Tth DNA polymerase mutant and application thereof

Abstract

The invention discloses a Tth DNA polymerase mutant. On the basis of Tth wild type polymerase, the mutant has a group of mutations as follows: S515K, I640F, S741G and L789F; a D734G unit and an M763T unit; or E617D, D734G, and M763T, and the like. The reverse transcription activity of the Tth DNA polymerase mutant disclosed by the invention is close to the activity of an M-MLV RT wild type under the condition that the Tth DNA polymerase mutant does not depend on manganese ions. The Tth DNA polymerase mutant provided by the invention is suitable for being prepared into a kit, and has a wide application prospect.

Description

[0001] This application is a divisional application of patent application 202211666064.6 entitled “Tth DNA polymerase mutants and their applications” filed by the applicant on December 23, 2022. Technical Field

[0002] The invention relates to the field of biotechnology, and in particular to a Tth DNA polymerase mutant and an application thereof. Background Art

[0003] Tth DNA polymerase is a single-subunit polymerase with a molecular weight of about 94KDa isolated from Thermus thermophilus. At present, Tth DNA polymerases are all imported from abroad and are expensive. Efficient production has not been achieved in China, which seriously hinders the development of my country's biological field. Tth DNA polymerase was initially extracted by fermentation culture of the natural strain Thermus thermophilus, but the optimal temperature for the growth of the bacterium is 75°C, the culture is difficult, the enzyme production is low, and the growth rate of the bacterium is extremely slow and the growth density is low. Therefore, some researchers cloned the gene of the polymerase using a gene library and then introduced it into the mesophilic microorganism Escherichia coli for expression. The enzyme activity is more than 12 times higher than that of the natural strain. There are few reports on the research of this enzyme in China. Researcher Jiang Yongqiang only cloned the Tth gene by two-stage PCR amplification, but failed to successfully express the gene. 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 reverse transcriptase activity. The enzyme is resistant to high temperatures, and its activity does not decrease after reacting for several hours at 70°C. Unlike Taq DNA polymerase, which also originates from Thermus, Tth DNA polymerase exhibits general DNA polymerase activity when only magnesium ions are present; but in the presence of manganese ions, the enzyme has higher reverse transcriptase activity and DNA polymerase activity, and the mismatch rate of the polymerase increases. Therefore, researchers quickly applied Tth DNA polymerase to reverse transcription-fluorescence quantitative PCR detection of RNA viruses such as hepatitis C and influenza virus. Researchers 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, the wild-type Tth DNA polymerase has defects such as low specific activity, inferior thermal stability to Taq DNA polymerase, and manganese ions affecting the fidelity of PCR amplification, which hinders its widespread application. Summary of the invention

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

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

[0007] In a second aspect, the present invention provides a nucleic acid molecule encoding the TthDNA polymerase mutant described in the first aspect.

[0008] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0009] In a fourth aspect, the present invention provides a host, wherein the host is transfected or transformed with the expression vector described in the third aspect.

[0010] In a fifth aspect, the present invention provides a kit comprising the Tth DNA polymerase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the host described in the fourth aspect.

[0011] In a sixth aspect, the present invention provides a method for DNA synthesis, comprising the step of using the Tth DNA polymerase mutant described in the first aspect as a DNA polymerase, or the step of using the kit described in the fifth aspect to perform DNA synthesis.

[0012] The reverse transcription activity of the Tth DNA polymerase mutant of the invention is close to that of the M-MLV RT wild type under the condition that the Tth DNA polymerase mutant does not depend on manganese ions. The Tth DNA polymerase mutant has a wide range of application values. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings involved in the embodiments are briefly introduced below. Obviously, the drawings described below are only part of the embodiments of the present invention, and for ordinary technicians in this field, other implementation schemes can be obtained based on these drawings without creative work.

[0014] Figure 1The electrophoresis diagram (Ni-NTA affinity chromatography) of DNA polymerase purification in the examples of the present invention, A: wild-type M-MLV RT; B: mutant THY1; C: mutant THY2; D: mutant THY3.

[0015] Figure 2 The electrophoresis diagrams (ion exchange method) of DNA polymerase purification in the examples of the present invention are as follows: A: wild-type M-MLVRT; B: mutant THY1; C: mutant THY2; D: mutant THY3.

[0016] Figure 3 The results of qPCR (Ni-NTA affinity chromatography) using Tth DNA polymerase mutants in the examples of the present invention.

[0017] Figure 4 This is the result of qPCR using Tth DNA polymerase mutants in the examples of the present invention (ion exchange method). DETAILED DESCRIPTION

[0018] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is only used to illustrate the present invention by way of example, and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims. In addition, those skilled in the art will understand that the technical solution of the present invention may be modified without departing from the spirit and purpose of the present invention. 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 this application have the meanings commonly understood by those skilled in the art. In order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0020] As used herein, the term "nucleic acid molecule" refers to a single-stranded or double-stranded RNA or DNA polymer.

[0021] As used herein, the term "degenerate sequence" refers to a sequence in which one or more codons in a nucleotide sequence are replaced by degenerate codons encoding the same amino acid.

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

[0023] In this article, the term "host" refers to a cell that can be used to express the protein encoded by the expression vector. The host cell can be a prokaryotic organism, such as Escherichia coli, or a eukaryotic organism, such as a unicellular eukaryotic organism (such as yeast or other fungi).

[0024] In this article, the term "transfection" refers to the process in which cells acquire new phenotypes by actively or passively introducing exogenous DNA fragments under certain conditions.

[0025] As used herein, the term "transformation" refers to the process of introducing plasmid DNA or recombinant DNA constructed using plasmid DNA as a vector into bacteria.

[0026] In this article, amino acids are represented by single-letter and three-letter abbreviations 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, the bases are represented by single letters known in the art, for example, A for adenine, G for guanine, C for cytosine, T for thymine, and U for uracil.

[0027] In this article, the method of expressing the mutation site of the amino acid sequence is the wild-type amino acid, site, and mutant amino acid, with no space between the three. For example, S515K means that the serine residue (S) at No. 515 in the amino acid sequence is replaced by a lysine residue (K), I640F means that the isoleucine residue (I) at No. 640 in the amino acid sequence is replaced by a phenylalanine residue (F), S741G means that the serine residue (S) at No. 741 in the amino acid sequence is replaced by a glycine residue (G), L789F means that the leucine residue (L) at No. 789 in the amino acid sequence is replaced by a phenylalanine residue (F), M763T means that the methionine residue (M) at No. 763 in the amino acid sequence is replaced by a threonine residue (T), D734G means that the aspartic acid residue (D) at No. 734 in the amino acid sequence is replaced by a glycine residue (G), and E617D means that the glutamic acid residue (E) at No. 617 in the amino acid sequence is replaced by an aspartic acid residue (D).

[0028] In this article, the STO structure refers to a nucleotide sequence that can enhance the reverse transcription performance, and its nucleotide sequence is ATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGAAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAAGGCACCGGCGGCAGCGGCGGCGGCAGC, and the corresponding amino acid sequence is MVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKKGTGGSGGGS.

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

[0030] Therefore, in the first aspect, the present invention provides a Tth DNA polymerase mutant, which has the following set of mutations based on the Tth wild-type polymerase: S515K, I640F, S741G and L789F (THY1); D734G and M763T (THY2); or E617D, D734G and M763T (THY3). Mutant THY3 includes an 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; the amino acid sequence of mutant THY3 is shown in SEQ ID NO:8.

[0031] In a second aspect, the present invention provides a nucleic acid molecule encoding the TthDNA polymerase mutant described in the first aspect. The nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO:3 or a degenerate sequence thereof, a nucleotide sequence as shown in SEQ ID NO:5 or a degenerate sequence thereof, and a nucleotide sequence as shown in SEQ ID NO:7 or a degenerate sequence thereof.

[0032] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect.

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

[0034] In a fourth aspect, the present invention provides a host, wherein the host is transfected or transformed with the expression vector described in the third aspect.

[0035] In some embodiments, the host is Escherichia 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 Escherichia coli, the culture temperature is 16° C., and the culture time is 20 h.

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

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

[0039] In a sixth aspect, the present invention provides a method for DNA synthesis, comprising the step of using the Tth DNA polymerase mutant described in the first aspect as a DNA polymerase, or the step of using the kit described in the fifth aspect to perform DNA synthesis.

[0040] Example

[0041] Expression and purification of Tth DNA polymerase mutants

[0042] The DNA sequence of the Tth DNA polymerase mutant was sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis. The nucleic acid molecule was connected to the commercial expression vector pET series based on the T7 promoter to construct an expression plasmid, which contained an operon sequence that responded to IPTG-induced expression to ensure efficient expression of the protein in Escherichia coli. The recombinant positive plasmid was transformed into Escherichia coli, and a single positive colony with good growth was selected for cultivation. When the OD 600 When the pH value is 0.6-0.8, IPTG is added for induction and the bacterial solution is collected; the cells are crushed by ultrasonic cell disruptor, and the supernatant is collected by high-speed centrifugation to obtain a crude enzyme solution; after balancing the Ni-NTA affinity chromatography column with buffer, the crude enzyme solution is passed through the chromatography column and eluted with elution buffer containing 20mM, 50mM, and 300mM imidazole, respectively, to collect the protein solution; and SDS-PAGE electrophoresis analysis is performed.

[0043] The target protein obtained by Ni-NTA affinity chromatography column was subjected to anion exchange chromatography to remove impurities and improve protein purity. Before use, the system was cleaned, the instrument was balanced with buffer, and then the ion exchange column (Q column) was connected. After the protein solution was loaded, high salt (1M NaCl) was used for gradient linear elution. Samples were collected according to the ultraviolet absorption detection peak. The target protein and purity were detected by SDS-PAGE electrophoresis, and finally dialyzed overnight to collect the target protein.

[0044] Ni-NTA affinity chromatography obtained Tth DNA polymerase with a molecular weight consistent with the expected value, but with low purity and impurity proteins ( Figure 1 After anion exchange chromatography, a highly pure Tth DNA polymerase with a molecular weight consistent with the expected value was finally obtained ( Figure 2 ).

[0045] Detection of reverse transcription efficiency of Tth DNA polymerase mutants

[0046] MSⅡ (purchased from Roche Group) was used as template RNA, with an amount of 1 ng, and reverse transcription was performed using random primers. The reverse transcription system and procedure are shown in Tables 1 and 2. The components of the buffer used in the reverse transcription were: 50 mM Tris-HCl, pH 8.8, 3 mM Mg 2+ , 50mM K +, 20 mM NH 4+ After the reverse transcription product cDNA was diluted 3 times, 1 μL was taken for real-time qPCR reaction, and 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 procedures 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℃ 10min 37℃-60℃ 15min 85℃ 5min

[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℃ 1min 95℃ 10s 60℃ 15s

[0059] Note: The last two steps consist of 40 cycles in total.

[0060] Here, the amount of PCR product is determined by comparing the Ct value, and then the efficiency of reverse transcription is determined. That is, the smaller the Ct value, the lower the number of cycles required to reach the threshold of the qPCR instrument, the higher the amount of initial reverse transcription product, the higher the reverse transcription efficiency of the enzyme, and the better the activity. The three Tth DNA polymerase mutants purified and separated by Ni-NTA affinity chromatography column and anion exchange column were tested for reverse transcription activity using RT-qPCR technology. Figure 3 As shown in the figure, the three mutants obtained by affinity chromatography were compared with the wild-type M-MLV reverse transcriptase. The results of qPCR 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 that of the control, among which THY2 had the best activity, followed by THY3, and THY1 had the worst activity. Figure 4As shown in the figure, the three Tth DNA polymerase mutants obtained by anion exchange method were compared with the wild-type M-MLV reverse transcriptase. The results of qPCR showed that the Ct values ​​of the three mutants were slightly higher than the control, indicating that the reverse transcription activity of the mutants was slightly lower than that of the control, among which THY3 had the best activity, followed by THY2, and THY1 had the worst activity. In general, under conditions independent of 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 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 Tth DNA polymerase mutant, characterized in that The mutant has the following set of mutations based on the Tth wild-type polymerase: E617D, D734G and M763T. The mutant also contains an STO structure that enhances reverse transcription performance. The amino acid sequence of the Tth wild-type 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 according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule is a nucleotide sequence as shown in SEQ ID NO: 7 or a degenerate sequence thereof.

4. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 2 or 3.

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

6. A host, characterized in that The host is transfected or transformed with the expression vector according to claim 4 or 5; preferably, the host is Escherichia coli, the culture temperature is 16°C, 24°C, 30°C or 37°C, and the culture time is 20h, 12h, 7h or 3h; more preferably, the host is Escherichia coli, the culture temperature is 16°C, and the culture time is 20h.

7. A kit, characterized in that: The kit comprises the Tth DNA polymerase mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, the expression vector according to claim 4 or 5, or the host according to claim 6.

8. The kit according to claim 7, characterized in that The kit also includes a buffer and primers required for DNA polymerization.

9. A method for DNA synthesis, characterized in that: The method comprises the step of using the Tth DNA polymerase mutant according to claim 1 as a DNA polymerase, or the step of using the kit according to claim 7 or 8 to perform DNA synthesis.

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