Pfu DNA polymerase mutant and use thereof in PCR
By substituting amino acids at the H680 and A684 sites of Pfu DNA polymerase, a mutant with higher substrate affinity and catalytic activity was developed, overcoming the shortcomings of existing Pfu DNA polymerases in substrate binding and catalytic activity, and improving the efficiency and specificity of PCR amplification.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- INNOVATION ACAD FOR PRECISION MEASUREMENT SCI & TECH CAS
- Filing Date
- 2025-03-24
- Publication Date
- 2026-07-30
AI Technical Summary
Existing Pfu DNA polymerases are insufficient in terms of substrate affinity and catalytic activity, making it difficult to meet the needs of synthetic biology and industrial production.
By substituting amino acids at the H680 and A684 sites of Pfu DNA polymerase, H680K, A684Q, and H680K+A684Q mutants were developed to enhance the enzyme's affinity for substrates and catalytic activity.
The mutant exhibited a lower Km value and higher catalytic activity, improved binding to DNA templates, and enhanced the specificity of PCR amplification and compatibility with high GC templates.
Smart Images
Figure CN2025084391_30072026_PF_FP_ABST
Abstract
Description
Pfu DNA polymerase mutants and their application in PCR Technical Field
[0001] This invention belongs to the field of PCR technology, specifically relating to Pfu DNA polymerase mutants and their application in PCR. Background Technology
[0002] PCR technology is the core of molecular biology, possessing powerful functions that enable efficient and rapid enzymatic amplification of specific DNA or RNA sequences from various sources in vitro. Pfu polymerase, discovered in the thermophilic archaeozoa genus *Pyrococcus*, possesses both 5'-3' polymerase and 3'-5' exonuclease activities. During in vivo DNA replication, it can correct erroneously incorporated bases during the polymerization reaction, exhibiting extremely high fidelity. However, with the development of emerging interdisciplinary fields such as synthetic biology, researchers are increasingly seeking polymerases with diverse properties. These include improved substrate affinity, the ability to amplify long fragments, compatibility with high-GC templates, and the capacity to directly extract DNA from crude extracts. Therefore, there is an urgent need to develop different Pfu polymerase mutants to meet the growing demands of laboratory and industrial production. Summary of the Invention
[0003] The purpose of this invention is to address the aforementioned problems in the prior art by providing a Pfu DNA polymerase mutant with strong substrate affinity and high catalytic activity, and its application in PCR.
[0004] To achieve the above objectives, 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 is formed by substitution at least one of the following amino acid sites in the amino acid sequence of wild-type Pfu DNA polymerase or wild-type Pfu DNA polymerase derivative: H680, A684.
[0006] Specifically, K is substituted at H680, and Q is substituted at A684.
[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, wherein the nucleotide sequences encoding wild-type Pfu DNA polymerase and its H680K mutant, A684Q mutant, and H680K+A684Q mutant are shown in SEQ ID NO. 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 NO.13-16, respectively.
[0011] The present invention also provides a recombinant vector comprising the aforementioned nucleotide sequence.
[0012] The present invention also provides a recombinant cell comprising the aforementioned nucleotide sequence or the aforementioned recombinant vector.
[0013] The present invention also provides the application of the aforementioned Pfu DNA polymerase mutant, the aforementioned nucleotide sequence, the aforementioned recombinant vector, and the aforementioned recombinant cells in the field of PCR.
[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0015] The H680K and A684Q mutants described in this invention have lower Km values than the unmutated Pfu DNA polymerase. A lower Km value indicates a higher affinity for the substrate, thereby enhancing the catalytic activity of the Pfu DNA polymerase. Furthermore, compared to the unmutated Pfu DNA polymerase, the H680K mutant mutates histidine at position 680 of the Pfu DNA polymerase to lysine, enhancing the electrostatic interaction with the DNA template by increasing the enzyme's surface electrostatic potential, thus improving amplification specificity. The A684Q mutant mutates alanine at position 684 of the Pfu DNA polymerase to glutamine. The glutamine side chain extends 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 salt tolerance, thus enhancing its compatibility with high-GC DNA templates. Attached Figure Description
[0016] Figure 1 shows the PCR amplification results obtained by using the H680K mutant of wild-type Pfu DNA polymerase derivative as a DNA template and upland cotton gDNA.
[0017] Figure 2 shows a structural comparison between the wild-type Pfu DNA polymerase derivative and its H680K mutant.
[0018] Figure 3 shows the PCR amplification results obtained by using the wild-type Pfu DNA polymerase derivative A684Q mutant with pet16b-derived plasmid DNA as a DNA template under potassium salt concentrations ranging from 10 mM to 100 mM.
[0019] Figure 4 shows the PCR amplification results obtained by using the wild-type Pfu DNA polymerase derivative A684Q mutant with Nipponbare rice gDNA as DNA template at 50mM and 100mM potassium salt concentrations.
[0020] Figure 5 shows a structural comparison between the wild-type Pfu DNA polymerase derivative and its A684Q mutant. Detailed Implementation
[0021] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.
[0022] The present invention provides a Pfu DNA polymerase mutant, which is a substitution of at least one of the following amino acid sites in the amino acid sequence of wild-type Pfu DNA polymerase or 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 wild-type Pfu DNA polymerase derivative was obtained by fusing a double-stranded DNA-binding protein sso7d to the C-terminus of the wild-type Pfu DNA polymerase. 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 nucleotide sequences encoding 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 mutant, A684Q mutant, and H680K+A684Q mutant 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] The application of the aforementioned Pfu DNA polymerase mutant, the aforementioned nucleotide sequence, the aforementioned recombinant vector, and the aforementioned recombinant cells in the field of PCR.
[0031] The following explanation uses the H680K mutant, A684Q mutant, and H680K+A684Q mutant of wild-type Pfu DNA polymerase derivatives as examples. The main materials and instruments used in this invention are shown in Table 1.
[0032] Table 1 Main Materials and Instruments
[0033] Example 1 Enzyme kinetic analysis of wild-type Pfu DNA polymerase derivative mutants
[0034] Enzyme kinetic analysis: Hairpin DNA was pretreated by heating at 98℃ for 5 min and annealing on ice for 30 min. 0.1–2 μM hairpin DNA was used as a substrate template, mixed with dNTPs, 1 nM wild-type Pfu DNA polymerase derivative or its mutant, and DNA synthesis was initiated by adding MgCl2 at 72℃. The reaction buffer contained 25 mM Tris-HCl (pH 8.8), 10 mM KCl, 2.5 mM MgCl2, 200 μM dNTPs, 0.05% Triton X-100, and 1X EvaGreen. Fluorescence curves were plotted by subtracting the fluorescence value from the background for each reaction. The first derivative of the fluorescence curves was calculated to obtain the initial rate of each reaction under different template amounts. The Michaelis-Menten kinetic equation was fitted using GraphPad Prism 8.0 software to obtain k. cat Value and K m Values. Three biological replicates were set up for each group. The base sequence of hairpin DNA was: 5'-CCAGCATTATGA AAGTGACACGTGCACCATTGGTGCACGTG-3'. The enzymatic kinetic analysis results of wild-type Pfu DNA polymerase derivatives and their A684Q, H680K, and A684Q+H680K mutants are shown in Table 2.
[0035] Table 2. Enzyme kinetic analysis results of wild-type Pfu DNA polymerase derivatives and their mutants.
[0036] As shown in Table 2, the Km value of the wild-type Pfu DNA polymerase derivative is 0.45±0.07, while the Km values of its A684Q and H680K mutants are 0.34±0.03 and 0.31±0.06, respectively. It can be seen that A684Q, H680K, and A684Q+H680K mutants all have lower Km values. The lower the Km value, the stronger the binding ability with the substrate. Among them, the Km value of the A684Q+H680K mutant is the lowest, which means that the A684Q+H680K mutant has the strongest binding ability with the substrate.
[0037] Example 2: Amplification performance analysis of wild-type Pfu DNA polymerase derivative mutants
[0038] PCR amplification was performed on different DNA templates using the A684Q and H680K mutants of wild-type Pfu DNA polymerase derivatives, respectively. The PCR amplification system included: 200 μmol / L dNTPs, 40 U / mL wild-type Pfu DNA polymerase derivative mutant, 200 pg / μL hsp20, 120 mmol / L Tris, 10 mmol / L KCl, 6 mmol / L (NH4)2SO4, 2 mmol / L MgCl2, 0.1% w / v Triton X-100, and 0.001% w / v BSA; pH 8.3–9.2. PCR amplification method: DNA templates, forward primers, and reverse primers were added to the aforementioned PCR amplification system, and the reaction was performed using a PCR instrument. The PCR amplification program is shown in Table 3. The A684Q mutant used a pet16b-derived plasmid DNA with a target length of 10 kbp and Nipponbare rice gDNA with a target length of 417 kbp as its DNA template. The Nipponbare rice gDNA was a high-GC template, and PCR amplification was performed at different concentrations of potassium salt ranging from 10 mM to 100 mM. The H680K mutant used upland cotton gDNA with a target length of 1.3 kbp as its DNA template. The corresponding forward and reverse primers are shown in Table 4.
[0039] Table 3 PCR amplification program
[0040] Table 4. Forward and Reverse Primers
[0041] Figure 1 shows the PCR amplification results obtained using upland cotton gDNA as a DNA template with the H680K mutant. 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 significantly improves the specificity of polymerase amplification. The structure of the wild-type Pfu DNA polymerase derivative and its H680K mutant was analyzed using Alphafold3 software. The results are shown in Figure 2. 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.
[0042] Figure 3 shows the PCR amplification results using the A684Q mutant with pet16b-derived plasmid DNA as a template. In the figure, M 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 wild-type Pfu DNA polymerase derivatives is generally 100 mM, indicating that the A684Q mutant has lower salt tolerance. Figure 4 shows the PCR amplification results using the A684Q mutant with Nipponbare rice gDNA as a template. 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 exhibits higher amplification performance for high-GC DNA templates at a salt concentration of 50 mM. The structure of Pfu DNA polymerase and its A684Q mutant was analyzed using Alphafold3 software. The results are shown in Figure 5. In the figure, A684 represents Pfu DNA polymerase and Q684 represents A684Q mutant. It can be seen that the glutamine side chain in A684Q mutant can be inserted more deeply into the double helix groove of the DNA template, making the binding tighter.
[0043] Example 3: Fidelity Analysis of Wild-Type Pfu DNA Polymerase Derivative Mutants
[0044] Fidelity analysis: The linearized pUC19 plasmid vector carrying the lacZ gene was amplified, and the amplification products were then subjected to Agrose gel electrophoresis. The linearized target band of the vector was recovered from the gel, and the recovered products were ligated using T4 ligase. The ligation products were then transformed into the α-complementary *E. coli* host strain Turbo. Aliquots of infected cells were inoculated onto upper agar plates containing 1 mg / mL X-gal and 1.5 mM IPTG LB plates and incubated overnight at 37°C. Standard blue-white screening was performed, and the number of blue-white colonies was counted. The apparent mutation frequency was calculated as: Apparent mutation frequency = (white colonies / total colonies) × 100. Three biological replicates were set up for each group. The fidelity analysis results are shown in Table 5.
[0045] Table 5. Fidelity analysis results of wild-type Pfu DNA polymerase derivatives and their A684Q and H680K mutants.
[0046] As shown in Table 5, the apparent mutation frequency of the wild-type Pfu DNA polymerase derivative was 0.41±0.04, while that of the A684Q mutant was 0.35±0.05. This indicates that the fidelity of the A684Q mutant was basically the same as that of the wild-type Pfu DNA polymerase derivative, while the error rate of the H680K mutant was slightly lower at 0.35±0.05. This suggests that the H680K mutant had better fidelity than the unmutated wild-type Pfu DNA polymerase derivative.
[0047] In summary, the two mutant wild-type Pfu DNA polymerase derivatives described in this invention maintain or have higher fidelity in PCR detection, while improving the Km value of wild-type Pfu DNA polymerase derivatives and enhancing the amplification performance of the enzyme in different scenarios, making them applicable to various cloning, functional analysis, and sequencing.
[0048] Example 4:
[0049] Nucleotide sequences containing Pfu DNA polymerase derivatives were constructed into an E. coli expression vector to form recombinant E. coli cells. The recombinant E. coli cells were lysed by a 6-minute pre-denaturation process during PCR, and PCR amplification was used to screen for NKK saturated mutant libraries at the H680 and A684 positions. Two beneficial mutants of the Pfu DNA polymerase derivative, namely the H680K mutant and the A684Q mutant, were identified.
Claims
1. A Pfu DNA polymerase mutant, characterized by: The Pfu DNA polymerase mutant is formed by substitution at at least one of the following amino acid sites in the amino acid sequence of wild-type Pfu DNA polymerase or wild-type Pfu DNA polymerase derivative: H680, A684.
2. The mutant Pfu DNA polymerase 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 NO. 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 NO.5-8, respectively.
4. The nucleotide sequence encoding the Pfu DNA polymerase mutant of 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 NO. 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 NO.13-16, respectively.
5. A recombinant vector comprising the nucleotide sequence as described in claim 4.
6. Recombinant cells comprising the nucleotide sequence of claim 4 or the recombinant vector of claim 5.
7. The application of a Pfu DNA polymerase mutant as described in any one of claims 1-3, the nucleotide sequence as described in claim 4, the recombinant vector as described in claim 5, and the recombinant cell as described in claim 6 in the field of PCR.