Mutant Taq DNA polymerase and application thereof

Abstract

The invention relates to the technical field of molecular biology, in particular to mutant Taq DNA polymerase and application thereof. On the basis of the wild 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 glutamic acid. Compared with a wild Taq DNA polymerase, the mutant Taq DNA polymerase has the advantages that the assembly error rate is reduced by 51.16%, the enzyme activity is improved to 1.64 times of that of the wild Taq DNA polymerase, and the mutant Taq DNA polymerase has important application value on a genome assembly technology and has important 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 in particular to a mutant Taq DNA polymerase and an application thereof. Background Art

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

[0003] Taq DNA polymerase is a DNA polymerase derived from aquatic thermophilic bacteria (Thermus aquaticus). It has high temperature stability and high catalytic activity. It has become an important tool for molecular biology research because of its key role in PCR reactions. Taq DNA polymerase is also often used in PCA gene assembly reactions, which are generally divided into two steps: the first step is that overlapping oligonucleotide fragments form alternating single-stranded and double-stranded DNA chains through the annealing process, and Taq DNA polymerase extends along the 5' end of the double-stranded DNA to complete the synthesis of double-stranded DNA of the target length; the second step is to add upstream and downstream primers of the target DNA, and Taq DNA polymerase amplifies the number of target DNA molecules through PCR reactions. Because Taq DNA polymerase has good heat resistance, it can maintain activity during multiple cycles of heating in PCA, thereby obtaining a certain number of target genes or DNA fragments for subsequent experiments.

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

[0005] In order to solve the above technical problems, the present invention first performs single-point mutation on the wild-type Taq DNA polymerase to reduce the assembly error rate of the 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, and R795E. It was 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 higher than those of the wild-type Taq DNA polymerase. Furthermore, the present invention attempts to modify wild-type Taq DNA polymerase by multiple mutations, 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 solution is proposed.

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

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

[0008] Preferably, based on the wild-type Taq DNA polymerase, only the following mutations exist: the 31st amino acid residue mutates from lysine to proline, the 186th amino acid residue mutates from threonine to arginine, and the 795th amino acid residue mutates from arginine to glutamic acid.

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

[0010] Preferably, the mutant Taq DNA polymerase has the following site mutation in addition to the above three site mutations: the 169th amino acid residue is mutated from tryptophan to glutamic acid.

[0011] The present invention found that when the above four site mutations exist, the assembly error rate is still low, but the enzyme activity decreases.

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

[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 bacteria or cells.

[0016] In a fourth aspect, the present invention provides a reagent or a 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 for polymerase cycle assembly.

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

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

[0021] In the specific implementation process, the reagent or kit may also include purified water to adjust the volume of the reaction system.

[0022] In the specific implementation process, the reagent or kit may also include a stabilizer, an inert dye, etc., for optimizing the reaction conditions.

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

[0024] In a specific implementation process, the reagent or kit is used for at least one of the following aspects: (1) Conventional PCR testing; (2) Multiplex PCR detection; (3) Sequencing library construction; (4) Genotype identification; (5) Assembly of gene fragments.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention obtains a mutant Taq DNA polymerase that can significantly reduce the assembly error rate by site-directed mutagenesis of the wild-type Taq DNA polymerase. 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 has great application potential in the fields of molecular biology and synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the result of agarose gel electrophoresis of cell lysate and purified protein, among which, Figure 1 A is the agarose gel electrophoresis result of cell lysate. Figure 1 B is the agarose gel electrophoresis result of the purified protein.

[0027] Figure 2 This is a statistical graph of the number of incorrect bases in assembling a 1.5k DNA fragment.

[0028] Figure 3 It is a statistical graph of Taq DNA polymerase activity.

[0029] Figure 4 This is the result of the EGFP fluorescence expression test. The scale bar is 275µm. DETAILED DESCRIPTION

[0030] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, those who do not indicate specific techniques or conditions are carried out according to the techniques or conditions described in the literature in this area, or according to the product specification. Those whose reagents or instruments are not indicated by the manufacturer are all conventional products that can be purchased through regular channels.

[0031] The present invention relates to molecular biology experiments. If not otherwise specified, reference can be made to the book Molecular Cloning (J. Sambrook, EF Fritsch, T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used reference books for those skilled in the art to guide the experimental operations related to molecular biology. In addition, according to different experimental purposes, those skilled in the art can complete the corresponding experiments under the guidance of the operating manuals attached to various commercial kits or entrust specialized companies to perform the experiments, such as gene sequencing, plasmid sequencing, and determination of molecular weight.

[0032] Example 1 Design and preparation of mutant Taq DNA polymerase 1. The amino acid sequence of wild-type Taq DNA polymerase is shown in SEQ ID No.1 (Taq WT ), the following five multi-site mutants were designed: (1) Taq ERE :W169E:T186R:R795E; (2) Taq PRE :K31P:T186R:R795E; (3) Taq PERE : K31P: W169E: T186R: R795E; (4) Taq ERHE :W169E:T186R:K354H:R795E; (5) Taq EREE :W169E:T186R:N415E:R795E.

[0033] 2. Construction of plasmid vector: Based on the pET-28a_6H-TAQ_E602D plasmid (P44831, Miaoling Biotechnology) (sequence see SEQID No. 2), wild-type and mutant expression plasmids were constructed. Primers were designed according to the mutation site, and the target fragment containing the mutation site was amplified. The target fragment was connected to the NdeI and EcoRI sites of the vector to complete the plasmid construction. ERE The primers used for the mutants were aF (SEQ ID No. 3), aR1 (SEQ ID No. 4), aF1 (SEQ ID No. 5), aR (SEQ ID No. 6), Taq PRE The primers used for the mutants were aF, bR1 (SEQ ID No.7), bF1 (SEQ ID No.8), bR2 (SEQ ID No.9), bF2 (SEQ ID No.10), aR, Taq PEREThe primers used for the mutants were aF, bR1, bF1, aR1, aF1, aR, Taq ERHE The primers used for the mutants were aF, aR1, aF1, cR1 (SEQ ID No. 11), cF1 (SEQ ID No. 12), aR, Taq EREE The primers used for the mutants were aF, aR1, aF1, dR1 (SEQ ID No. 13), dF1 (SEQ ID No. 14), and aR. Sequencing confirmed that they were consistent with the design of wild-type and mutant Taq DNA polymerases.

[0034] 3. Prepare solution: (1) Isopropyl β-D-thiogalactoside (IPTG) solution (1 M): Weigh 2.38 g IPTG and add it to 10 mL enzyme-free water. After dissolving, filter using a 0.22 µm filter membrane and store at -20°C until needed.

[0035] (2) Lysis buffer: 50 mM Tris HCl (pH 8.0), 500 mM NaCl, 0.1% NP-40 and 0.1% Triton X-100.

[0036] (3) Imidazole stock solution (2 M): Weigh 1.36 g of imidazole and add it to 10 mL of enzyme-free water. After dissolving, adjust the pH to 8 with hydrochloric acid and store at 4°C until use.

[0037] (4) Gradient elution buffer: 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; 20 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 20 mM imidazole; 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; 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.

[0038] (5) Dialysate / protein storage solution: 50 mM Tris HCl (pH 8.0), 100 mM NaCl.

[0039] 4. Protein induced expression: The plasmids encoding wild-type and mutant Taq DNA polymerase were transformed into BL21 (DE3) competent cells respectively, and the transformed bacterial solution was spread on a solid LB culture plate containing kanamycin, cultured overnight at 37°C, and a single clone was picked and inoculated into 200 mL of LB culture medium containing 50 μg / mL kanamycin, and cultured at 37°C and 220 rpm with shaking until the OD 600 When the value reaches 0.8-1.0, add IPTG solution to the culture medium to a final concentration of 1 mM. Continue to induce the culture medium at 37°C for 2 hours, and collect the cells by centrifugation.

[0040] 5. Cell lysis: Wash the cells twice with PBS, centrifuge and discard the supernatant, add 4mL of lysis buffer to the cell pellet, fully resuspend and add 20µL of 100mg / mL lysozyme solution, mix well. Place the mixture in a four-dimensional rotating mixer and rotate at room temperature for 15min. Heat the cell suspension after enzymatic hydrolysis in a 75℃ water bath for 1h, centrifuge at 16904g for 10min (4℃), collect the supernatant, filter with a 0.22µm filter membrane, add 400µL of glycerol and 1.4µL of 100mM benzamidine to the filtrate, mix well and store at -80℃ for later use.

[0041] 6. Protein purification: Use nickel column (Ni-NTA) for purification. Use lysate to balance the nickel column, add 20µL imidazole storage solution (2M) to the cell lysate, mix well and combine it with the nickel column. Use gradient elution buffer with concentrations from low to high to elute the target protein.

[0042] 7. Protein analysis and quantification: The eluted fractions were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Based on the staining results, the fractions with high purity of the target protein were collected for overnight dialysis. After the dialysis, a 50 kDa ultrafiltration tube was used for ultrafiltration and concentration, and the concentrate was collected for BCA quantification.

[0043] 8. Protein identification: Mass spectrometry analysis confirmed that the mutant Taq DNA polymerase only underwent corresponding multi-site mutations at the designed sites compared to the wild-type Taq DNA polymerase.

[0044] Example 2: Assembling 1.5k DNA fragments with mutant Taq DNA polymerase 1. Use DNAWorks to design and synthesize oligonucleotide (Oligo) sequences based on the 1.5k DNA fragment (SEQ ID No.15), as shown in SEQ ID No.16~SEQ ID No.53, and take 5µL of each to mix as Oligo Mix (10µM).

[0045] 2. Prepare 10×PCR Buffer: 100mM Tris-HCl (pH8.9), 500mM KCl, 15mM MgCl2.

[0046] 3. The cell lysate and purified protein in Example 1 were used to perform PCA to assemble a 1.5k DNA fragment.

[0047] The first round of PCA system was: 2.0μL 10×PCR Buffer, 1.6μL dNTP (2.5mM), 2mg cell lysate / 0.4mg purified protein, 4.0μL Oligos Mix, ddH2O to 20μL. The reaction program was: 95℃ 3min; 98℃ 10s, 60℃ 20s, 72℃ 2min / cycles, 30 cycles in total; 72℃ 5min; 4℃ ∞. The second round of PCR system was: 5.0μL 10×PCR Buffer, 4.0μL dNTP (2.5mM), 5mg cell lysate / 1mg purified protein, 1µL first round PCA product, 1.5µL Primer F (10µM) (SEQ ID No.16), 1.5µL Primer R (10µM) (SEQ ID No.53), ddH2O to 50μL, the reaction program was the same as the first round.

[0048] The second round of PCR products were subjected to agarose gel electrophoresis and recovered, and the DNA concentration was determined using a Qubit 4.0 fluorometer. Figure 1 As shown in A, among the five mutant Taq DNA polymerases, only Taq PRE and Taq PERE The results of agarose gel electrophoresis of the purified protein are as follows Figure 1 As shown in B, Taq PRE and Taq PERE All assembled successfully.

[0049] Furthermore, the recovered product was connected with the vector pUC57 (Bomed, CL118) to construct a plasmid. The connection system was: 5.0μL 2×NEB Builder Buffer, 100ng DNA, 1.0μL pUC57, and ddH2O was added to 10.0μL. The connection product was transformed into DH5α competent cells in a 50℃ metal bath for 1h, and cultured on a solid LB culture plate containing kanamycin at 37℃ for 12-16h. The monoclonal colony was picked for sequencing, and the statistical graph of the number of incorrect bases is shown in the figure. Figure 2 As shown, the statistical error rates are as follows: Taq WT 0.86%, Taq PRE 0.42%, Taq PERE The error rates of the two mutant Taq DNA polymerases were significantly lower than those of the wild type. PRE The error rate of Taq PERE The error rate was reduced by 1.72 times compared with the wild type.

[0050] Further, 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 fluorescent polymerase activity assay kit (Biotium, 29051). The statistical results of Taq DNA polymerase activity are shown in Figure 3 Shown: Taq WT 130.10mU / µL, Taq PRE 213.56 mU / µL, Taq PERE is 72.14mU / µL, of which Taq PRE Activity compared to Taq WT Increased by 1.64 times.

[0051] Example 3: Assembling EGFP gene by mutant Taq DNA polymerase 1. Use DNAWorks to design and synthesize oligonucleotide (Oligo) sequences according to the EGFP gene (SEQ ID No.54), as shown in SEQ ID No.55~SEQ ID No.74, and take 5µL of each to mix as Oligo Mix (10µM).

[0052] 2. Prepare 10×PCR Buffer: 100mM Tris-HCl (pH8.9), 500mM KCl, 15mM MgCl2.

[0053] 3. PCA was performed using the purified protein in Example 1 to assemble the EGFP gene. Taq WT For assembly using wild-type Taq DNA polymerase, TaqPRE and Taq PERE Two mutant Taq DNA polymerases were used for assembly. In the experiment of measuring protein expression, the control group (Control) was untreated cells, and Taq WT , Taq PRE and Taq PERE The groups were wild type, PRE mutant, and PERE mutant Taq DNA polymerase-assembled EGFP gene products transfected into cells.

[0054] The first round of PCA system was: 2.0μL 10×PCR Buffer, 1.6μL dNTP (2.5mM), 0.4mg purified protein, 4.0μL Oligos Mix, ddH2O to 20μL. The reaction program was: 95℃ 3min; 98℃ 10s, 60℃ 20s, 72℃ 1min / cycles, 30 cycles in total; 72℃ 5min; 4℃∞. The second round of PCR system was: 5.0μL 10×PCR Buffer, 4.0μL dNTP (2.5mM), 1mg purified protein, 1µL first round PCA product, 1.5µL Primer F (10µM) (SEQ ID No.55), 1.5µL Primer R (10µM) (SEQ ID No.74), ddH2O to 50μL, the reaction program was the same as the first round.

[0055] The second round of PCR products were subjected to agarose gel electrophoresis and recovered, and the DNA concentration was determined using a Qubit 4.0 fluorescence instrument. The eukaryotic cell expression vector pmScarlet-C1 plasmid (Miaoling Biotechnology, P0918) was double-digested with XhoI / AgeI to obtain a linearized empty vector containing a eukaryotic expression promoter. The recovered product linearized empty vector was connected to construct a gene transfection plasmid. The ligation reaction system was: 5.0μL 2×NEB Builder Buffer, 0.09pmol DNA, 0.03pmol pUC57, and ddH2O was supplemented to 10.0μL. In a 50℃ metal bath for 1h, the ligation product was transformed into DH5α competent cells, inoculated into 15mL LB medium containing kanamycin for overnight culture, and after extracting the plasmid, it was transferred into eukaryotic cells HEK293T to observe the fluorescence expression and calculate the relative fluorescence intensity.

[0056] The results are as follows Figure 4 As shown, Taq PRE and Taq PERE The EGFP fluorescence expression level of the assembled protein was significantly higher than that of the wild type.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions 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 Based on the wild-type Taq DNA polymerase, the following site mutations exist: the 31st amino acid residue mutates from lysine to proline, the 186th amino acid residue mutates from threonine to arginine, and the 795th amino acid residue mutates from arginine to glutamic acid.

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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