A mutant ExoⅢ and its application

Abstract

The present invention relates to the field of molecular biology technology, and specifically to a mutant Exo III and an application thereof. Compared with the amino acid sequence of wild-type Exo III, the mutant Exo III includes a core site mutation combination; the core site mutation combination includes: G113N and R189N; the mutant Exo III also includes an auxiliary mutation combination 1 and / or an auxiliary mutation combination 2; the present application optimizes the characteristics of Exo III by introducing the above-mentioned at least one group of site-directed mutation combinations into the amino acid sequence of wild-type Exo III, so that the mutant Exo III obtained after site-directed mutagenesis has excellent specificity and thermosensitivity; improves the mutant Exo III's ability to recognize AP sites and reduces its non-specific cleavage of normal double-stranded DNA; and improves the mutant Exo III's specific cleavage performance on templates in RPA reactions, thereby improving the RPA nucleic acid amplification efficiency; and solves the problems of poor specificity, insufficient thermosensitivity, and limited scope of application of the existing wild-type Exo III.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a mutant ExoⅢ and an application thereof. Background Art

[0002] ExoⅢ (ExonucleaseⅢ) is an exonuclease that acts on double-stranded DNA, catalyzing the gradual removal of single nucleotides in the 3'→5' direction. ExoⅢ primarily targets DNA structures with gaps or abnormalities, such as AP sites and oxidative damage sites. It can be widely used in DNA repair research and molecular diagnostics, such as RPA technology, the preparation of strand-specific probes, and the preparation of single-stranded substrates for dideoxy sequencing. However, wild-type ExoⅢ still has many shortcomings. For example, when used in recombinase polymerase amplification (RPA) technology, nonspecific cleavage may occur. That is, in the RPA reaction, wild-type ExoⅢ has high binding and cleavage activity for normal double-stranded DNA, which can easily lead to nonspecific degradation, thereby affecting its amplification efficiency and detection specificity. Recombinase polymerase amplification technology is one of the isothermal nucleic acid amplification techniques (INAATs). It is also a nucleic acid amplification method that can perform amplification quickly, simply and sensitively to make up for the shortcomings of PCR technology in complex equipment and time consumption. In addition, the core of RPA technology lies in the use of multiple recombinases, single-strand binding proteins and strand-displacing DNA polymerases to specifically amplify the target sequence under constant temperature conditions. Therefore, ExoⅢ plays an important role in the RPA amplification process, especially in fluorescent probe RPA. The shortcomings of ExoⅢ will also limit the application scope of RPA amplification technology.

[0003] Furthermore, the thermosensitive properties of wild-type ExoⅢ are also insufficient. Under lower temperature conditions, such as 25-35°C, wild-type ExoⅢ still maintains high activity, which limits its application in temperature-controlled reactions.

[0004] The present invention provides a mutant Exo III and its application, so as to solve the problems existing in the prior art of wild-type Exo III, such as poor specificity, insufficient thermosensitivity, and limited scope of application. Summary of the Invention

[0005] The purpose of the present invention is to provide a mutant ExoⅢ and its application to solve the problems existing in the prior art such as poor specificity, insufficient thermosensitivity and limited scope of application of wild-type ExoⅢ.

[0006] The technical solution of the present invention is: a mutant ExoⅢ, which comprises a core site mutation combination compared with the amino acid sequence of wild-type ExoⅢ;

[0007] The core site mutation combination includes: G113N, R189N.

[0008] Preferably, the mutant Exo III further comprises auxiliary mutation combination 1 and / or auxiliary mutation combination 2 compared with the amino acid sequence of the wild-type Exo III;

[0009] The auxiliary mutation combination 1 includes: P145S, W186K;

[0010] The auxiliary mutation combination 2 includes: K121E, D132N.

[0011] Preferably, the mutant ExoⅢ includes a core site mutation combination, an auxiliary mutation combination 1 and an auxiliary mutation combination 2 compared with the amino acid sequence of the wild-type ExoⅢ.

[0012] Preferably, the amino acid sequence of the mutant ExoⅢ is shown in SEQ ID No.1.

[0013] The present invention also provides applications of the mutant ExoⅢ, including applications of the mutant ExoⅢ in RPA nucleic acid amplification technology, applications in DNA repair technology, applications as a thermosensitive nuclease in nucleic acid processing reactions, and the like.

[0014] Preferably, the DNA repair technology includes AP site recognition and repair.

[0015] Compared with the prior art, the advantages of the present invention are:

[0016] (1) The present invention provides a mutant ExoⅢ and its application. By using genetic engineering technology to introduce a core site mutation combination, an auxiliary mutation combination 1 and / or an auxiliary mutation combination 2 into the amino acid sequence of the wild-type ExoⅢ, the characteristics of ExoⅢ are optimized, so that the mutant ExoⅢ obtained after site-directed mutagenesis has excellent specificity and thermosensitivity; thereby enhancing the mutant ExoⅢ's ability to recognize AP sites and reducing the mutant ExoⅢ's non-specific cutting of normal double-stranded DNA; and, enhancing the mutant ExoⅢ's ability to recognize specific DNA defect sites, improving the mutant ExoⅢ's template-specific cutting performance in the RPA reaction, and significantly improving the RPA nucleic acid amplification efficiency, thereby improving the application range of the mutant ExoⅢ in the RPA technology; at the same time, it can also optimize the flexibility and stability of the mutant ExoⅢ, so that the mutant ExoⅢ can quickly lose activity below 35°C, so that the mutant ExoⅢ can be used in nucleic acid processing reactions that require temperature control, and expand the application range of the mutant ExoⅢ; solve the problems of poor specificity, insufficient thermosensitivity, and limited application range of the wild-type ExoⅢ in the prior art.

[0017] (2) The present invention provides a mutant ExoⅢ and its application. Compared with the amino acid sequence of the wild-type ExoⅢ, the amino acid sequence of the mutant ExoⅢ includes at least one of a group of mutation combinations such as G113N and R189N, P145S and W186K, K121E and D132N; wherein, the glycine at position 113 is mutated to asparagine, which can improve the binding performance of ExoⅢ with metal ions and the geometric adaptability with substrates; the arginine at position 189 is mutated to asparagine, which can weaken the electrostatic attraction of ExoⅢ and enhance the recognition and specific cleavage of ExoⅢ to the AP site; the proline at position 145 is mutated to serine. Acid can enhance the thermosensitivity and protein flexibility of ExoⅢ; the mutation of tryptophan at position 186 to lysine can change the hydrophobic region of ExoⅢ and reduce the affinity of ExoⅢ to double-stranded DNA; the mutation of lysine at position 121 to glutamate can regulate the binding performance of the active center of ExoⅢ with metal ions through charge changes; the mutation of aspartic acid at position 132 to asparagine can improve the adaptability of ExoⅢ to DNA defect sites, which is used to enhance the efficiency of RPA nucleic acid amplification; therefore, by introducing the above-mentioned site-directed mutations into the amino acid sequence of wild-type ExoⅢ, the specificity and thermosensitivity of ExoⅢ can be optimized, and the application range of ExoⅢ can be expanded. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0019] Figure 1 The SDS-PAGE protein electrophoresis results of the target protein of the present invention after nickel column affinity chromatography;

[0020] Figure 2 The SDS-PAGE protein electrophoresis detection result of the target protein of the present invention after elution with an ion column;

[0021] Figure 3 The results are the test results of the cleavage performance of the mutant ExoⅢ and wild-type ExoⅢ in the RPA reaction;

[0022] Figure 4 The performance test results of the mutant ExoⅢ of the present invention in the RPA reaction at a temperature of 30°C;

[0023] Figure 5 The performance test results of the mutant ExoⅢ described in the present invention in the RPA reaction at a temperature of 35°C;

[0024] Figure 6 The performance test results of the mutant ExoⅢ of the present invention in the RPA reaction at a temperature of 40°C;

[0025] Wherein: M, protein marker; 1, supernatant after fragmentation; 2, flow-through; 3, 40mM imidazole eluent; 4, 100mM first imidazole eluent; 5, 100mM second imidazole elution; 6, nickel column collection solution; 7, linear elution flow-through; 8a, 8b, 8c are all 100mM-500mM NaCl linear eluents. DETAILED DESCRIPTION

[0026] The present invention will be described in further detail below with reference to specific embodiments:

[0027] In response to the shortcomings of the existing wild-type ExoⅢ, the present invention first predicts and screens multiple key sites through homology modeling and molecular docking methods; then, the amino acid sequence of the wild-type ExoⅢ, such as that from Escherichiacoli, is extracted, and multiple key site mutations are designed and introduced by site-directed mutagenesis; these key site mutations include at least one group of G113N and R189N, P145S and W186K, K121E and D132N, etc.; among them, mutating glycine at position 113 to asparagine and mutating arginine at position 189 to asparagine are core site mutation combinations, which are used to change the geometric configuration of the active center of ExoⅢ, enhance the cutting ability of ExoⅢ to the AP site, and weaken the binding of ExoⅢ to ordinary double-stranded DNA; mutating proline at position 145 to asparagine is a key site mutation combination. Auxiliary mutation combination 1 is to mutate amino acid to serine and tryptophan at position 186 to lysine, which is used to enhance the thermosensitivity of ExoⅢ and reduce ExoⅢ's binding ability to double-stranded DNA; auxiliary mutation combination 2 is to mutate lysine at position 121 to glutamate and aspartic acid at position 132 to asparagine, which is used to regulate ExoⅢ's binding performance with metal ions and substrate adaptation performance by introducing additional charges, optimize the active center conformation of ExoⅢ, and enhance the specificity and efficiency of ExoⅢ in DNA treatment in RPA reaction; then, the mutated amino acid sequence is cloned into the expression vector (pET28a), efficiently expressed in Escherichia coli (BL21), and purified using a nickel column to obtain a highly pure mutant ExoⅢ protein; finally, the function of the mutant ExoⅢ protein was verified through experiments.

[0028] 1. Construction and inducible expression of mutant ExoⅢ

[0029] The amino acid sequence of the wild-type ExoⅢ from Escherichia coli was extracted, and six sites, G113N, P145S, W186K, R189N, K121E and D132N, were introduced by site-directed mutagenesis. A professional company (Suzhou Hongxun Biotechnology Co., Ltd.) was commissioned to construct a vector (pET28a) of the mutant sequence to obtain plasmid vector dry powder.

[0030] First, centrifuge the plasmid powder and dissolve it in 100 μl of sterile water. Mix thoroughly to obtain a plasmid solution. Next, remove 100 μL of E. coli competent cells BL21 (DE3) and place on ice. Once thawed, add 2 μL of the above plasmid solution to the competent E. coli cells and gently flick them with your fingers to mix thoroughly. Place on ice for 30 minutes. Then, heat shock the cells at 42°C for 90 seconds, place them on ice for 2 minutes, and add 500 μL of resistance-free LB medium. Incubate the cells at 37°C, 180 rpm for 1 hour, and centrifuge them at 3000 rpm for 2 minutes. Discard 250 μL of the supernatant and gently pipette to mix thoroughly. Then, spread the solution onto LB solid medium containing Kan resistance and incubate them inverted overnight at 37°C in a 37°C incubator to obtain a single colony. LB medium is the abbreviation for Luria-Bertani medium.

[0031] A single colony after transformation was picked and inoculated into 10 mL of LB liquid culture medium containing Kan resistance (100 μg / mL), and cultured in a shaking incubator at 37°C and 180 rpm for 4 h; then, the strain was preserved using 40% glycerol; and then 1% of the inoculum was inoculated into 1 L of LB culture medium containing Kan resistance (100 μg / mL), and cultured at 37°C and 180 rpm until the OD600 was 0.6-0.8, 0.1-1 mM IPTG was added, and expression was induced at 20-30°C for 10-15 h to obtain a fermentation broth.

[0032] 2. Protein purification of mutant ExoⅢ

[0033] 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; the resuspended bacteria were then disrupted 1 to 2 times using an ATS high-pressure homogenizer at 1000 MPa and 4°C to form a disruption liquid; thereafter, the centrifuge was turned on and pre-cooled, and the disruption liquid was centrifuged at 4°C and 7000 rpm after pre-cooling. The liquid after centrifugation was affinity chromatographed using a nickel column, and eluted with buffer B and buffer C in sequence, and the elution peak was collected; 20 μL of the elution peak was sampled for SDS-PAGE protein electrophoresis detection; after elution, the target protein was dialyzed into buffer D for further purification. Among them, buffer A is: 25mM Tris·HCl (pH8.0), 10mM imidazole, 500mM sodium chloride; buffer B is: 25mM Tris·HCl (pH8.0), 40mM imidazole, 500mM sodium chloride; buffer C is: 25mM Tris·HCl (pH8.0), 100mM imidazole, 500mM sodium chloride; buffer D is: 25mMTris (pH8.0), 50mM sodium chloride, 1mM DTT, 1mM EDTA.

[0034] After affinity chromatography using nickel column, Figure 1 As shown, the SDS-PAGE protein electrophoresis detection results showed that, compared with the protein marker, at 35KDa, the supernatant 1 and flow-through 2 after fragmentation almost did not contain the target protein, while the 40mM imidazole eluent 3, the 100mM first imidazole eluent 4, and the 100mM second imidazole eluent 5 all clearly contained the target protein, and the position was correct; among them, the target proteins in the 40mM imidazole eluent 3 and the 100mM imidazole second eluent 5 were both of high purity and could be used for further purification of the target protein; that is, the residual nucleic acid in the target protein was removed by the ion column (Q column) to further purify the target protein.

[0035] After dialysis, the 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 8.0), 1 M sodium chloride, 1 mM DTT, 1 mM EDTA; buffer F is 25 mM Tris (pH 8.0), 50 mM sodium chloride, 1 mM DTT, 1 mM EDTA, and 40% glycerol.

[0036] After linear elution using an ion column, Figure 2As shown, the results of SDS-PAGE protein electrophoresis showed that the linear elution flowthrough 7 did not contain the target protein, and the nickel column collection solution 6 and the 100mM~500mM NaCl linear elution solutions 8a, 8b, and 8c all clearly contained the target protein with high purity and correct position; further indicating that after the above two-step purification, a relatively high purity target protein was obtained, namely the mutant ExoⅢ; and compared with the amino acid sequence of the wild-type ExoⅢ, the amino acid sequence of the mutant ExoⅢ also includes a core site mutation combination, an auxiliary mutation combination 1, and an auxiliary mutation combination 2. Among them, the amino acid sequence of the mutant ExoⅢ is shown in SEQ ID No. 1, and the amino acid sequence of the wild-type ExoⅢ is shown in SEQ ID No. 2.

[0037] 3. Comparison of the cleavage performance of mutant ExoⅢ and wild-type ExoⅢ in RPA reaction

[0038] Using HBV as the research object, the cleavage performance of mutant ExoⅢ and wild-type ExoⅢ in the RPA reaction was investigated. In addition, the recognition, cleavage specificity, and cleavage efficiency of mutant ExoⅢ at the AP site during the RPA reaction were also investigated. The specific experimental design and experimental results are as follows:

[0039] (1) The primers and probes used in the RPA reaction are shown in Table 1;

[0040] Table 1. Primers and probes used in RPA reactions

[0041]

[0042] (2) 50 μL RPA reaction system (pH 7.6) including: RPA particles, 1000 nM HBV-RPA-F, 1000 nM HBV-RPA-R, 500 nM HBV-RPA-P, 5% PEG 20000, 14 mM magnesium acetate, and 5 μL HBV template (103 copies / mL);

[0043] (3) The amplification procedure was: 50°C, 60 seconds; 40°C, 20 minutes, and the fluorescence signal value was collected every 30 seconds.

[0044] The test results of the cleavage performance of mutant ExoⅢ and wild-type ExoⅢ in RPA reaction are as follows Figure 3As shown in the figure, after 20 cycles of wild-type ExoⅢ, the collected fluorescence signal value began to decrease significantly, indicating that the wild-type ExoⅢ had a higher activity in the early stage and produced more non-specific cutting, resulting in a decrease in its activity in the late cycle, a gradual decrease in cutting efficiency, and a downward trend in fluorescence; while the mutant ExoⅢ had a fluorescence value of 3049.5 after 5 cycles, and in the subsequent cycles, its fluorescence value was still able to maintain around 3049.5, with no obvious downward trend; this further indicates that in the entire reaction process, the mutant ExoⅢ and other enzymes have higher activity and higher cutting efficiency; and the mutant ExoⅢ can specifically recognize and cut the AP site of THF; thus It is shown that by using the site-directed mutagenesis method to introduce six sites, G113N, P145S, W186K, R189N, K121E and D132N, into the amino acid sequence of the wild-type ExoⅢ, the ability of ExoⅢ to cut double-stranded DNA can be significantly reduced, and the recognition and cutting specificity of ExoⅢ for AP sites can be improved; and the template recognition ability and amplification efficiency of ExoⅢ in the RPA reaction are optimized; the mutant ExoⅢ can be used as an optimized nuclease in the field of recombinase polymerase amplification technology, suitable for clinical diagnosis and rapid detection; it can also be used in the field of AP site recognition and repair, for studying DNA repair pathways and high-specificity DNA degradation experiments.

[0045] 4. Performance test of mutant ExoⅢ in RPA reaction under different temperature conditions

[0046] This test also used HBV as the research object and used the RPA primers and probes listed in Table 1 and the above-mentioned 50μL RPA reaction system (pH 7.6) to examine the effect of mutant ExoⅢ on RPA reaction efficiency at 30°C, 35°C, and 40°C. The specific experimental design and results are as follows:

[0047] The amplification procedures are:

[0048] 1) 50°C, 60 seconds; 30°C, 20 minutes, collecting fluorescence signal values ​​every 30 seconds;

[0049] 2) 50°C, 60 seconds; 35°C, 20 minutes, collecting fluorescence signal values ​​every 30 seconds;

[0050] 3) 50°C, 60 seconds; 40°C, 20 minutes, collecting fluorescence signal values ​​every 30 seconds.

[0051] The test results of the effect of mutant ExoⅢ on RPA reaction efficiency at 30℃, 35℃ and 40℃ are as follows: Figure 4 、 Figure 5 、 Figure 6As shown, at a temperature of 40°C, the mutant ExoⅢ always has good activity; when the temperature is reduced to 35°C, the mutant ExoⅢ has almost no activity; at a temperature of 30°C, the mutant ExoⅢ has completely lost its activity; this further indicates that the mutant ExoⅢ has excellent thermosensitive properties; the mutant ExoⅢ has almost no activity at a temperature of 35°C, which helps to avoid the phenomenon that the RPA reaction has already started at low temperatures, affecting the detection efficiency and result determination; the mutant ExoⅢ can be used as a thermosensitive nuclease, suitable for nucleic acid processing reactions that require temperature control, and improve the specificity of nucleic acid processing experiments through rapid inactivation at low temperatures.

[0052] 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.

[0053]

[0054]

Claims

1. A mutant ExoⅢ, characterized in that: Compared with the amino acid sequence of wild-type ExoⅢ, the mutant ExoⅢ has mutation sites G113N, R189N, P145S, W186K, K121E and D132N based on the amino acid sequence shown in SEQ ID No.

2.

2. The mutant Exo III according to claim 1, characterized in that: The amino acid sequence of the mutant ExoⅢ is shown in SEQ ID No.

1.

3. The use of the mutant Exo III according to claim 1 or 2, characterized in that: This includes the application of the mutant ExoⅢ in RPA nucleic acid amplification technology and DNA repair technology, as well as the application as a thermosensitive nuclease in nucleic acid processing reactions.

4. The use of the mutant Exo III according to claim 3, characterized in that: The DNA repair technology includes AP site recognition and repair.

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