase derivative, and AQ represents the A684Q mutant of the wild-type Pfu DNA polymerase derivative. It can be seen that the A684Q mutant can exhibit higher amplification performance for high-GC DNA templates at a salt ion concentration of 50 mM. The PfuDNA polymerase and its A684Q mutant were subjected to structural analysis using the Alphafold3 software, and the analysis results are as Figure 5 shown. In the figure, A684 represents Pfu DNA polymerase, and Q684 represents the A684Q mutant. It can be seen that the glutamine side chain in the A684Q mutant can insert more deeply into the double helix groove of the DNA template, making the binding tighter.

[0048] Example 3 Fidelity Analysis of Mutants of Wild-Type Pfu DNA Polymerase Derivatives

[0049] Fidelity analysis: The linearized pUC19 plasmid vector carrying the lacZ gene was amplified, and then the amplification product was subjected to agarose gel electrophoresis. The target band of the linearized vector was recovered from the gel, and the gel recovery product was ligated using T4 ligase. The ligation product was transformed into the α-complementary Escherichia coli host strain Turbo. An aliquot of the infected cells was inoculated onto an LB plate containing 1 mg / mL X-gal and 1.5 mM IPTG in the upper agar and cultured overnight at 37°C. Standard blue-white colony screening was performed, and the number of blue and white colonies was counted to calculate the apparent mutation frequency: Apparent mutation frequency = (number of white colonies / total number of colonies) × 100. Three biological replicates were set for each group. The fidelity analysis results are shown in Table 5.

[0050] Table 5 Fidelity Analysis Results of Wild-Type Pfu DNA Polymerase Derivatives and Their A684Q and H680K Mutants

[0051]

[0052]

[0053] As can be seen from Table 5, the apparent mutation frequency of the wild-type Pfu DNA polymerase derivative is 0.41±0.04, and the apparent mutation frequency of its A684Q mutant is 0.35±0.05. This indicates that the fidelity of the A684Q mutant is basically the same as that of the wild-type Pfu DNA polymerase derivative, while the error rate of the H680K mutant is slightly reduced to 0.35±0.05, suggesting that the H680K mutant has better fidelity than the unmutated wild-type Pfu DNA polymerase derivative.

[0054] In summary, the mutants of the two wild-type Pfu DNA polymerase derivatives described in the present invention can improve the Km value of the wild-type Pfu DNA polymerase derivative while maintaining or having higher fidelity in PCR detection, and enhance the amplification performance of the enzyme under different scenarios, and can be applied to various cloning, functional analysis and sequencing.

[0055] Example 4:

[0056] The nucleotide sequence containing the Pfu DNA polymerase derivative was constructed into an Escherichia coli expression vector to form an Escherichia coli recombinant cell. By pre-denaturation for 6 min during PCR, the Escherichia coli recombinant cell was lysed, and the NKK saturation mutation library at positions H680 and A684 was screened by PCR amplification, and two beneficial mutants of the Pfu DNA polymerase derivative, namely the H680K mutant and the A684Q mutant, were screened out.

Claims

1. A Pfu DNA polymerase mutant, characterized in that: The Pfu DNA polymerase mutant is obtained by substitution of at least one amino acid site selected from the following: H680, A684 in the amino acid sequence of the wild-type Pfu DNA polymerase or a derivative of the wild-type Pfu DNA polymerase.

2. The Pfu DNA polymerase mutant according to claim 1, characterized in that: The substitution at H680 is K and the substitution at A684 is Q.

3. The Pfu DNA polymerase mutant according to claim 2, characterized in that: The amino acid sequences of the wild-type Pfu DNA polymerase and its H680K mutant, A684Q mutant, and H680K+A684Q mutant are shown in SEQ ID NOs. 1-4, respectively; The amino acid sequences of the wild-type Pfu DNA polymerase derivative and its H680K mutant, A684Q mutant, and H680K+A684Q mutant are shown in SEQ ID NOs. 5-8, respectively.

4. The nucleotide sequence encoding the Pfu DNA polymerase mutant according to claim 3, characterized in that: The nucleotide sequences encoding the wild-type Pfu DNA polymerase and its H680K mutant, A684Q mutant, and H680K+A684Q mutant are shown in SEQ ID NOs. 9-12, respectively; The nucleotide sequences encoding the wild-type Pfu DNA polymerase derivative and its H680K mutant, A684Q mutant, and H680K+A684Q mutant are shown in SEQ ID NOs. 13-16, respectively.

5. A recombinant vector comprising the nucleotide sequence according to claim 4.

6. A recombinant cell comprising the nucleotide sequence according to claim 4 or the recombinant vector according to claim 5.

7. Use of the Pfu DNA polymerase mutant according to any one of claims 1 to 3, the nucleotide sequence according to claim 4, the recombinant vector according to claim 5, and the recombinant cell according to claim 6 in the field of PCR.

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