Optimized SSo7d structural domain, improved Taq DNA polymerase and application of optimized SSo7d structural domain and improved Taq DNA polymerase

Abstract

The invention relates to the technical field of molecular biology, in particular to an optimized SSo7d structural domain, an improved Taq DNA polymerase and application of the improved Taq DNA polymerase, and the N end and / or the C end of the improved Taq DNA polymerase are / is fused with the optimized SSo7d structural domain; moreover, compared with the amino acid sequence of the wild type Taq DNA polymerase, the improved Taq DNA polymerase at least comprises any one of a catalytic efficiency optimized mutation combination, a DNA binding optimized mutation combination, a stable mutation combination, a sample tolerance mutation combination and a specific optimized mutation combination. The improved Taq DNA polymerase has higher DNA binding capacity and amplification efficiency, excellent specificity and sensitivity and better heat resistance and stability; the problems that in the prior art, existing Taq DNA polymerase is insufficient in amplification specificity, sensitivity, inhibitor tolerance and the like are solved.

Description

Technical Field

[0001] The 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] Polymerase chain reaction (PCR) is a molecular biology technique for amplifying specific DNA fragments, which is widely used in molecular biology, medical diagnosis, forensic identification and other fields. The core of polymerase chain reaction technology is to use a class of heat-resistant DNA polymerases to simulate the natural replication process of DNA in vitro, and rapidly amplify specific DNA fragments millions of times through the three-step cycle of "denaturation-annealing-extension"; among them, the most commonly used heat-resistant DNA polymerase is a Taq DNA polymerase derived from thermophilic bacteria. However, although conventional Taq DNA polymerase can meet the needs of general polymerase chain reaction, it still has deficiencies in amplification specificity, sensitivity, inhibitor tolerance, etc.; these deficiencies limit or affect the application of polymerase chain reaction technology in complex sample detection, multiple amplification, etc. to a certain extent. Therefore, the development of a new type of Taq DNA polymerase with high efficiency, specificity and strong tolerance is of great significance for expanding the application of PCR technology and improving detection performance.

[0003] In recent years, studies have found 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 much attention due to its unique DNA binding ability and thermal stability. Studies have shown that fusing the SSo7d DNA-binding protein to Taq DNA polymerase can improve the enzyme's DNA binding ability, replication fidelity and heat resistance, thereby improving the performance of the polymerase chain reaction; however, there is still room for further optimization of the natural SSo7d DNA-binding protein, especially in terms of DNA binding ability and synergy with polymerase. Therefore, before fusing the SSo7d DNA-binding protein to Taq DNA polymerase, the 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, so as to solve the problem that the existing Taq DNA polymerase in the prior art has deficiencies in amplification specificity, sensitivity, inhibitor tolerance and the like, which results in the limitation of 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 applications thereof, 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 comprises a mutation combination 1 compared with the amino acid sequence of the wild-type SSo7d domain; wherein the mutation combination 1 comprises the following mutation sites:

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

[0008] Mutation site 2: K22R, i.e., lysine at position 22 mutates 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 as shown in 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 is 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., the glutamic acid at position 507 mutates to lysine; R587Q, i.e., the arginine at position 587 mutates to glutamine; D578N, i.e., the aspartic acid at position 578 mutates 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., the isoleucine at position 707 mutates to leucine; E708K, i.e., the glutamic acid at position 708 mutates to lysine; P685K, i.e., the proline at position 685 mutates to lysine; L459M, i.e., the leucine at position 459 mutates to methionine;

[0016] The specific optimized mutation combination includes the following mutation sites: R630Q, i.e., the arginine at position 630 is mutated to glutamine; E694D, i.e., 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 applications thereof. 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 endowed with 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, etc. of the Taq DNA polymerase itself 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; has good hot start characteristics, so that it can avoid non-specific amplification to the greatest extent during the amplification reaction; has high DNA binding force and processivity, thereby broadening its efficient target length; 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; solves the existing Taq DNA polymerase existing in the prior art. 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 in conjunction with the accompanying drawings and embodiments:

[0025] Figure 1 The SDS-PAGE protein electrophoresis detection result 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 by an ion column;

[0027] Figure 3 The test results of the fluorescence quantitative PCR amplification performance 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 is further described in detail below in conjunction with specific embodiments:

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

[0032] In view of the shortcomings of the existing Taq DNA polymerase, the present invention firstly predicts and screens out 8 key sites through homology modeling and molecular docking methods, and introduces site-directed mutations at these sites by using site-directed mutagenesis, for example, 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 introduces site-directed mutations at these sites by using site-directed mutagenesis, for example, 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 protein was introduced into the N-terminus and C-terminus of the DNA polymerase to construct a fusion protein containing the SSo7d domain; and DNA binding enhancing mutations (K21R, K22R) and hydrophobic interaction optimizing mutations (W24F) were introduced into the SSo7d binding protein; thereby a novel hot-start improved Taq DNA polymerase with improved inhibition resistance and overall improved performance fused with the optimized SSo7d domain was obtained; and the improved Taq DNA polymerase was applied to the 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 of sterile water to prepare a plasmid solution; at the same time, a 100 μL aliquot of E. 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 E. coli competent cells BL21, and Use your fingers to gently flick to mix and place on ice for 30 minutes; then, heat shock at 42°C for 90 seconds, place on ice for 2 minutes, and add 500 μL of LB medium without resistance; then, culture at 37°C and 180 rpm for 1 hour, centrifuge at 3000 rpm for 2 minutes, discard 250 μL of supernatant and gently blow to mix; finally, spread it on LB solid medium containing Kan resistance, culture it upside down in a 37°C incubator overnight to obtain a single colony.

[0036] Pick a single colony after transformation and inoculate it into 10mL LB liquid medium containing Kan resistance (100μg / mL), and culture it in a shaking incubator at 37°C and 180rpm for 4h. Use 40% glycerol to preserve the strain, and then inoculate it into 1L LB medium containing Kan resistance (100μg / mL) at a 1% inoculum, and culture it at 37°C and 180rpm until OD600 is 0.8-1, add 0.5-1mm IPTG, and induce expression at 20-30°C for 10-15h to obtain 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, the supernatant was discarded and the bacteria were collected; the bacteria were resuspended with disruption buffer A; and the resuspended bacteria were disrupted 1-2 times with an ATS high-pressure homogenizer at 1000 MPa and 4°C to form a disruption solution; wherein the disruption buffer A was 25 mM Tris·HCl (pH 7.4), 10 mM imidazole, and 500 mM sodium chloride.

[0039] Turn on the centrifuge, precool and centrifuge the broken liquid at 4°C and 7000rpm. Use nickel column for affinity chromatography on the liquid after centrifugation, use buffer B and buffer C for elution in turn, and collect the elution peak; sample 20μL of the elution peak for SDS-PAGE protein electrophoresis detection; after elution, dialyze the target protein into buffer D for further purification. Among them, buffer B is 25mM Tris·HCl (pH7.4), 100mM imidazole, 500mM sodium chloride; buffer C is 25mM Tris·HCl (pH7.4), 200mM imidazole, 500mM sodium chloride; buffer D is 25mMTris (pH7.4), 50mM sodium chloride, 1mM DTT, 1mMEDTA.

[0040] After affinity chromatography using a 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 elution solution, and the elution peak was collected; 20 μL of the elution peak was sampled for SDS-PAGE protein electrophoresis detection; the target protein obtained after elution was dialyzed again into buffer F and frozen in a -80°C refrigerator for use; wherein buffer E was 20 mM Tris (pH 7.4), 1 M sodium chloride, 1 mM DTT, and 1 mM EDTA; buffer F was 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 detection showed that the target protein was obviously contained in the 100mMNaCl eluate, and the purity was high and the position was correct; 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 at 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 (SEQID No.1); Taq[mutation](1-290): is the exonuclease domain; PSTPPSPGGGGS is the flexible linker protein linker1; GGGGS is the end rigid linker2; 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, the same method as in the first embodiment is used to construct, induce expression, and purify a common Taq DNA polymerase; the difference between the common Taq DNA polymerase and the improved Taq DNA polymerase is that the N-terminus and the C-terminus of the common Taq DNA polymerase are not connected to the optimized SSo7d domain. The amino acid sequence of the common Taq DNA polymerase is shown in SEQ ID No.4.

[0046] The performance of the improved Taq DNA polymerase, the common Taq DNA polymerase and the wild-type Taq DNA polymerase purified above 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 amplification performance of different types of Taq DNA polymerases on complex sample fluorescence quantitative PCR

[0048] Taking blood samples as an example, the amplification performance of improved Taq DNA polymerase, ordinary Taq DNA polymerase and wild-type Taq DNA was investigated under direct sample addition conditions. The improved Taq DNA polymerase, ordinary Taq DNA polymerase and wild-type Taq DNA polymerase were amplified under the same system conditions to compare the amplification effects; JEV (Japanese swine encephalitis virus) was used as the research object, and 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] 25μL RT-PCR common liquid reaction system (pH 8.3) includes: 20mM Tris, 2.5mM MgCl2, 10mM (NH4)2SO4, 500mM KCl, 20ng / mL BSA, 0.1% Triton X-100, 250nM each dNTP, 50ng / mL UDG, 5U mutant Taq DNA polymerase, 0.2μg reverse transcriptase, 300nM JEV-F, 300nM JEV-R, 100nM JEV-P, 5μL JEVRNA template blood sample. Amplification program: 55℃3min, 95℃2min; 95℃15s, 60℃40s, a total of 40 cycles.

[0053] The results of the amplification test are as follows Figure 3 As shown in the figure; 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; further indicating that the improved Taq DNA polymerase containing double SSo7d domains and multiple site mutations performs better in harsh sample PCR; thus indicating 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 Taq DNA polymerase itself, has stronger DNA binding ability and amplification efficiency, and more excellent specificity (processivity) and sensitivity; enabling it to effectively overcome the interference of complex background of clinical samples; and the improvement of DNA binding ability and specificity broadens 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, so that the heat resistance and stability of the improved Taq DNA polymerase are improved, and it can adapt to more harsh reaction conditions.

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

[0055] Similarly, JEV (Japanese swine 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] 25 μL RT-PCR common liquid reaction system (pH 8.3) includes: 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 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, 5 μL JEV RNA template blood sample. Amplification program: 55 ° C 3 min, 95 ° C 2 min; 95 ° C 15 s, 60 ° C 40 s, a total of 40 cycles.

[0057] Hot start test results: Figure 4 As shown in the figure, the CT value of the common Taq DNA polymerase amplification is 26.2, and the CT value of the improved Taq DNA polymerase amplification is 25.1; this further illustrates 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 a better amplification effect, and can be completed in less time when amplifying fragments of the same size, which can effectively improve the PCR amplification efficiency; compared with the improved Taq DNA polymerase, the hot start characteristics of the common Taq DNA polymerase are poor, which makes the common Taq DNA polymerase have more non-specific amplification, resulting in lower fluorescence intensity during the amplification process, delayed CT value of amplification, and significantly lower amplification effect than 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 used in 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 cannot be used 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 features 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 limited by the attached claims rather than the above description, and it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention.

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Claims

1. An optimized SSo7d domain, characterized in that The amino acid sequence of the optimized SSo7d domain was compared with that of the wild-type SSo7d domain, including mutation combination 1; The mutation combination 1 includes the following mutation sites: Mutation site 1: K21 R; Mutation site 2: K22R; Mutation site 3: W24F.

2. An optimized SSo7d domain according to claim 1, characterized in that: The amino acid sequence of the optimized SSo7d domain is shown in SEQ ID No.

1.

3. An improved Taq DNA polymerase, characterized in that: The N-terminus and / or C-terminus of the improved Taq DNA polymerase is fused with the optimized SSo7d domain described in claim 2.

4. The improved Taq DNA polymerase according to claim 3, characterized in that: Compared with the amino acid sequence of the wild-type Taq DNA polymerase, the improved Taq DNA polymerase includes 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; The catalytic efficiency optimization mutation combination includes the following mutation sites: E507K, R587Q, D578N; The DNA binding optimized mutation combination includes the following mutation sites: F667Y, A661 E, R659Y; The stability mutation combination includes the following mutation sites: L781 M, D732N; The sample tolerance mutation combination includes the following mutation sites: I 707L, E708K, P685K, L459M; The specific optimized mutation combination includes the following mutation sites: R630Q, E694D.

5. An improved Taq DNA polymerase according to claim 4, characterized in that: The N-terminus and C-terminus of the improved Taq DNA polymerase are both fused with the optimized SSo7d domain; 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.

6. The improved Taq DNA polymerase according to claim 5, characterized in that: The amino acid sequence of the improved Taq DNA polymerase is shown in SEQ ID NO:

2.

7. Use of the improved Taq DNA polymerase according to any one of claims 3 to 6, characterized in that: The invention comprises the application of the improved Taq DNA polymerase in PCR technology.

8. The use of an improved Taq DNA polymerase according to claim 7, characterized in that: 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.

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