Method for preparing formamidopyrimidine DNA glycosylase

Abstract

Provided is a method for preparing formamidopyrimidine DNA glycosylase. Specifically, recombinant formamidopyrimidine DNA glycosylase is expressed by means of genetically engineered Escherichia coli. The recombinant formamidopyrimidine DNA glycosylase achieves high-level soluble expression in the Escherichia coli system, and has strong catalytic activity. Thus, the present invention has the advantages of short production cycle, easy purification of expression products, low cost and the like, and can achieve industrial production of recombinant formamidopyrimidine DNA glycosylase.

Description

Method for preparing formamidopyrimidine DNA glycosylase Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for preparing formamidopyrimidine DNA glycosylase. Background Art

[0002] DNA is subject to deamination, oxidation, and hydrocarbonation reactions within cells, resulting in a large number of damaged bases. This damage can cause mismatches during DNA replication and is a major cause of cancer and neurodegenerative diseases. An endogenous base excision repair mechanism (BER) exists within biological cells. Initially, DNA glycosylases are responsible for locating and removing most endogenously damaged bases.

[0003] Formamidopyrimidine DNA glycosylase (Fpg) is a glycosylase found in Escherichia coli. Fpg possesses both N-terminal glycosylase and AP lyase activities. Its excision repair process consists of two steps. The first step, through the N-terminal glycosylase activity, cleaves the damaged base from the double-stranded DNA, forming an apurinic (AP) site. The second step, through the AP lyase activity, cleaves the 3' or 5' end of the AP site, removing the AP site and creating a gap between the 3' and 5' phosphate groups. Fpg enzymes catalyze a wide range of substrates, including 7,8-dihydroxy-8-oxoguanine (8-oxoguanine), 8-hydroxyadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine, and 5-hydroxyuracil. 8-Oxoguanine is a hallmark substrate recognized by Fpg proteins. Due to the diversity of Fpg's substrates, it plays a crucial role in stabilizing the base excision repair system within cells and maintaining the integrity of the genome.

[0004] Due to its central role in eliminating DNA damage in vivo, Fpg has been widely used in a variety of assays to assess oxidative DNA damage in cells and in vitro. Although commercially available Fpg is available, its high price hinders widespread basic research and application. Limited research has been conducted on constructing genetically engineered expression systems for Fpg. In 1987, Serge Boiteux et al. expressed and purified Fpg. Limited by the current state of biotechnology, the purification process involved four steps, requiring 5-6 days to complete. This long production cycle made it difficult to scale up production. Intracellular overexpression of Fpg can have toxic effects on the host, potentially limiting its industrial production using genetically engineered bacteria. Therefore, achieving high expression and activity of Fpg using prokaryotic expression systems is crucial. The Escherichia coli prokaryotic expression system offers advantages such as ease of operation, high yield, short growth cycle, clear research background, excellent product stability, ease of purification, and amenable to mass production via transfection. These advantages offer significant advantages over the current four-step purification method.

[0005] Therefore, those skilled in the art are committed to transforming and recombinantly expressing the Fpg enzyme in order to prepare a low-cost Fpg enzyme that is suitable for industrial production and can maintain high reaction activity.

[0006] Summary of the Invention

[0007] The object of the present invention is to provide a method for preparing formamidopyrimidine DNA glycosylase.

[0008] In the first aspect of the present invention, a recombinant formamide pyrimidine DNA glycosylase is provided, the amino acid sequence of the recombinant formamide pyrimidine DNA glycosylase is shown in SEQ ID NO.1, or the amino acid sequence of the recombinant formamide pyrimidine DNA glycosylase has at least 80% homology compared with SEQ ID NO.1; more preferably, it has at least 90% homology; most preferably, it has at least 95% homology; such as at least 96%, 97%, 98%, or 99% homology.

[0009] The second aspect of the present invention provides a polynucleotide molecule encoding the recombinant formamidopyrimidine DNA glycosylase described in the first aspect of the present invention.

[0010] In another preferred embodiment, the polynucleotide molecule sequence is shown as SEQ ID NO.4.

[0011] The third aspect of the present invention provides a vector, wherein the vector contains the nucleic acid molecule described in the second aspect of the present invention.

[0012] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the first aspect of the present invention or the chromosome is integrated with the nucleic acid molecule described in the second aspect of the present invention.

[0013] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0014] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0015] The fifth aspect of the present invention provides a method for preparing the recombinant formamidopyrimidine DNA glycosylase according to the first aspect of the present invention, comprising the steps of:

[0016] (i) culturing the host cell according to the fourth aspect of the present invention under suitable conditions to express the recombinant formamidopyrimidine DNA glycosylase; and

[0017] (ii) isolating the recombinant formamidopyrimidine DNA glycosylase.

[0018] In another preferred embodiment, the temperature for culturing the host cells in step (i) is 20°C-40°C; preferably 25°C-37°C, such as 37°C.

[0019] In another preferred embodiment, the host cell in step (i) is an Escherichia coli cell.

[0020] The sixth aspect of the present invention provides a kit comprising the recombinant formamidopyrimidine DNA glycosylase described in the first aspect of the present invention.

[0021] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the expression test results of recombinant formamidopyrimidine DNA glycosylase;

[0023] Figure 2 is the electrophoresis diagram of enzyme activity detection. DETAILED DESCRIPTION

[0024] Through extensive and in-depth research, the inventors have developed a method for expressing recombinant formamidopyrimidine DNA glycosylase in genetically engineered Escherichia coli. The recombinant formamidopyrimidine DNA glycosylase achieves soluble expression in the E. coli system, with high protein yield and high catalytic activity. This method offers advantages such as a short production cycle, easy purification of the expressed product, and low cost, enabling industrialized production of the recombinant formamidopyrimidine DNA glycosylase. This is the basis for the completion of the present invention.

[0025] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0027] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0028] In a preferred embodiment of the present invention, the amino acid sequence of the recombinant formamidopyrimidine DNA glycosylase according to the present invention (SEQ ID NO.1) is as follows:

[0029] In a preferred embodiment of the present invention, the gene sequence of the recombinant formamidopyrimidine DNA glycosylase is as follows (optimized for synonymous codon preference in Escherichia coli, SEQ ID NO. 4):

[0030] Those skilled in the art can obtain the recombinase gene sequence of the present invention using conventional methods, such as complete artificial synthesis or PCR synthesis. A preferred synthesis method is asymmetric PCR. Asymmetric PCR uses unequal amounts of a pair of primers to produce a large amount of single-stranded DNA (ssDNA) after PCR amplification. These primers are referred to as the non-restricting primer and the restricting primer, respectively, and their ratio is generally 50-100:1. In the first 10-15 cycles of the PCR reaction, the amplified product is primarily double-stranded DNA. However, once the restricting primer (low-concentration primer) is consumed, PCR guided by the non-restricting primer (high-concentration primer) produces a large amount of single-stranded DNA. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods, such as gel electrophoresis.

[0031] The mutant enzyme of the present invention can be expressed or produced by conventional recombinant DNA technology, comprising the steps of:

[0032] (1) transforming or transducing a suitable host cell with a polynucleotide encoding the protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0033] (2) culturing the host cells in a suitable culture medium;

[0034] (3) Isolate and purify the target protein from the culture medium or cells to obtain the target enzyme.

[0035] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the enzyme of the present invention and appropriate transcriptional / translational control signals, preferably the commercially available vector pET28. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcriptional terminator. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

[0036] The recombinant vector comprises, in the 5' to 3' direction: a promoter, a target gene, and a terminator. If necessary, the recombinant vector may further comprise the following elements: a protein purification tag; a 3' polyadenylation signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a selectable marker (antibiotic resistance gene, fluorescent protein, etc.); an enhancer; or an operator.

[0037] Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host.

[0038] Those skilled in the art can construct vectors containing the promoter of the present invention and / or target gene sequence using well-known methods, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

[0039] The expression vectors of the present invention can be used to transform appropriate host cells to cause the host to transcribe the target RNA or express the target protein. The host cells can be prokaryotes such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, or Agrobacterium; lower eukaryotic cells such as yeast cells; or higher eukaryotic cells such as plant cells. Those skilled in the art will appreciate how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques familiar to those skilled in the art. When the host is a prokaryotic organism (such as Escherichia coli), CaCl2 treatment or electroporation can be used. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Plants can also be transformed using methods such as Agrobacterium transformation or gene gun transformation, for example, the leaf disc method, the embryo transformation method, and the flower bud immersion method. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants.

[0040] The term "operably linked" means that the target gene to be transcribed and expressed is linked to its control sequence in a conventional manner in the art so as to be expressed.

[0041] Cultivation of engineered bacteria and fermentation production of target protein

[0042] After obtaining the engineered cells, the engineered cells can be cultured under suitable conditions to express the protein encoded by the gene sequence of the present invention. Depending on the host cell, the culture medium used can be selected from various conventional culture media, and the cells are cultured under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

[0043] In the present invention, conventional fermentation conditions can be used. Representative conditions include (but are not limited to):

[0044] (a) Regarding temperature, the fermentation and induction temperature of the enzyme is maintained at 25-37°C;

[0045] (b) With regard to the pH value during the induction period, the pH during the induction period is controlled at 3-9;

[0046] (c) Dissolved oxygen (DO) is controlled at 10-90%. This can be maintained by introducing an oxygen / air mixture.

[0047] (d) As for feed, feed types should preferably include carbon sources such as glycerol, methanol, and glucose, which can be fed individually or in combination;

[0048] (e) With respect to the IPTG concentration during the induction period, conventional induction concentrations can be used in the present invention, and the IPTG concentration is usually controlled at 0.1-1.5 mM;

[0049] (f) The induction time is not particularly limited, but is usually 2 to 20 hours, preferably 5 to 15 hours.

[0050] The target protein of the present invention is present intracellularly in Escherichia coli cells. The host cells are collected by centrifugation and then disrupted by high pressure, mechanical force, enzymatic cell lysis, or other cell disruption methods to release the recombinant protein. High pressure is preferred. The host cell lysate can be initially purified by methods such as flocculation, salting out, and ultrafiltration, followed by purification by chromatography, ultrafiltration, or directly by chromatography.

[0051] Chromatographic techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc. Commonly used chromatography methods include:

[0052] 1. Anion exchange chromatography:

[0053] Anion exchange chromatography media include (but are not limited to) Q-Sepharose and DEAE-Sepharose. If the fermentation sample has a high salt concentration that affects binding to the ion exchange media, the salt concentration should be reduced before ion exchange chromatography. The sample can be equilibrated with a buffer exchanger using dilution, ultrafiltration, dialysis, gel filtration chromatography, or other methods until the equilibration buffer system matches that of the corresponding ion exchange column. The sample can then be loaded and eluted using a salt concentration or pH gradient.

[0054] 2. Hydrophobic chromatography:

[0055] Hydrophobic chromatography media include (but are not limited to) phenyl-sepharose, butyl-sepharose, and octyl-sepharose. The sample is loaded with a solution containing a salt concentration increased by adding NaCl, (NH₄)₂SO₄, or other methods. Elution is then performed by decreasing the salt concentration. Hydrophobic chromatography removes contaminating proteins with significantly different hydrophobicities.

[0056] 3. Gel Filtration Chromatography

[0057] Hydrophobic chromatography media include (but are not limited to): Sephacryl, Superdex, Sephadex, etc. Gel filtration chromatography can be used to replace the buffer system or further purify the product.

[0058] 4. Affinity chromatography

[0059] Affinity chromatography media include (but are not limited to): HiTrap TM HeparinHPColumns.

[0060] 5. Membrane filtration

[0061] Ultrafiltration media include organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, and metal membranes. Purification and concentration can be achieved through membrane filtration.

[0062] The main advantages of the present invention are:

[0063] (1) The recombinant Fpg enzyme of the present invention can be expressed in large quantities in an E. coli expression system, is easy to purify with a high yield, and has high enzyme activity.

[0064] (2) The present invention provides a method for industrial production of Fpg enzyme, which uses genetically engineered Escherichia coli to recombinantly express Fpg enzyme. The purification step is simple, the output enzyme activity is high, the protein expression level is high, and the production cost is low, which is suitable for large-scale production.

[0065] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, are generally performed according to conventional conditions such as those described in "Molecular Cloning Laboratory Manual" by Sambrook.J et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0066] Example 1 Construction, expression and purification of E. coli Fpg enzyme plasmid

[0067] 1) Using the protein sequence of the Escherichia coli Fpg enzyme provided by NCBI as a reference and in combination with the experimental design requirements of the present invention, its structure was optimized by computer molecular simulation. After optimization of the synonymous codon preference of Escherichia coli, the vector was connected to pET-28a(+) and the C-terminal fusion expression (His)6 tag was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0068] The protein sequence of the Escherichia coli Fpg enzyme obtained by optimization screening is as follows (SEQ ID NO.1):

[0069] The gene sequence 1 of Escherichia coli with optimized synonymous codon preference is as follows (SEQ ID NO.2):

[0070] The gene sequence 2 optimized for synonymous codon preference in Escherichia coli is as follows (SEQ ID NO.3):

[0071] The gene sequence 3 of Escherichia coli with optimized synonymous codon preference is as follows (SEQ ID NO.4):

[0072] 2) Transformation of recombinant plasmid into Escherichia coli BL21(DE3)

[0073] Take 1 μL of plasmid and add it to 30 μL of competent E. coli BL21(DE3) on ice. Place the culture on ice for 20 minutes, heat shock the culture in a 42°C water bath for 45 seconds, and immediately place it on ice for 2 minutes. Add 400 μL of antibiotic-free SOC medium and incubate the culture at 37°C with shaking at 220 rpm for 50 minutes. Spread 100 μL of the culture evenly onto an LB plate containing 100 μg / mL kanamycin and incubate the culture overnight at 37°C.

[0074] 3) Target protein expression

[0075] Pick the single clone from step 2) and inoculate it in TB medium containing 100 μg / mL kanamycin aseptically, and culture it at 37°C with shaking at 220 rpm until the OD 600 The cells were induced with IPTG (final concentration 0.1 mM) at 0.6-0.8, and cultured overnight with shaking at 37°C and 18°C, respectively. The group without IPTG was used as the control and cultured at 37°C for 3 h. Each experiment was repeated once.

[0076] Sampling was performed by ultrasonication and SDS-PAGE analysis. The results showed that Fpg enzyme was soluble and expressed in the supernatant of TB culture medium at both 37°C and 18°C. The protein had a molecular weight of approximately 30 kDa, which is consistent with the protein size predicted by the Expasy website (30 kDa).

[0077] Figure 1 shows the electrophoresis results of Fpg enzymes expressed from different gene sequences, where lane 1 is a molecular weight marker, lane 2 is the optimized gene sequence 1, lane 3 is the optimized gene sequence 2, lane 4 is the optimized gene sequence 3, and lane 5 is the wild-type Fpg enzyme.

[0078] 4) Purification of Fpg enzyme

[0079] Culture 1.5 L of bacterial liquid in a shake flask with TB medium. Expression conditions are consistent with those for the target protein expression in step 3). Collect the cells by centrifugation. Weigh approximately 30 g of cells and resuspend them in 230 ml of Lysis Buffer on ice. Disintegrate with a homogenizer and centrifuge at 8000 rpm at 4°C for 30 min. Remove the supernatant and filter through a 0.22 μm needle filter to obtain the supernatant. The supernatant is then subjected to Ni-column affinity chromatography using a HisTrap column. TMHP, 0-80% Buffer B for linear elution, take the main peak eluate for ion exchange elution, the purification column used is HisTrap TM For Q-HP, the target protein was eluted with 50% Buffer C. After overnight dialysis in dialysate, the concentration was determined by SDS method. The expression level of the target protein was calculated.

[0080] The concentrations of the solutions used are as follows:

[0081] BufferA: 50mM Tris, 50mM NaCl, 5% Glycerol, pH9.0

[0082] BufferB: 50mM Tris, 50mM NaCl, 500mM Midazole, 5% Glycerol, pH9.0

[0083] BufferC: 100mM Tris, 1M NaCl, 10% Glycerol, pH9.0

[0084] Lysisbuffer: 50mM Tris, 300mM NaCl, 5% Glycerol, pH9.0

[0085] Fpg dialysate: 50 mM NaCl, 20 mM Tris-HCl, 0.5 mM EDTA, 50% Glycerol, 200 μg / ml BSA, pH 8.0

[0086] Calculations showed that the protein yield of the strain with optimized gene sequence 1 was 5.1 mg / g bacteria; the protein content of the strain with optimized gene sequence 2 was 8.6 mg / g bacteria; and the protein content of the strain with optimized gene sequence 3 was 18.2 mg / g bacteria, indicating a very high protein yield.

[0087] Example 2 Determination of recombinant Fpg enzyme activity

[0088] 1. Experimental Purpose

[0089] The Fpg enzyme activity in the self-produced Fpg enzyme of the present invention and the commercially available benchmark reagent was calibrated.

[0090] 2. Experimental Materials

[0091] 1) Samples: Fpg enzyme purified sample, Fpg enzyme positive reference (NEB).

[0092] 2) Equipment: PCR amplification instrument, chemiluminescence fluorescence imaging system.

[0093] 3) Substrate Fpg-Test2: AAGACGGCATACGAG / i8oxodG / ATAACTATCAAGAC, with a FAM group added to the 5' end and purified by PAGE.

[0094] 4) Reagent: NEBuffer TM 1 (NEB), Fpg (NEB), Purified BSA (NEB), 6X DNA Loading Buffer, 15% urea PAGE gel.

[0095] 5) Fpg enzyme diluent: 20 mM Tris-HCl, 50 mM NaCl, 0.5 mM EDTA, 200 μg / ml BSA, 50% Glycerol, pH 8 @ 25°C.

[0096] 3. Experimental steps

[0097] NEB positive enzyme was diluted as shown in the following table:

[0098] The Fpg enzyme prepared in Example 1 was diluted to the same protein concentration, and then diluted as shown in the table below.

[0099] Reaction system configuration (single reaction system)

[0100] The above system was prepared at a volume of (N+3), where N is the number of diluted samples. 6 μL / well of the prepared reaction system was added to an 8-well tube strip. 4 μL of each test sample was then added to each tube. The system was gently flicked to mix and centrifuged until bubbles were essentially eliminated. A separate NTC group was set up, where 4 μL of Fpg enzyme dilution was added to 6 μL of the reaction system.

[0101] The PCR amplification instrument was set to a reaction program of 37°C for 30 min and 95°C for 10 min, and 8-well plates were added until the reaction was completed.

[0102] Add 2 μL of 6X DNA Loading Buffer to the reaction system, mix well, and perform urea PAGE electrophoresis (voltage: 120 V, time: 40-50 min). After electrophoresis, scan the urea PAGE gel in the blue light channel on a ChampChemi 910PLUS chemiluminescence fluorescence imaging system.

[0103] Experimental results:

[0104] The test results showed that the Fpg enzyme produced by the strain with optimized gene sequence 1 and the Fpg enzyme produced by the strain with optimized gene sequence 2 had almost no activity.

[0105] The electrophoresis results of the Fpg enzyme activity assay of strain 3 with optimized gene sequence and the control Fpg enzyme are shown in FIG2 .

[0106] The trend of the Fpg enzyme produced by the optimized gene sequence 3 strain after dilution 4 times was consistent with that of the control enzyme after dilution 4 times, and the activity was consistent with that of the positive enzyme, which was 8*4=32U / μL.

[0107] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A recombinant formamidopyrimidine DNA glycosylase, characterized in that: The amino acid sequence of the recombinant formamidopyrimidine DNA glycosylase is shown in SEQ ID NO.1, or the amino acid sequence of the recombinant formamidopyrimidine DNA glycosylase has at least 95% homology with SEQ ID NO.

1.

2. A polynucleotide molecule, characterized in that The polynucleotide molecule encodes the recombinant formamidopyrimidine DNA glycosylase according to claim 1.

3. The polynucleotide molecule according to claim 2, characterized in that The sequence of the polynucleotide molecule is shown in SEQ ID NO.

4.

4. A carrier, characterized in that The vector contains the polynucleotide molecule according to claim 2.

5. A host cell, characterized in that The host cell contains the vector of claim 4 or the polynucleotide molecule of claim 2 is integrated into the chromosome.

6. The host cell according to claim 5, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

7. A method for preparing the recombinant formamidopyrimidine DNA glycosylase according to claim 1, characterized in that: Includes steps: (i) culturing the host cell according to claim 5 under suitable conditions to express the recombinant formamidopyrimidine DNA glycosylase; and (ii) isolating the recombinant formamidopyrimidine DNA glycosylase.

8. The method according to claim 7, characterized in that The temperature of culturing the host cells in step (i) is 20°C-40°C 9. The method according to claim 7, characterized in that The host cell in step (i) is an Escherichia coli cell.

10. A kit, characterized in that: The kit comprises the recombinant formamidopyrimidine DNA glycosylase according to claim 1.

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