A mutant Taq DNA polymerase and its application

Abstract

The present invention relates to the technical field of molecular biology, and particularly to a mutant Taq DNA polymerase and its application. Based on the wild-type Taq DNA polymerase, the mutant Taq DNA polymerase has the following site mutations: the 31st amino acid residue is mutated from lysine to proline, the 186th amino acid residue is mutated from threonine to arginine, and the 795th amino acid residue is mutated from arginine to glutamate. Compared with the wild-type Taq DNA polymerase, the mutant Taq DNA polymerase of the present invention has a 51.16% reduction in the assembly error rate and its enzyme activity is increased to 1.64 times that of the wild-type, which has important application value for gene assembly technology and great application potential in the fields of molecular biology and synthetic biology.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a mutant Taq DNA polymerase and its application. Background Art

[0002] Polymerase cycling assembly (PCA) is an efficient, rapid, and flexible seamless gene assembly method based on DNA polymerase, and has wide applications in the fields of genetic engineering, protein engineering, synthetic biology, etc. The PCA technology assembles chemically synthesized overlapping oligonucleotide fragments into a complete gene or DNA fragment through the polymerase chain reaction (PCR). Among them, DNA polymerase is the core of the PCR reaction and the PCA technology.

[0003] Taq DNA polymerase is a DNA polymerase derived from Thermus aquaticus, which has high temperature stability and high catalytic activity, and has become an important tool for molecular biology research due to its key role in the PCR reaction. Taq DNA polymerase is also often used in the PCA gene assembly reaction, which generally includes two steps: First, the overlapping oligonucleotide fragments form single-stranded and double-stranded alternating DNA strands through the annealing process. Taq DNA polymerase extends along the 5' end of the double strand to complement and synthesize the double-stranded DNA of the target length. Second, the upstream and downstream primers of the target DNA are added, and Taq DNA polymerase amplifies the number of target DNA molecules through the PCR reaction. Since Taq DNA polymerase has good heat resistance, it can maintain its activity during multiple cycles of heating in PCA, so as to obtain a certain number of target genes or DNA fragments for subsequent experiments.

[0004] However, the wild-type Taq DNA polymerase has problems such as insufficient fidelity, reduced specificity and enzyme activity during multiple thermal cycles of PCA. In particular, there is a problem of high assembly error rate of wild-type Taq DNA polymerase in the existing PCA gene assembly technology, which limits the application of the PCA gene assembly technology. Summary of the Invention

[0005] To solve the above technical problems, the present invention first performs single-point mutations on wild-type Taq DNA polymerase in order to reduce the assembly error rate of wild-type Taq DNA polymerase. For example, the present invention has tried single-point mutations such as W318E, W299P, M317L, M236R, W169E, M317P, M236L, Y161K, N415L, W318K, H283R, W645R, Y161R, T186R, W243R, L322E, N415E, D58E, Y339E, R795E. It is found that only 9 mutant Taq DNA polymerases, namely W169E, M236L, N415L, Y161R, T186R, W243R, L322E, Y339E, and R795E, have assembly activity, but the assembly error rates are all higher than that of wild-type Taq DNA polymerase. Further, the present invention attempts to perform multi-point mutations to transform wild-type Taq DNA polymerase, and finally obtains a mutant Taq DNA polymerase with assembly activity and capable of effectively reducing the assembly error rate. Based on this, the following technical solutions are proposed.

[0006] In the first aspect, the present invention provides a mutant Taq DNA polymerase, which has the following site mutations on the basis of wild-type Taq DNA polymerase: the 31st amino acid residue is mutated from lysine to proline, the 186th amino acid residue is mutated from threonine to arginine, and the 795th amino acid residue is mutated from arginine to glutamate.

[0007] Preferably, the amino acid sequence of the wild-type Taq DNA polymerase is as shown in SEQ ID No.1.

[0008] Preferably, on the basis of wild-type Taq DNA polymerase, there are only the following site mutations: the 31st amino acid residue is mutated from lysine to proline, the 186th amino acid residue is mutated from threonine to arginine, and the 795th amino acid residue is mutated from arginine to glutamate.

[0009] The above mutant Taq DNA polymerase not only has a lower assembly error rate, but also has a significantly improved enzyme activity, and the enzyme activity is 1.64 times that of wild-type Taq DNA polymerase.

[0010] Preferably, on the basis of the above three-site mutations of the mutant Taq DNA polymerase, there are also the following site mutations: the 169th amino acid residue is mutated from tryptophan to glutamate.

[0011] The present invention discovers that when there are the above four-site mutations, the assembly error rate is still relatively low, but the enzyme activity decreases.

[0012] Preferably, based on wild-type Taq DNA polymerase, there are only the following site mutations: the 31st amino acid residue is mutated from lysine to proline, the 169th amino acid residue is mutated from tryptophan to glutamic acid, the 186th amino acid residue is mutated from threonine to arginine, and the 795th amino acid residue is mutated from arginine to glutamic acid.

[0013] In a second aspect, the present invention provides a nucleic acid sequence encoding the mutant Taq DNA polymerase.

[0014] In a third aspect, the present invention provides a biological material containing the mutant Taq DNA polymerase or the nucleic acid sequence.

[0015] Preferably, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacterium or cell.

[0016] In a fourth aspect, the present invention provides a reagent or kit containing the mutant Taq DNA polymerase or the nucleic acid sequence.

[0017] Preferably, the reagent or kit is used for PCR reaction.

[0018] Preferably, the reagent or kit is used for polymerase cycling assembly.

[0019] Preferably, the reagent or kit includes the following components: the mutant Taq DNA polymerase, reaction buffer and dNTP mixture.

[0020] In a specific implementation process, the reagent or kit may further include oligonucleotide primers designed according to the target sequence and oligonucleotide fragments for assembling the target DNA fragment.

[0021] In a specific implementation process, the reagent or kit may further include purified water for adjusting the volume of the reaction system.

[0022] In a specific implementation process, the reagent or kit may further include stabilizers, inert dyes, etc. for optimizing the reaction conditions.

[0023] In a fifth aspect, the present invention provides the application of the mutant Taq DNA polymerase or the nucleic acid sequence in the preparation of a reagent or kit.

[0024] In a specific implementation process, the reagent or kit is used for at least one of the following aspects:

[0025] (1) Conventional PCR detection;

[0026] (2) Multiplex PCR detection;

[0027] (3) Sequencing library construction;

[0028] (4) Genotype identification;

[0029] (5) Gene fragment assembly.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] By site-directed mutagenesis of wild-type Taq DNA polymerase, the present invention obtains a mutant Taq DNA polymerase that can significantly reduce the assembly error rate. Compared with the wild-type Taq DNA polymerase, the assembly error rate is reduced by more than 1.72 times. The mutant Taq DNA polymerase of the present invention has important application value for gene assembly technology and great application potential in the fields of molecular biology and synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 are the results of agarose gel electrophoresis of cell lysate and purified protein. Among them, Figure 1 A is the result of agarose gel electrophoresis of cell lysate, Figure 1 B is the result of agarose gel electrophoresis of purified protein.

[0033] Figure 2 is a statistical chart of the number of incorrect bases in assembling 1.5k DNA fragments.

[0034] Figure 3 is a statistical chart of Taq DNA polymerase activity.

[0035] Figure 4 is a test result chart of EGFP fluorescence expression level, and the scale bar is 275 µm. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention. In the embodiments provided in this specification, those not specifying specific techniques or conditions are carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. Those reagents or instruments not indicating the manufacturer can be obtained as conventional products through regular channels.

[0037] The present invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book "Molecular Cloning" (written by J. Sambrook, E.F. Fritsch, and T. Maniatis, published by Science Press in 1994). This book and its subsequent published versions are the most commonly used and guiding reference books for those skilled in the art when conducting experiments related to molecular biology. In addition, depending on different experimental purposes, those skilled in the art complete corresponding experiments under the guidance of the operation manuals attached to various commercial kits or entrust professional companies to do so, such as gene sequencing, plasmid sequencing, and determining molecular weight, etc.

[0038] Example 1 Design and Preparation of Mutant Taq DNA Polymerase

[0039] 1. The amino acid sequence of wild-type Taq DNA polymerase is shown in SEQ ID No.1 (Taq WT ), and the following 5 multi-site mutants were designed:

[0040] (1) Taq ERE : W169E: T186R: R795E;

[0041] (2) Taq PRE : K31P: T186R: R795E;

[0042] (3) Taq PERE : K31P: W169E: T186R: R795E;

[0043] (4) Taq ERHE : W169E: T186R: K354H: R795E;

[0044] (5) Taq EREE : W169E: T186R: N415E: R795E.

[0045] 2. Plasmid vector construction: Based on the pET-28a_6H-TAQ_E602D plasmid (P44831, Miaoling Biology) (the sequence is shown in SEQ ID No.2), wild-type and mutant expression plasmids were constructed. Primers were designed according to the mutation sites, and the target fragments containing the mutation sites were amplified. The target fragments were ligated into the NdeI and EcoRI sites of the vector to complete plasmid construction. Among them, the primers used for Taq ERE mutants were aF (SEQ ID No.3), aR1 (SEQ ID No.4), aF1 (SEQ ID No.5), and aR (SEQ ID No.6), and for Taq PREThe primers used for the mutants are aF, bR1 (SEQ ID No.7), bF1 (SEQ ID No.8), bR2 (SEQ ID No.9), bF2 (SEQ ID No.10), aR, Taq PERE The primers used for the mutants are aF, bR1, bF1, aR1, aF1, aR, Taq ERHE The primers used for the mutants are aF, aR1, aF1, cR1 (SEQ ID No.11), cF1 (SEQ IDNo.12), aR, Taq EREE The primers used for the mutants are aF, aR1, aF1, dR1 (SEQ ID No.13), dF1 (SEQ IDNo.14), aR. It was verified by sequencing to meet the design of wild-type and mutant Taq DNA polymerase.

[0046] 3. Prepare solutions:

[0047] (1) Isopropyl-β-D-thiogalactoside (IPTG) solution (1M): Weigh 2.38 g of IPTG and add it to 10 mL of enzyme-free water. After dissolution, filter it with a 0.22 µm filter membrane and store it at -20 °C for later use.

[0048] (2) Lysis buffer: 50 mM Tris HCl (pH 8.0), 500 mM NaCl, 0.1% NP-40 and 0.1% Triton X-100.

[0049] (3) Imidazole stock solution (2M): Weigh 1.36 g of imidazole and add it to 10 mL of enzyme-free water. After dissolution, adjust the pH to 8 with hydrochloric acid and store it at 4 °C for later use.

[0050] (4) Gradient elution buffer:

[0051] 10 mM elution buffer: 50 mM Tris HCl (pH 8.0), 500 mM NaCl / 100 mM NaCl, 0.05% NP-40, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM benzamidine and 10 mM imidazole;

[0052] 20 mM elution buffer: 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 0.05% NP-40, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM benzamidine and 20 mM imidazole;

[0053] 50 mM eluent: 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 0.05% NP-40, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM benzamidine, and 50 mM imidazole;

[0054] 300 mM eluent: 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 0.05% NP-40, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM benzamidine, and 300 mM imidazole.

[0055] (5)Dialysis buffer / protein storage solution: 50 mM Tris HCl (pH 8.0), 100 mM NaCl.

[0056] 4. Protein induction and expression: Plasmids encoding wild-type and mutant Taq DNA polymerase were separately transformed into BL21(DE3) competent cells. The transformed bacterial solutions were spread on solid LB culture plates containing kanamycin and cultured overnight at 37 °C. Single colonies were picked and inoculated into 200 mL of LB medium containing 50 μg / mL kanamycin, and cultured with shaking at 37 °C and 220 rpm until the OD 600 value reached 0.8 - 1.0. IPTG solution was added to the culture medium to a final concentration of 1 mM. The bacterial solution was continued to be induced and cultured at 37 °C for 2 h, and the cells were collected by centrifugation.

[0057] 5. Cell lysis: The cells were washed twice with PBS, and the supernatant was discarded by centrifugation. 4 mL of lysis buffer was added to the cell pellet, and after thorough resuspension, 20 μL of 100 mg / mL lysozyme solution was added and mixed well. The mixture was placed in a four-dimensional rotary mixer and rotated at room temperature for 15 min. The enzymatically digested cell suspension was placed in a 75 °C water bath and heated for 1 h, centrifuged at 16904 g for 10 min (4 °C), the supernatant was collected, filtered through a 0.22 μm filter membrane, 400 μL of glycerol and 1.4 μL of 100 mM benzamidine were added to the filtrate, and after mixing, it was stored at -80 °C for later use.

[0058] 6. Protein purification: Purification was performed using a nickel column (Ni-NTA). The nickel column was equilibrated with lysis buffer. 20 μL of imidazole stock solution (2 M) was added to the cell lysate, and after mixing, it was bound to the nickel column. The target protein was eluted successively with gradient elution buffers of increasing concentration.

[0059] 7. Protein analysis and quantification: The eluted fractions were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. According to the staining situation, fractions with high purity of the target protein were collected for overnight dialysis. After dialysis, ultrafiltration concentration was performed using a 50 kDa ultrafiltration tube, and the concentrated solution was collected for BCA quantification.

[0060] 8. Protein identification: It was determined by mass spectrometry analysis that the mutant Taq DNA polymerase had corresponding multiple-site mutations only at the designed sites compared with the wild-type Taq DNA polymerase.

[0061] Example 2 Assembly of 1.5k DNA Fragment by Mutant Taq DNA Polymerase

[0062] 1. Use DNAWorks to design and synthesize oligonucleotide (Oligo) sequences according to the 1.5k DNA fragment (SEQ ID No.15), as shown in SEQ ID No.16 to SEQ ID No.53 respectively. Take 5 μL of each and mix them into Oligo Mix (10 μM).

[0063] 2. Prepare 10×PCR Buffer: 100 mM Tris-HCl (pH 8.9), 500 mM KCl, 15 mM MgCl2.

[0064] 3. Use the cell lysate and purified protein in Example 1 respectively to perform PCA to assemble the 1.5k DNA fragment.

[0065] The system for the first round of PCA was: 2.0 μL of 10×PCR Buffer, 1.6 μL of dNTP (2.5 mM), 2 mg of cell lysate / 0.4 mg of purified protein, 4.0 μL of Oligos Mix, and ddH2O was added to make up 20 μL. The reaction program was: 95°C for 3 min; 98°C for 10 s, 60°C for 20 s, 72°C for 2 min / cycles, a total of 30 cycles; 72°C for 5 min; 4°C ∞. The system for the second round of PCR was: 5.0 μL of 10×PCR Buffer, 4.0 μL of dNTP (2.5 mM), 5 mg of cell lysate / 1 mg of purified protein, 1 μL of the product of the first round of PCA, 1.5 μL of Primer F (10 μM) (SEQ ID No.16), 1.5 μL of Primer R (10 μM) (SEQ ID No.53), and ddH2O was added to make up 50 μL. The reaction program was the same as that of the first round.

[0066] Perform agarose gel electrophoresis on the product of the second round of PCR and recover it. Use a Qubit 4.0 fluorometer to measure the DNA concentration. The results of agarose gel electrophoresis of the cell lysate are as Figure 1 shown in A. Among the 5 mutant Taq DNA polymerases, only Taq PRE and Taq PERE had assembly activity. The results of agarose gel electrophoresis of the purified protein are as Figure 1 shown in B. Taq PRE and TaqPERE All were successfully assembled.

[0067] Furthermore, the recycled product was ligated with vector pUC57 (Bomed, CL118) to construct a plasmid. The ligation system was as follows: 5.0 μL of 2×NEB Builder Buffer, 100 ng of DNA, 1.0 μL of pUC57, and ddH2O was added to make up to 10.0 μL. Incubate in a metal bath at 50 °C for 1 h. The ligation product was transformed into DH5α competent cells and cultured at 37 °C for 12 - 16 h on a solid LB culture plate containing kanamycin. Single colonies were picked for sequencing. The statistical chart of the number of incorrect bases is as shown in Figure 2 and the statistical error rates are as follows: Taq WT was 0.86%, Taq PRE was 0.42%, Taq PERE was 0.50%. The error rates of both mutant Taq DNA polymerases were significantly lower than that of the wild type. The error rate of Taq PRE was reduced by 2.05 times compared to the wild type, and the error rate of Taq PERE was reduced by 1.72 times compared to the wild type.

[0068] Furthermore, a part of the purified protein was uniformly diluted to 0.01 μg / μL, and the enzyme activity of Taq DNA polymerase was detected using a fluorescence polymerase activity assay kit (Biotium, 29051). The statistical results of Taq DNA polymerase activity are as shown in Figure 3 : Taq WT was 130.10 mU / µL, Taq PRE was 213.56 mU / µL, Taq PERE was 72.14 mU / µL. Among them, the activity of Taq PRE was increased by 1.64 times compared to Taq WT .

[0069] Example 3 Assembly of EGFP Gene by Mutant Taq DNA Polymerase

[0070] 1. Use DNAWorks to design and synthesize oligonucleotide (Oligonucleotide, Oligo) sequences according to the EGFP gene (SEQ ID No.54), which are shown in SEQ ID No.55 - SEQ ID No.74 respectively. Take 5 µL of each and mix them to form Oligo Mix (10 µM).

[0071] 2. Prepare 10×PCR Buffer: 100 mM Tris - HCl (pH8.9), 500 mM KCl, 15 mM MgCl2.

[0072] 3. The purified protein in Example 1 was used to assemble the EGFP gene by PCA, and Taq WT was assembled using wild-type Taq DNA polymerase, and Taq PRE and Taq PERE were assembled using two mutant Taq DNA polymerases. In the experiment for measuring protein expression, the control group (Control) was untreated cells, and Taq WT , Taq PRE and Taq PERE groups were the products of assembling the EGFP gene with wild-type, PRE mutant, and PERE mutant Taq DNA polymerases transfected into cells respectively.

[0073] The first-round PCA system was: 2.0 μL 10×PCR Buffer, 1.6 μL dNTP (2.5 mM), 0.4 mg purified protein, 4.0 μL Oligos Mix, and ddH2O was added to make up 20 μL. The reaction program was: 95°C for 3 min; 98°C for 10 s, 60°C for 20 s, 72°C for 1 min / cycles, for a total of 30 cycles; 72°C for 5 min; 4°C ∞. The second-round PCR system was: 5.0 μL 10×PCR Buffer, 4.0 μL dNTP (2.5 mM), 1 mg purified protein, 1 μL of the first-round PCA product, 1.5 μL Primer F (10 μM) (SEQ ID No. 55), 1.5 μL Primer R (10 μM) (SEQ ID No. 74), and ddH2O was added to make up 50 μL. The reaction program was the same as the first round.

[0074] The second-round PCR product was subjected to agarose gel electrophoresis and recovered, and the DNA concentration was measured using a Qubit 4.0 fluorometer. The eukaryotic expression vector pmScarlet-C1 plasmid (Miaoling Biology, P0918) was double-digested with XhoI / AgeI to obtain a linearized empty vector containing the eukaryotic expression promoter. The recovered product was ligated with the linearized empty vector to construct a gene transfection plasmid. The ligation reaction system was: 5.0 μL 2×NEB Builder Buffer, 0.09 pmol DNA, 0.03 pmol pUC57, and ddH2O was added to make up to 10.0 μL. Incubate in a 50°C metal bath for 1 h. The ligation product was transformed into DH5α competent cells, inoculated into 15 mL of LB medium containing kanamycin and cultured overnight. After plasmid extraction, it was transferred into eukaryotic cells HEK293T to observe the fluorescence expression and statistical relative fluorescence intensity.

[0075] The results were as Figure 4 shown, Taq PRE and Taq PEREThe assembled EGFP fluorescence expression level was significantly higher than that of the wild type.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant Taq DNA polymerase, characterized in that The wild-type Taq DNA polymerase has the following mutations: the 31st amino acid residue is mutated from lysine to proline, the 186th amino acid residue is mutated from threonine to arginine, and the 795th amino acid residue is mutated from arginine to glutamic acid; the amino acid sequence of the wild-type Taq DNA polymerase is shown in SEQ ID No.

1.

2. The mutant Taq DNA polymerase according to claim 1, characterized in that The following mutations also occurred: the amino acid residue at position 169 mutated from tryptophan to glutamic acid.

3. A nucleic acid sequence encoding the mutant Taq DNA polymerase according to claim 1 or 2.

4. A biological material containing the mutant Taq DNA polymerase according to claim 1 or 2 or the nucleic acid sequence according to claim 3.

5. The biomaterial according to claim 4, characterized in that The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or cell.

6. A reagent or kit comprising the mutant Taq DNA polymerase according to claim 1 or 2 or the nucleic acid sequence according to claim 3.

7. The reagent or kit according to claim 6, characterized in that The reagent or kit is used for PCR reaction.

8. The reagent or kit according to claim 7, characterized in that The reagents or kits are used for polymerase cycle assembly.

9. The reagent or kit according to claim 8, characterized in that The reagent or kit comprises the following components: the mutant Taq DNA polymerase according to claim 1 or 2, a reaction buffer and a dNTP mixture.

10. Use of the mutant Taq DNA polymerase according to claim 1 or 2 or the nucleic acid sequence according to claim 3 in the preparation of a reagent or a kit.

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