Tth dna polymerase mutants and uses thereof

Abstract

The present application discloses Tth DNA polymerase mutants, which have the following mutations on the basis of Tth wild-type polymerase: S515K, I640F, S741G and L789F; D734G and M763T; or E617D, D734G and M763T. The Tth DNA polymerase mutants of the present application have reverse transcription activity close to the activity of M-MLV RT wild-type under manganese ion-independent conditions. The Tth DNA polymerase mutants of the present application are suitable for being prepared into kits, and have wide application prospects.

Description

[0001] This application is a divisional application of patent application 202211666064.6 entitled "Tth DNA polymerase mutants and applications thereof" filed by the applicant on December 23, 2022. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular to Tth DNA polymerase mutants and applications thereof. BACKGROUND

[0003] Tth DNA polymerase is a single subunit polymerase with a molecular weight of about 94 KDa isolated from Thermus thermophilus. Currently, Tth DNA polymerase is imported from abroad, which is expensive, and high-efficiency production has not been achieved in China, which seriously hinders the development of the biological field in China. Tth DNA polymerase was initially extracted by fermentation culture of natural strain Thermus thermophilus, but the optimal growth temperature of the strain is 75℃, the culture is difficult, the enzyme yield is low, and the growth rate of the strain is extremely slow and the growth density is low. Therefore, researchers cloned the gene of the polymerase using a gene library and then introduced it into mesophilic microorganism Escherichia coli for expression, and the enzyme activity was more than 12 times higher than that of the natural strain. There are few reports on the enzyme in China. Researcher Jiang Yongqiang only amplified and cloned Tth gene in two segments by PCR, but failed to express the gene successfully. Wang Qiao et al successfully expressed active Tth DNA polymerase, but the yield, purity and activity were not high.

[0004] Tth DNA polymerase not only has DNA polymerase activity, but also has reverse transcriptase activity, and the enzyme is resistant to high temperature, and the activity does not decrease after reacting for several hours at 70℃. Unlike Taq DNA polymerase also derived from Thermus, Tth DNA polymerase exhibits general DNA polymerase activity in the presence of only magnesium ions; but in the presence of manganese ions, the enzyme has high reverse transcriptase activity and DNA polymerase activity, and the mispairing rate of the polymerase increases. Therefore, researchers quickly applied Tth DNA polymerase to the reverse transcription-fluorescence quantitative PCR detection of RNA viruses such as hepatitis C and influenza virus. Researchers have also found that Tth DNA polymerase has strong tolerance to PCR inhibitor components contained in biological tissues such as blood and muscle, and is very suitable for rapid detection of crude samples. However, wild-type Tth DNA polymerase has low specific activity, lower thermal stability than Taq DNA polymerase, and manganese ions affect the fidelity of PCR amplification, which hinders its wide application. SUMMARY

[0005] The present application aims to provide a Tth DNA polymerase mutant, which has reverse transcription activity close to that of the wild type M-MLV RT under manganese ion-independent conditions.

[0006] In a first aspect, the present application provides a Tth DNA polymerase mutant, which has the following set of mutations on the basis of a Tth wild type polymerase: S515K, I640F, S741G and L789F; D734G and M763T; or E617D, D734G and M763T, the amino acid sequence of the Tth wild type polymerase being shown as SEQ ID NO: 2.

[0007] In a second aspect, the present application provides a nucleic acid molecule, which encodes the Tth DNA polymerase mutant of the first aspect.

[0008] In a third aspect, the present application provides an expression vector, which contains the nucleic acid molecule of the second aspect.

[0009] In a fourth aspect, the present application provides a host, which is transfected or transformed with the expression vector of the third aspect.

[0010] In a fifth aspect, the present application provides a kit, which contains the Tth DNA polymerase mutant of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect or the host of the fourth aspect.

[0011] In a sixth aspect, the present application provides a DNA synthesis method, which comprises the step of using the Tth DNA polymerase mutant of the first aspect as a DNA polymerase, or the step of using the kit of the fifth aspect to synthesize DNA.

[0012] The Tth DNA polymerase mutant of the present application has reverse transcription activity close to that of the wild type M-MLV RT under manganese ion-independent conditions. The Tth DNA polymerase mutant has wide application value. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings involved in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the embodiments of the present application, and for those skilled in the art, other embodiments can also be obtained from these drawings without creative labor.

[0014] Figure 1Figure 6 shows the results of a DNA polymerase purification electrophoresis (Ni-NTA affinity chromatography) for the embodiments of the present application, A: wild-type M-MLV RT; B: mutant THY1; C: mutant THY2; D: mutant THY3.

[0015] Figure 2 Figure 7 shows the results of a DNA polymerase purification electrophoresis (ion exchange method) for the embodiments of the present application, A: wild-type M-MLV RT; B: mutant THY1; C: mutant THY2; D: mutant THY3.

[0016] Figure 3 Figure 8 shows the results of qPCR using Tth DNA polymerase mutants (Ni-NTA affinity chromatography) for the embodiments of the present application.

[0017] Figure 4 Figure 9 shows the results of qPCR using Tth DNA polymerase mutants (ion exchange method) for the embodiments of the present application. DETAILED DESCRIPTION

[0018] Hereinafter, the present application will be described in detail with reference to the accompanying drawings. It is to be understood that the following description is merely illustrative of the present application and does not in any way limit the scope of the present application as defined in the appended claims. And it is to be understood that those skilled in the art can make modifications to the technical solutions of the present application without departing from the spirit and scope of the present application. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0019] Unless otherwise specified, the scientific and technical terms used in the present application have the meanings commonly understood by those skilled in the art. In order to better understand the present application, the definitions and explanations of the relevant terms are provided below.

[0020] In the present application, the term "nucleic acid molecule" refers to a single-stranded or double-stranded RNA or DNA polymer.

[0021] In the present application, the term "degenerate sequence" refers to a sequence in which one or more codons in a nucleotide sequence are replaced by degenerate codons that encode the same amino acid.

[0022] In the present document, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression regulatory sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises cis-acting elements sufficient for expression; other elements for expression can be provided by a host cell or in an in vitro expression system. In the present invention, the expression vector can include all expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses incorporating a recombinant polynucleotide, such as lentivirus, retrovirus, adenovirus, herpes simplex virus, and adeno-associated virus, but is not limited thereto.

[0023] In the present document, the term "host" refers to a cell that can be used to express a protein encoded by an expression vector. The host cell can be a prokaryote, such as Escherichia coli, or a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi).

[0024] In the present document, the term "transfection" refers to a process in which a cell is actively or passively introduced with an exogenous DNA fragment under certain conditions to obtain a new phenotype.

[0025] In the present document, the term "transformation" refers to a process in which plasmid DNA or a recombinant DNA constructed using the same is introduced into a bacterial body.

[0026] In the present document, amino acids are represented by one-letter and three-letter abbreviations well known in the art. For example, glycine is represented by G or Gly, alanine is represented by A or Ala, valine is represented by Val or V, leucine is represented by Leu or L, isoleucine is represented by Ile or I, proline is represented by Pro or P, phenylalanine is represented by Phe or F, tyrosine is represented by Tyr or Y, tryptophan is represented by Trp or W, serine is represented by Ser or S, threonine is represented by Thr or T, cysteine is represented by Cys or C, methionine is represented by Met or M, asparagine is represented by Asn or N, glutamine is represented by Gln or Q, aspartic acid is represented by Asp or D, glutamic acid is represented by Glu or E, lysine is represented by Lys or K, arginine is represented by Arg or R, and histidine is represented by His or H. Similarly, bases are also represented by one-letter abbreviations well known in the art, for example, adenine is represented by A, guanine is represented by G, cytosine is represented by C, thymine is represented by T, and uracil is represented by U.

[0027] In this text, the representation method of amino acid sequence mutation site is wild type amino acid, site, mutant amino acid, without space between the three, for example, S515K represents that the 515th serine residue (S) in the amino acid sequence is replaced by lysine residue (K), I640F represents that the 640th isoleucine residue (I) in the amino acid sequence is replaced by phenylalanine residue (F), S741G represents that the 741st serine residue (S) in the amino acid sequence is replaced by glycine residue (G), L789F represents that the 789th leucine residue (L) in the amino acid sequence is replaced by phenylalanine residue (F), M763T represents that the 763rd methionine residue (M) in the amino acid sequence is replaced by threonine residue (T), D734G represents that the 734th aspartic acid residue (D) in the amino acid sequence is replaced by glycine residue (G), and E617D represents that the 617th glutamic acid residue (E) in the amino acid sequence is replaced by aspartic acid residue (D).

[0028] In this text, the STO structure refers to a nucleotide sequence capable of enhancing reverse transcription performance, and the nucleotide sequence is ATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGAAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAAGGCACCGGCGGCAGCGGCGGCGGCAGC, and the corresponding amino acid sequence is MVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKKGTGGSGGGS.

[0029] In this text, Tth DNA polymerase is a single subunit polymerase with a molecular weight of about 94 KDa isolated from Thermus thermophilus. The nucleotide sequence of the wild type Tth polymerase is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The present inventors screened a mutant by point mutation of the wild type Tth polymerase, and the mutant has reverse transcription activity close to that of the wild type M-MLV RT under manganese ion-independent conditions.

[0030] Accordingly, in a first aspect, the present application provides a Tth DNA polymerase mutant, which has the following set of mutations on the basis of Tth wild-type polymerase: S515K, I640F, S741G and L789F (THY1); D734G and M763T (THY2); or E617D, D734G and M763T (THY3). Mutant THY3 includes a STO structure (located at the 5' end) to enhance reverse transcription performance. The amino acid sequence of mutant THY1 is shown in SEQ ID NO: 4; the amino acid sequence of mutant THY2 is shown in SEQ ID NO: 6; and the amino acid sequence of mutant THY3 is shown in SEQ ID NO: 8.

[0031] In a second aspect, the present application provides a nucleic acid molecule encoding the Tth DNA polymerase mutant of the first aspect. The nucleic acid molecule comprises: a nucleotide sequence shown in SEQ ID NO: 3 or a degenerate sequence thereof, a nucleotide sequence shown in SEQ ID NO: 5 or a degenerate sequence thereof, or a nucleotide sequence shown in SEQ ID NO: 7 or a degenerate sequence thereof.

[0032] In a third aspect, the present application provides an expression vector containing the nucleic acid molecule of the second aspect.

[0033] In some embodiments, the construction of the expression vector is achieved by linking the nucleic acid molecule into a T7 promoter-based expression vector pET series.

[0034] In a fourth aspect, the present application provides a host transfected or transformed with the expression vector of the third aspect.

[0035] In some embodiments, the host is E. coli, the culture temperature is 16°C, 24°C, 30°C or 37°C, and the culture time is 20h, 12h, 7h or 3h.

[0036] In some preferred embodiments, the host is E. coli, the culture temperature is 16°C, and the culture time is 20h.

[0037] In a fifth aspect, the present application provides a kit comprising the Tth DNA polymerase mutant of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the host of the fourth aspect.

[0038] In some embodiments, the kit further comprises buffers, primers required for DNA polymerization.

[0039] In the sixth aspect, the present application provides a DNA synthesis method, which comprises the step of using the mutant Tth DNA polymerase of the first aspect as a DNA polymerase, or the step of using the kit of the fifth aspect to perform DNA synthesis.

[0040] Examples

[0041] Expression and purification of the mutant Tth DNA polymerase

[0042] The DNA sequence of the mutant Tth DNA polymerase was sent to Beijing Genki Biotechnology Co., Ltd. for synthesis, and the nucleic acid molecule was connected to a commercial expression vector pET series based on the T7 promoter to construct an expression plasmid, which contains an operator sequence for IPTG-induced expression, ensuring efficient expression of the protein in E. coli. The recombinant positive plasmid was transformed into E. coli, and well-grown positive single colonies were selected for culture. When the OD 600 was 0.6-0.8, IPTG was added for induction, and the bacterial solution was collected. The cells were broken by ultrasonic cell disrupter, and the supernatant was collected by high-speed centrifugation to obtain crude enzyme solution. After equilibrating the Ni-NTA affinity chromatography column with buffer, the crude enzyme solution was passed through the chromatography column, and the protein solution was collected by elution with elution buffer containing 20 mM, 50 mM, and 300 mM imidazole, respectively. SDS-PAGE electrophoresis analysis was performed.

[0043] The target protein obtained from the Ni-NTA affinity chromatography column was subjected to anion exchange chromatography to remove impurities and improve protein purity. Before use, the system was cleaned with a buffer equilibration instrument, and then the ion exchange column (Q column) was connected. After loading the protein solution, linear gradient elution was performed with high salt (1 M NaCl), and the sample was collected according to the ultraviolet absorption detection peak. SDS-PAGE electrophoresis was used to detect the target protein and purity, and finally the target protein was collected after overnight dialysis.

[0044] The Tth DNA polymerase obtained by Ni-NTA affinity chromatography had a molecular weight consistent with the expected value, but the purity was low, with impurities Figure 1 ). After anion exchange chromatography, high-purity Tth DNA polymerase with a molecular weight consistent with the expected value was obtained Figure 2 ).

[0045] Detection of reverse transcription efficiency of the mutant Tth DNA polymerase

[0046] MS II (purchased from Roche Group) was used as the template RNA, and the amount used was 1 ng. Random primers were used for reverse transcription, and the reverse transcription system and procedure are shown in Tables 1 and 2. The composition of the buffer used in the reverse transcription is: 50 mM Tris-HCl, pH 8.8, 3 mM Mg 2+ , 50 mM K +, 20 mM NH 4+ The cDNA was diluted 3 times, and 1 μL was used for real-time qPCR reaction. Two pairs of primers were used to detect the amplification efficiency. The qPCR specific primers are shown in Table 3, and the qPCR reaction system and procedure are shown in Table 4 and Table 5.

[0047] Table 1: Reverse transcription reaction system

[0048]

[0049]

[0050] Table 2: Reverse transcription reaction procedure

[0051] Temperature Time 25℃ 10 min 37℃-60℃ 15 min 85℃ 5 min

[0052] Table 3: qPCR specific primers

[0053]

[0054] Table 4: qPCR reaction system

[0055]

[0056]

[0057] Table 5: qPCR reaction procedure

[0058] Temperature Time 95℃ 1 min 95℃ 10s 60℃ 15s

[0059] Note: The last two procedures are 40 cycles in total.

[0060] Here, the PCR product amount is compared by Ct value, and then the reverse transcription efficiency is judged, that is, the smaller the Ct value, the lower the cycle number required to reach the threshold of the qPCR instrument, the higher the initial reverse transcription product amount, the higher the reverse transcription efficiency of the enzyme and the better the activity. The three Tth DNA polymerase mutants obtained by Ni-NTA affinity chromatography and anion exchange column purification and separation were detected for reverse transcription activity by RT-qPCR technology. As shown in Table 6, the three mutants obtained by affinity chromatography were compared with the wild type M-MLV reverse transcriptase as a control, and the qPCR results showed that the Ct values of the three mutants were slightly higher than the control, with a difference of about 1 Ct, indicating that the reverse transcription activity of the mutants was slightly lower than the control. Among them, the activity of THY2 was the best, followed by THY3, and the activity of THY1 was the worst. Figure 3 Figure 4 ​As shown, the results of qPCR showed that the Ct values of the three mutant Tth DNA polymerases were slightly higher than that of the wild-type M-MLV reverse transcriptase, indicating that the reverse transcription activity of the mutants was slightly lower than that of the control, and the activity of THY3 was the best, followed by THY2, and THY1 was the worst. Overall, under the condition of not relying on manganese ions, the reverse transcription activity of the three Tth DNA polymerase mutants was very close to that of the wild-type M-MLV reverse transcriptase.

[0061] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A Tth DNA polymerase mutant, characterized in that, The mutant has the following set of mutations based on the wild-type Tth polymerase: E617D, D734G and M763T. The mutant also contains an STO structure. The amino acid sequence of the wild-type Tth polymerase is shown in SEQ ID NO:2, and the amino acid sequence of the mutant is shown in SEQ ID NO:

8.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the Tth DNA polymerase mutant of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecule is a nucleotide sequence or a degenerate sequence thereof as shown in SEQ ID NO:

7.

4. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 2 or 3.

5. The expression vector according to claim 4, characterized in that, The expression vector was constructed by linking the nucleic acid molecule into the pET series of expression vectors based on the T7 promoter.

6. A host, characterized in that, The host is transfected or transformed with the expression vector of claim 4 or 5, wherein the host is not an animal or plant species.

7. The host according to claim 6, characterized in that, The host is Escherichia coli, and the culture temperature is 16℃, 24℃, 30℃ or 37℃, and the culture time is 20h, 12h, 7h or 3h.

8. The host according to claim 7, characterized in that, The host was Escherichia coli, and the culture temperature was 16°C for 20 hours.

9. A reagent kit, characterized in that, The kit comprises the Tth DNA polymerase mutant of claim 1, the nucleic acid molecule of claim 2 or 3, the expression vector of claim 4 or 5, or the host of any one of claims 6-8.

10. The reagent kit according to claim 9, characterized in that, The kit also includes buffers and primers required for DNA polymerization.

11. A method for DNA synthesis, characterized in that, The method includes the step of using the Tth DNA polymerase mutant of claim 1 as a DNA polymerase, or the step of using the kit of claim 9 or 10 to synthesize DNA.

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