An optimized SSo7d structural domain, improved Taq DNA polymerase and its application

Abstract

The present invention relates to the field of molecular biology technology, and specifically to an optimized SSo7d domain, an improved Taq DNA polymerase, and applications thereof. The improved Taq DNA polymerase is fused with an optimized SSo7d domain at its N-terminus and / or C-terminus. Furthermore, compared to the amino acid sequence of a wild-type Taq DNA polymerase, the improved Taq DNA polymerase includes at least one of a catalytic efficiency optimization mutation combination, a DNA binding optimization mutation combination, a stability mutation combination, a sample tolerance mutation combination, and a specificity optimization mutation combination. The improved Taq DNA polymerase has stronger DNA binding ability and amplification efficiency, excellent specificity and sensitivity, and good heat resistance and stability. It solves the problems of existing Taq DNA polymerases in the prior art, such as deficiencies in amplification specificity, sensitivity, and inhibitor tolerance.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to an optimized SSo7d structural domain, an improved TaqDNA polymerase and applications thereof. Background Art

[0002] The polymerase chain reaction (PCR) is a molecular biology technique for amplifying specific DNA fragments and is widely used in molecular biology, medical diagnosis, forensic identification, and other fields. The core of PCR technology lies in the use of a class of thermostable DNA polymerases to simulate the natural DNA replication process in vitro. Through a three-step cycle of "denaturation-annealing-extension," specific DNA fragments can be rapidly amplified millions of times. The most commonly used thermostable DNA polymerase is Taq DNA polymerase, derived from Thermobacterium thermophilum. However, while conventional Taq DNA polymerase meets the requirements of general PCR, it still has shortcomings in amplification specificity, sensitivity, and inhibitor tolerance. These shortcomings, to a certain extent, limit or hinder the application of PCR technology in complex sample testing and multiplex amplification. Therefore, the development of a new Taq DNA polymerase that is efficient, specific, and highly tolerant is of great significance for expanding the application of PCR technology and improving detection performance.

[0003] In recent years, studies have revealed that some single-stranded DNA-binding proteins (SSBs) can significantly enhance the amplification efficiency and specificity of DNA polymerases. Among them, the SSo7d DNA-binding protein from the archaeon Sulfolobus solfataricus has attracted considerable attention due to its unique DNA-binding ability and thermostability. Studies have shown that fusing the SSo7d DNA-binding protein to Taq DNA polymerase enhances the enzyme's DNA binding affinity, replication fidelity, and thermostability, thereby improving polymerase chain reaction (PCR) performance. However, further optimization of the native SSo7d DNA-binding protein remains, particularly in terms of DNA binding affinity and synergistic effects with the polymerase. Therefore, prior to fusing the SSo7d DNA-binding protein to Taq DNA polymerase, key functional sites of the SSo7d DNA-binding protein need to be optimized.

[0004] The present invention provides an optimized SSo7d DNA binding protein, an improved Taq DNA polymerase and applications thereof, to solve the problem in the prior art that the existing Taq DNA polymerase has deficiencies in amplification specificity, sensitivity, inhibitor tolerance, etc., which limits the application of polymerase chain reaction technology. Summary of the Invention

[0005] The purpose of the present invention is to provide an optimized SSo7d domain, an improved Taq DNA polymerase and their applications, so as to solve the problem that the existing Taq DNA polymerase in the prior art has deficiencies in amplification specificity, sensitivity, inhibitor tolerance, etc., which limits the application of polymerase chain reaction technology.

[0006] The technical solution of the present invention is: an optimized SSo7d domain, which includes mutation combination 1 compared with the amino acid sequence of the wild-type SSo7d domain; wherein the mutation combination 1 includes the following mutation sites:

[0007] Mutation site 1: K21R, i.e. lysine at position 21 mutates to arginine;

[0008] Mutation site 2: K22R, where lysine at position 22 is mutated to arginine;

[0009] Mutation site 3: W24F, that is, the tryptophan at position 24 mutates to phenylalanine.

[0010] Preferably, the amino acid sequence of the optimized SSo7d domain is shown as SEQ ID No.1.

[0011] The present invention also provides an improved Taq DNA polymerase, wherein the N-terminus and / or the C-terminus of the improved Taq DNA polymerase are fused with the above-mentioned optimized SSo7d domain.

[0012] Preferably, the improved Taq DNA polymerase comprises at least any one of a catalytic efficiency optimization mutation combination, a DNA binding optimization mutation combination, a stability mutation combination, a sample tolerance mutation combination, and a specificity optimization mutation combination compared to the amino acid sequence of the wild-type Taq DNA polymerase; wherein the catalytic efficiency optimization mutation combination comprises the following mutation sites: E507K, i.e., mutation of glutamic acid at position 507 to lysine; R587Q, i.e., mutation of arginine at position 587 to glutamine; D578N, i.e., mutation of aspartic acid at position 578 to asparagine;

[0013] The DNA binding optimized mutation combination includes the following mutation sites: F667Y, i.e., the phenylalanine at position 667 is mutated to tyrosine; A661E, i.e., the alanine at position 661 is mutated to glutamic acid; R659Y, i.e., the arginine at position 659 is mutated to tyrosine;

[0014] The stability mutation combination includes the following mutation sites: L781M, i.e., the leucine at position 781 is mutated to methionine; D732N, i.e., the aspartic acid at position 732 is mutated to asparagine;

[0015] The sample tolerance mutation combination includes the following mutation sites: I707L, i.e., isoleucine at position 707 mutates to leucine; E708K, i.e., glutamic acid at position 708 mutates to lysine; P685K, i.e., proline at position 685 mutates to lysine; L459M, i.e., leucine at position 459 mutates to methionine;

[0016] The specific optimized mutation combination includes the following mutation sites: R630Q, that is, the arginine at position 630 is mutated to glutamine; E694D, that is, the glutamic acid at position 694 is mutated to aspartic acid.

[0017] Preferably, the N-terminus and C-terminus of the improved Taq DNA polymerase are both fused with the optimized SSo7d domain;

[0018] Compared with the amino acid sequence of wild-type Taq DNA polymerase, the improved Taq DNA polymerase includes a catalytic efficiency optimization mutation combination, a DNA binding optimization mutation combination, a stability mutation combination, a sample tolerance mutation combination and a specificity optimization mutation combination (processivity optimization mutation combination).

[0019] Preferably, the amino acid sequence of the improved Taq DNA polymerase is shown in SEQ ID NO: 2.

[0020] The invention also discloses the application of the improved Taq DNA polymerase, including the application of the improved Taq DNA polymerase in PCR technology.

[0021] Preferably, the application of the improved Taq DNA polymerase in PCR technology includes application in complex sample PCR detection, application in difficult-to-amplify region PCR, application in trace DNA amplification, application in direct sample PCR detection, and application in GC-rich template amplification.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] (1) The present invention provides an optimized SSo7d domain, an improved Taq DNA polymerase and its application. By optimizing the key functional sites on the SSo7d domain and fusing the optimized SSo7d domain to the N-terminus and C-terminus of the Taq DNA polymerase, the Taq DNA polymerase is given a stronger DNA binding ability and amplification efficiency. In addition, the optimized hydrophobic interface also makes the molecular structure of the Taq DNA polymerase more compact and stable. At the same time, the catalytic efficiency optimization mutation, DNA binding optimization mutation, stability mutation, inhibitor tolerance mutation and the like of the Taq DNA polymerase are superimposed, so that the improved Taq DNA polymerase has excellent specificity and sensitivity, so that it can effectively overcome the interference of the complex background of clinical samples during the amplification reaction; it has good hot start characteristics, so that it can avoid non-specific amplification to the greatest extent during the amplification reaction; it has high DNA binding force and processivity, thereby broadening its efficient target length; it has good heat resistance and stability, so that it can adapt to more severe reaction conditions; thereby expanding the application range of the improved Taq DNA polymerase; solving the problems existing in the prior art of the existing Taq DNA polymerase has deficiencies in amplification specificity, sensitivity, inhibitor tolerance, etc., which limits the application of polymerase chain reaction technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0025] Figure 1 The SDS-PAGE protein electrophoresis results of the target protein of the present invention after nickel column affinity chromatography;

[0026] Figure 2 The SDS-PAGE protein electrophoresis detection result of the target protein of the present invention after elution with an ion column;

[0027] Figure 3 The results of the fluorescent quantitative PCR amplification performance test of different types of Taq DNA polymerases on complex samples described in the present invention;

[0028] Figure 4 The hot start characteristics test results of different types of Taq DNA polymerases described in the present invention;

[0029] Wherein: M, protein marker; 1, supernatant after fragmentation; 2, flow-through; 3: 100mM imidazole elution; 4: 200mM imidazole elution; 5: linear elution flow-through; 6: 40mM NaCl elution; 7: 100mM NaCl elution of target protein. DETAILED DESCRIPTION

[0030] The present invention will be described in further detail below with reference to specific embodiments:

[0031] It should be noted that the specific implementation manner or the drawings in the specification Figure 1-Figure 4 The improved Taq enzyme, ordinary Taq enzyme, and wild-type Taq enzyme described in the reference are improved Taq DNA polymerase, ordinary Taq DNA polymerase, and wild-type Taq DNA polymerase, respectively.

[0032] In response to the shortcomings of existing Taq DNA polymerase, the present invention first predicts and screens 8 key sites through homology modeling and molecular docking methods, and introduces site-directed mutations at these sites using site-directed mutagenesis, such as catalytic efficiency optimization mutations (E507K, R587Q, D578N), DNA binding optimization mutations (F667Y, A661E, R659Y), stability mutations (L781M, D732N), inhibitor tolerance mutations (I707L, E708K, P685K, L459M), processivity optimization mutation combination (R630Q, E694D), etc. On this basis, in order to further improve the performance of Taq DNA polymerase, the present invention first predicts and screens 8 key sites through homology modeling and molecular docking methods, and introduces site-directed mutations at these sites using site-directed mutagenesis, such as catalytic efficiency optimization mutations (E507K, R587Q, D578N), DNA binding optimization mutations (F667Y, A661E, R659Y), stability mutations (L781M, D732N), inhibitor tolerance mutations (I707L, E708K, P685K, L459M), processivity optimization mutation combination (R630Q, E694D), etc. SSo7d binding proteins were introduced into the N- and C-termini of the DNA polymerase to construct a fusion protein containing the SSo7d domain. Furthermore, DNA binding-enhancing mutations (K21R, K22R) and a hydrophobic interaction-optimizing mutation (W24F) were introduced into the SSo7d binding protein. This resulted in a novel hot-start Taq DNA polymerase with improved inhibition resistance and overall enhanced performance fused with the optimized SSo7d domain. The Taq DNA polymerase was then applied to PCR technology to maximize its specificity, sensitivity, robustness, and adaptability during the PCR reaction.

[0033] Implementation Method 1

[0034] (1) Construction and induced expression of improved Taq DNA polymerase

[0035] First, a professional company was commissioned to construct a mutant sequence vector (pET28a) using a site-directed mutagenesis method to obtain pET28a plasmid vector powder; then, the pET28a plasmid powder was centrifuged and dissolved with 100 μL sterile water to prepare a plasmid solution; at the same time, 100 μL of Escherichia coli competent cells BL21 (DE3) was taken out and placed on ice for thawing. After thawing, 2 μL of the above plasmid solution was added to the Escherichia coli competent cells BL21 and Gently flick the mixture with your fingers and place it on ice for 30 minutes; then, heat shock it at 42°C for 90 seconds, place it on ice for 2 minutes, and add 500 μL of LB medium without resistance; then, culture it at 37°C and 180 rpm for 1 hour, centrifuge it at 3000 rpm for 2 minutes, discard 250 μL of supernatant and gently pipette to mix; finally, spread it on LB solid medium containing Kan resistance, culture it upside down in a 37°C incubator overnight, and obtain single colonies.

[0036] Pick a single transformed colony and inoculate it into 10 mL of LB liquid medium containing Kan resistance (100 μg / mL) and culture it in a shaker at 37°C and 180 rpm for 4 hours. Use 40% glycerol to preserve the strain, then inoculate 1% of the inoculum into 1 L of LB medium containing Kan resistance (100 μg / mL). Cultivate it at 37°C and 180 rpm until the OD600 reaches 0.8-1. Then add 0.5-1 mM IPTG and induce expression at 20-30°C for 10-15 hours to obtain a fermentation broth.

[0037] (2) Protein purification of improved Taq DNA polymerase

[0038] The fermentation broth was centrifuged at 4°C and 5000 rpm for 30 min, and the supernatant was discarded and the cells were collected; the cells were resuspended in disruption buffer A; and the resuspended cells were disrupted 1-2 times using an ATS high-pressure homogenizer at 1000 MPa and 4°C to form a disruption solution; wherein, the disruption buffer A is 25 mM Tris·HCl (pH 7.4), 10 mM imidazole, and 500 mM sodium chloride.

[0039] Start the centrifuge, pre-cool, and centrifuge the disrupted liquid at 4°C and 7000 rpm. Apply affinity chromatography to a nickel column, eluting with buffer B and then buffer C, and collect the elution peaks. Sample 20 μL of each elution peak for SDS-PAGE protein electrophoresis. After elution, dialyze the target protein into buffer D for further purification. Buffer B consists of 25 mM Tris·HCl (pH 7.4), 100 mM imidazole, and 500 mM sodium chloride; buffer C consists of 25 mM Tris·HCl (pH 7.4), 200 mM imidazole, and 500 mM sodium chloride; and buffer D consists of 25 mM Tris (pH 7.4), 50 mM sodium chloride, 1 mM DTT, and 1 mM EDTA.

[0040] After affinity chromatography using nickel column, Figure 1 As shown, the SDS-PAGE protein electrophoresis detection results show that the 100mM imidazole eluate clearly contains the target protein and is in the correct position, which can be used for further purification of the target protein; that is, the residual nucleic acid in the target protein is removed by an ion column (Q column) to further purify the target protein.

[0041] The dialyzed target protein was linearly eluted using an ion column (Q column) with buffer D as the equilibration solution and buffer E as the eluent, and the elution peak was collected; 20 μL of the elution peak was sampled and detected by SDS-PAGE protein electrophoresis; the target protein obtained after elution was dialyzed again into buffer F and frozen in a -80°C refrigerator until use; wherein, buffer E is 20 mM Tris (pH 7.4), 1 M sodium chloride, 1 mM DTT, 1 mM EDTA; buffer F is 25 mM Tris (pH 7.4), 50 mM sodium chloride, 1 mM DTT, 1 mM EDTA, and 40% glycerol.

[0042] After linear elution using an ion column, Figure 2 As shown, the results of SDS-PAGE protein electrophoresis showed that the 100mMNaCl eluate clearly contained the target protein with high purity and correct location; further indicating that the target protein with relatively high purity was obtained after the above two-step purification, namely the improved Taq DNA polymerase; and, compared with the amino acid sequence of the wild-type Taq DNA polymerase, the improved Taq DNA polymerase includes a catalytic efficiency optimization mutation combination, a DNA binding optimization mutation combination, a stability mutation combination, a sample tolerance mutation combination and a processivity optimization mutation combination in its amino acid sequence, and the optimized SSo7d domain is fused to both the N-terminus and the C-terminus of its amino acid sequence; wherein, the amino acid sequence of the optimized SSo7d domain is shown in SEQ ID No. 1.

[0043] The amino acid sequence of the improved Taq DNA polymerase is shown in SEQ ID No. 2, and the complete sequence of the improved Taq DNA polymerase can be expressed as: SSo7d1-Taq[mutation](1-290)-PSTPPSPGGGGS-SSo7d2-GGGGS-Taq[mutation](291-832)-GGSHis6; wherein SSo7d1 and SSo7d2 are both optimized SSo7d sequences (SEQ ID No. 1); Taq[mutation](1-290): is the exonuclease domain; PSTPPSPGGGGS is the flexible linker protein linker 1; GGGGS is the terminal rigid linker 2; Taq[mutation](291-832) is the Taq core enzyme domain; GGSHis6 is the C-terminal His tag. Meanwhile, the amino acid sequence of the wild-type Taq DNA polymerase is shown in SEQ ID No. 3.

[0044] Implementation Method 2

[0045] In this embodiment, a conventional Taq DNA polymerase is constructed, induced for expression, and purified using the same methods as in Embodiment 1. This conventional Taq DNA polymerase differs from the improved Taq DNA polymerase in that it lacks the optimized SSo7d domain at either the N-terminus or the C-terminus. The amino acid sequence of the conventional Taq DNA polymerase is shown in SEQ ID No. 4.

[0046] The performance of the improved Taq DNA polymerase, the conventional Taq DNA polymerase and the wild-type Taq DNA polymerase obtained by the above purification was tested. The performance of the different types of Taq DNA polymerases was tested. The specific tests are as follows:

[0047] 1. Comparison of the performance of different types of Taq DNA polymerases in fluorescent quantitative PCR amplification of complex samples

[0048] Using blood samples as an example, the amplification performance of the improved Taq DNA polymerase, standard Taq DNA polymerase, and wild-type Taq DNA was investigated under direct sample loading conditions. The improved Taq DNA polymerase, standard Taq DNA polymerase, and wild-type Taq DNA polymerase were amplified under the same system conditions to compare their amplification results. Japanese porcine encephalitis virus (JEV) was used as the target of study. The amplification primers and probes are shown in Table 1.

[0049] Table 1. Primers and probes for quantitative PCR amplification.

[0050]

[0051] The specific operation of PCR amplification is as follows:

[0052] A 25 μL RT-PCR standard liquid reaction system (pH 8.3) consisted of 20 mM Tris, 2.5 mM MgCl₂, 10 mM (NH₄)₂SO₄, 500 mM KCl, 20 ng / mL BSA, 0.1% Triton X-100, 250 nM of each dNTP, 50 ng / mL UDG, 5 U of mutant Taq DNA polymerase, 0.2 μg of reverse transcriptase, 300 nM JEV-F, 300 nM JEV-R, 100 nM JEV-P, and 5 μL of JEV RNA template blood sample. The amplification program was: 55°C for 3 min, 95°C for 2 min, 95°C for 15 s, 60°C for 40 s, for a total of 40 cycles.

[0053] The amplification test results are as follows Figure 3 As shown in the figures, under direct sample addition conditions, the improved Taq DNA polymerase provided by the present invention has significantly better tolerance to whole blood samples than ordinary Taq DNA polymerase and wild-type Taq DNA polymerase. This further indicates that the improved Taq DNA polymerase containing two SSo7d domains and multiple mutations performs better in PCR with harsh samples. This shows that the improved Taq DNA polymerase obtained by introducing the optimized SSo7d domains at the N-terminus and C-terminus of Taq DNA polymerase, respectively, and then superimposing the catalytic, heat-resistant, and tolerance-optimized mutations of the Taq DNA polymerase itself, has stronger DNA binding ability and amplification efficiency, and better specificity (processivity) and sensitivity; it can effectively overcome the interference of complex background of clinical samples; and the improved DNA binding ability and specificity broaden the effective target length (efficient target length) of the improved Taq DNA polymerase. At the same time, the optimized hydrophobic interface on the optimized SSo7d domain can also make the molecular structure of the improved Taq DNA polymerase more compact and stable, thereby improving the heat resistance and stability of the improved Taq DNA polymerase and enabling it to adapt to more harsh reaction conditions.

[0054] (2) Testing the hot start characteristics of different types of Taq DNA polymerases

[0055] Similarly, JEV (Japanese porcine encephalitis virus) was used as the research object, and the primers and probes in Table 1 were used as the primers and probes for this detection.

[0056] A 25 μL RT-PCR standard liquid reaction system (pH 8.3) consisted of 20 mM Tris, 2.5 mM MgCl₂, 10 mM (NH₄)₂SO₄, 500 mM KCl, 20 ng / mL BSA, 0.1% Triton X-100, 250 nM of each dNTP, 50 ng / mL UDG, 5 U Taq DNA polymerase, 0.2 μg reverse transcriptase, 300 nM JEV-F, 300 nM JEV-R, 100 nM JEV-P, and 5 μL JEV RNA template blood sample. The amplification program was: 55°C for 3 min, 95°C for 2 min, 95°C for 15 s, 60°C for 40 s, for a total of 40 cycles.

[0057] Hot start test results: Figure 4 As shown in the figure, the CT value of the amplification by ordinary Taq DNA polymerase is 26.2, and the CT value of the amplification by improved Taq DNA polymerase is 25.1; this further demonstrates that the improved Taq DNA polymerase has excellent hot start characteristics; there is less non-specific amplification during the amplification process; and the improved Taq DNA polymerase has better amplification effect, and can complete the amplification of fragments of the same size in less time, which can effectively improve the PCR amplification efficiency; compared with the improved Taq DNA polymerase, the hot start characteristics of ordinary Taq DNA polymerase are poor, resulting in more non-specific amplification of ordinary Taq DNA polymerase, resulting in lower fluorescence intensity during the amplification process, delayed CT value of amplification, and significantly lower amplification effect than that of the improved Taq DNA polymerase.

[0058] It can be seen that by introducing mutations in key performance such as catalysis, binding, and tolerance, and fusing the optimized double SSo7d domain, the performance of the improved Taq DNA polymerase can be comprehensively improved, making it widely applicable to conventional, multiplex, high-sensitivity PCR, trace DNA amplification, direct sample PCR detection, GC-rich template amplification, etc.; it can provide a key core tool enzyme for the in-depth expansion of PCR technology in basic research and application fields; thus, PCR technology can have significant application value and broad application prospects in in vitro diagnosis, genetic analysis, molecular breeding, ancient DNA research, forensic evidence, food safety, environmental monitoring and other fields.

[0059] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly, and they are not intended to limit the scope of protection of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

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Claims

1. An improved Taq DNA polymerase, characterized in that: The amino acid sequence of the improved Taq DNA polymerase is shown in SEQ ID NO:

2.

2. The use of the improved Taq DNA polymerase according to claim 1, characterized in that: The invention also includes the application of the improved TaqDNA polymerase in PCR technology.

3. The use of the improved Taq DNA polymerase according to claim 2, characterized in that: The applications of the improved TaqDNA polymerase in PCR technology include applications in complex sample PCR detection, applications in difficult-to-amplify region PCR, applications in trace DNA amplification, applications in direct sample PCR detection, and applications in GC-rich template amplification.

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