Method for producing uracil glycosylase and double vector system for use in the method

Abstract

The present application relates to the field of biotechnology, and more particularly, to a method for producing uracil glycosylase (UDG) and a double vector system used in the method. The method can significantly improve the solubility of UDG, thereby increasing the expression amount and yield of UDG, while ensuring the activity, stability and purity of UDG.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for producing uracil glycosylation enzyme and a dual-carrier system for the method. Background Technology

[0002] Uracil glycosylase (UDG) is a hydrolase that specifically degrades DNA containing uracil bases, primarily used for controlling residual contamination in PCR. Based on the thermal stability of the UDG protein, it is classified into thermostable UDG and thermosensitive UDG (not thermostable). Thermostable UDG, due to its good thermal stability, is difficult to completely inactivate by heat during use, thus affecting the sensitivity of downstream detection technologies (such as qPCR, multiplex PCR, and in vitro diagnostic kits). Therefore, thermosensitive UDG is more suitable for compatibility with high-sensitivity detection technologies such as qPCR.

[0003] Currently, thermosensitive UDGs mainly originate from psychrophilic marine organisms, such as Atlantic cod and Antarctic bacteria. UDGs derived from these organisms have poor heat resistance, easily forming inclusion bodies during expression, leading to low solubility. Furthermore, they are prone to activity reduction and loss during purification, ultimately resulting in low UDG yields. Therefore, the discovery of UDG genes, the construction of expression vectors, and the expression and purification processes are all technical challenges in developing this enzyme.

[0004] Therefore, there is an urgent need in the field for a method for producing UDG that can significantly improve the solubility of UDG, increase the expression level and yield of UDG, while ensuring the activity, stability and purity of UDG. Summary of the Invention

[0005] As mentioned above, existing UDG production methods result in low UDG yields due to the low solubility of UDG during expression and the tendency for activity reduction and loss during purification. Therefore, there is an urgent need in the art for a UDG production method that can significantly improve UDG solubility, thereby increasing UDG expression levels and yields, while ensuring UDG activity, stability, and purity.

[0006] In view of this, in a first aspect, the present invention provides a method for producing uracil glycosylation enzyme (UDG), the method comprising the following steps:

[0007] a) Provides an expression cell comprising a first vector capable of expressing the UDG and a second vector capable of expressing the molecular chaperone system GroEL-GroES, wherein the amino acid sequence of the UDG is shown in SEQ ID NO: 1, and the amino acid sequences of the GroEL and GroES are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively;

[0008] b) Under the first induction condition, the expressing cells are induced to express the molecular chaperone system GroEL-GroES;

[0009] c) Under the second induction condition, the expressing cells are induced to simultaneously express the molecular chaperone system GroEL-GroES and the UDG.

[0010] In a second aspect, the present invention provides a dual-carrier system, the dual-carrier system comprising:

[0011] (1) A first vector capable of expressing uracil glycosylation enzyme (UDG), the amino acid sequence of which is shown in SEQ ID NO: 1; and

[0012] (2) A second vector capable of expressing the molecular chaperone system GroEL-GroES, wherein the amino acid sequences of GroEL and GroES are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0013] In a third aspect, the present invention provides an expression cell comprising the dual-vector system of the second aspect of the present invention.

[0014] In a fourth aspect, the present invention provides a kit comprising: expression cells according to the third aspect of the present invention, and instructions on how to use the kit to produce uracil glycosylase (UDG).

[0015] The beneficial effects of the present invention include one or more of the following:

[0016] 1) A method for producing uracil glycosylation enzyme (UDG) is provided, which can significantly improve the solubility of UDG, thereby increasing the expression level and yield of UDG;

[0017] 2) A method for producing temperature-sensitive UDG is provided, which ensures the activity and stability of the temperature-sensitive UDG through low-temperature induction and low-temperature purification; Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other implementation schemes can be obtained based on these drawings without creative effort.

[0019] Figure 1 The phylogenetic tree is shown, constructed using psychrophilic UDGs and commonly used UDGs as objects.

[0020] Figure 2 The gel electrophoresis results of UDG (U1-U8) expression in *E. coli* containing a single plasmid system are shown. Before induction: lysed cells without inducer; Whole cells: lysed cells after induction; Supernatant: supernatant sample from whole cells; Dashed line represents UDG protein.

[0021] Figure 3 The gel electrophoresis results of UDG(U1-U8) expression in *E. coli* containing a single plasmid system are shown under different induction conditions at different temperatures and inducing agent concentrations. Before induction: lysed cells without inducing agent; Whole cells: lysed cells after induction agent addition; Supernatant: supernatant sample from whole cells; Dashed lines represent UDG protein.

[0022] Figure 4 The image shows the gel electrophoresis results of UDG (U3 and U4) expressed by Escherichia coli containing a dual plasmid system under the same induction conditions via simultaneous and stepwise induction.

[0023] Figure 5 Further, gel electrophoresis results of U3 expression in Escherichia coli containing single-plasmid or dual-plasmid systems under the same induction conditions are shown.

[0024] Figure 6 The image shows the gel electrophoresis results of U3 expressed in the dual plasmid system via simultaneous induction (A) and stepwise induction (B) after affinity chromatography. Detailed Implementation

[0025] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be clearly and completely described below in conjunction with its embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments that can be obtained by those skilled in the art based on the embodiments of this invention are within the scope of protection of this invention.

[0026] As mentioned above, existing UDG production methods result in low UDG yields due to the low solubility of UDG during expression and the tendency for activity reduction and loss during purification. Therefore, the purpose of this invention is to provide a novel UDG production method that improves UDG solubility, thereby increasing UDG expression levels and yields, while simultaneously ensuring UDG activity, stability, and purity.

[0027] Molecular chaperones are a class of proteins that assist in intracellular molecular assembly and protein folding. There are three main categories of molecular chaperones: chaperonins, the heat shock protein 70 family, and the heat shock protein 90 family. GroEL and its accessory protein GroES (GroEL-GroES) are typical chaperone proteins in *E. coli*. GroEL-GroES together form a barrel-shaped complex under the action of ATP, providing a suitable microenvironment for protein folding. During experiments, the inventors unexpectedly discovered that GroEL-GroES can also promote the folding of UDG. Therefore, the inventors hypothesized whether GroEL-GroES could be used to improve the solubility of UDG, thereby increasing UDG yield.

[0028] The inventors discovered through experiments that if the gene sequences expressing UDG and GroEL-GroES are constructed in the same vector and used to transform host cells, the resulting transformed cells cannot be induced stepwise. However, if the gene sequences expressing UDG and GroEL-GroES are constructed in two separate vectors (a dual-vector system), and these two vectors are used to transform host cells, the solubility of expressed UDG in the resulting transformed cells does not significantly improve during simultaneous induction. But during stepwise induction, the solubility of one type of UDG (i.e., U3 and U4 in this invention) significantly improves, becoming completely soluble. Thus, the inventors completed this invention.

[0029] Therefore, in a first aspect, the present invention provides a method for producing uracil glycosylation enzyme (UDG), the method comprising the following steps:

[0030] a) Provides an expression cell comprising a first vector capable of expressing the UDG and a second vector capable of expressing the molecular chaperone system GroEL-GroES, wherein the amino acid sequence of the UDG is shown in SEQ ID NO: 1, and the amino acid sequences of the GroEL and GroES are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively;

[0031] b) Under the first induction condition, the expressing cells are induced to express the molecular chaperone system GroEL-GroES;

[0032] c) Under the second induction condition, the expressing cells are induced to simultaneously express the molecular chaperone system GroEL-GroES and the UDG.

[0033] The expression cells mentioned in step a) can be pre-existing expression cells, or they can be obtained by transforming the same host cell with a first vector capable of expressing the UDG and a second vector capable of expressing the molecular chaperone system GroEL-GroES, respectively. Therefore, in some embodiments, in step a), the expression cells are obtained by transforming the same host cell with the first and second vectors, respectively.

[0034] The term "transformation" refers to the process of directly introducing a recombinant vector containing a foreign gene, such as a plasmid, into a prokaryotic cell, such as bacteria, to impart new genetic characteristics. In one embodiment, the first vector and the second vector transform the host cell using different transformation methods. Commonly used transformation methods include heat shock and electroporation. Therefore, in a preferred embodiment, the first vector transforms the host cell using heat shock, and the second vector transforms the host cell using electroporation. However, it should be understood that the transformation methods are not limited to these methods, and those skilled in the art can select the most suitable transformation method based on the type of vector and host cell.

[0035] To deliver a target gene into a biological cell (recipient cell) using genetic engineering, a carrier vehicle is needed to carry the foreign gene into the recipient cell. This carrier vehicle is called a vector. The vectors used in genetic engineering are a type of self-replicating DNA molecule. A segment of DNA is removed from the vector without affecting its replication. This vector can be used to replace or insert foreign (target) DNA to bring the target DNA into the host cell.

[0036] In this invention, the term "vector" refers to a self-replicating DNA molecule used in recombinant DNA technology to transfer exogenous gene fragments into host cells. Commonly used vectors include plasmids, phages, and viruses. In some embodiments, plasmids can be used as both the first and second vectors. In some embodiments, plasmids containing an UDG-encoding gene, enabling the expression of the UDG protein in suitable host cells, such as pET21a, pHUE, pET20b, pET-22b, pET-28a, or pET-32a plasmids (preferably pET21a plasmid), can be used as the first vector. As another example, pBAD33 plasmids containing an encoding gene for the molecular chaperone system GroEL-GroES, enabling the expression of this molecular chaperone in suitable host cells, can be used as the second vector. These are merely examples; those skilled in the art will understand that any modified vector capable of expressing the molecular chaperone system GroEL-GroES or UDG can be used in this invention.

[0037] A host cell is a cell that receives a foreign gene during transformation and transduction (infection). For transformation purposes, the host cell is typically a prokaryotic cell, such as a bacterial cell, with *Escherichia coli* being the most commonly used. In some embodiments, the host cell is a prokaryotic cell, such as a bacterial cell like *Escherichia coli*.

[0038] As described in the background section, "UDG" in this article, or uracil glycosylation enzyme, is a specific DNA hydrolase that degrades uracil-containing bases. It is mainly used for controlling residual contamination in PCR and can be classified into thermostable UDG and thermosensitive UDG based on its thermal stability. In a preferred embodiment, the inventors selected a thermosensitive UDG from the UDG branch originating from psychrophilic bacteria based on bioinformatics homology alignment and phylogenetic analysis. Its amino acid sequence is shown in SEQ ID NO: 1.

[0039] In step b), the expression cells are induced to express the molecular chaperone system GroEL-GroES using a first induction condition, which is induction at 16°C to 37°C for 0.5 to 5 hours using a first inducer at a concentration of 0.001% to 1%. Those skilled in the art will understand that in recombinant expression systems, the inducer is determined by the expression vector. In step b), GroEL-GroES is first induced to increase the intracellular baseline molecular chaperone content, ensuring that UDG is more fully folded during subsequent induction, thereby improving UDG solubility. As an example, when the first vector is a pET21a plasmid expressing the UDG, the first inducer can be arabinose. In some specific embodiments, the first induction condition is incubation at 25°C for 3 hours using 0.4% arabinose. In the context of this invention, the concentration % of the first and second inducers refers to a mass percentage concentration, that is, a concentration expressed as a percentage of the mass of the solute to the total mass of the solution.

[0040] In step c), a second induction condition is used to induce the expression cells to simultaneously express the molecular chaperone system GroEL-GroES and the UDG. The second induction condition involves inducing the expression cells at 16°C to 20°C for 16 to 20 hours using both the second and third inducing agents. The second inducing agent induces the expression cells to express the molecular chaperone system GroEL-GroES, and the third inducing agent induces the expression cells to express the UDG. The second inducing agent, like the first inducing agent, is used to induce the expression of the molecular chaperone system GroEL-GroES; they are essentially the same inducing agent. In step c), inducing the expression of the molecular chaperone system GroEL-GroES simultaneously with the induction of UDG is to maintain the intracellular GroEL-GroES content and further improve the solubility of UDG. Similarly, as an example, the second inducing agent can be arabinose. The concentration of the second inducer can be from 0.001% to 1%, for example, 0.001%, 0.05%, 0.2%, 0.4%, 0.6%, 0.8%, or 1.0%; the final concentration of the third inducer can be from 0.1 mM to 1.0 mM, for example, 0.1 mM, 0.2 mM, 0.5 mM, or 1.0 mM. In one specific embodiment, the second induction conditions are: using 0.4% arabinose as the second inducer and 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) as the third inducer, induction at 16°C for 16 to 20 hours.

[0041] To obtain high-purity UDG suitable for practical use, further purification of the expressed UDG is required after inducing UDG expression. Therefore, in some embodiments, the method further includes step d): disrupting the expressing cells obtained in step c) and purifying the UDG by affinity chromatography at a temperature of 4°C to 10°C.

[0042] Cell disruption can be performed using conventional cell disruption methods, including mechanical disruption (such as high-pressure homogenization, bead milling, impact disruption, and ultrasonic disruption), chemical and biochemical disruption (such as acid-base treatment, chemical reagent treatment, and enzymatic dissolution), and physical disruption (such as osmotic pressure shock and freeze-thaw cycles), but is not limited thereto. In one embodiment of the invention, the cell disruption is performed using ultrasonic disruption.

[0043] After cell disruption and before protein purification, preferably, totipotent nucleases can be used to treat the disrupted UDG-expressing cells. The purpose of using totipotent nucleases is to effectively control nucleic acid residue.

[0044] The protein purification can be performed using affinity chromatography. In one embodiment, the first vector also expresses an affinity tag, such as a His tag, FLAG tag, HAT tag, HPC tag, MBP tag, NusA tag, UB tag, or GST tag, in which case the affinity chromatography can be performed using the affinity tag. In a specific embodiment, the affinity tag can be a 6×His tag. As mentioned above, some UDGs are heat-sensitive, so it is best to perform the purification process at low temperatures. Therefore, in one embodiment, the entire purification process is performed at a temperature of 4°C to 10°C, for example, in a cryo-chromatograph.

[0045] In a second aspect, the present invention provides a dual-carrier system, the dual-carrier system comprising:

[0046] (1) A first vector capable of expressing uracil glycosylation enzyme (UDG), the amino acid sequence of which is shown in SEQ ID NO: 1; and

[0047] (2) A second vector capable of expressing the molecular chaperone system GroEL-GroES, wherein the amino acid sequences of GroEL and GroES are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0048] The vectors of this aspect of the invention have the same or similar properties as the vectors described in the first aspect of the invention. For example, in some embodiments, plasmids can be used as both the first and second vectors. For example, the pET21a plasmid or pHUE plasmid, which contains an UDG-encoding gene to express the UDG protein in a suitable host cell, can be used as the first vector. As another example, the pBAD33 plasmid, which contains an UGEN system GroEL-GroES-encoding gene to express the UGEN system in a suitable host cell, can be used as the second vector. These are merely examples, and those skilled in the art will understand that any modified vector capable of expressing the UGEN system GroEL-GroES or UDG can be used in this invention. For example, the vector can also be a plasmid such as pET20b, pET-22b, pET-28a, or pET-32a.

[0049] In addition, to facilitate the purification of UDG proteins obtained using this dual-vector system, in some specific embodiments, the first vector may also express an affinity tag, such as a His tag, FLAG tag, HAT tag, HPC tag, MBP tag, NusA tag, UB tag, or GST tag. In a preferred embodiment, the affinity tag is a 6×His tag.

[0050] In a third aspect, the present invention provides an expression cell comprising the dual-vector system of the second aspect of the present invention.

[0051] In this invention, the expressing cell can be a prokaryotic cell, such as a bacterial cell, like *Escherichia coli*. However, those skilled in the art will understand that any other cell suitable for expressing UDG protein using the method of this invention can also be used in this invention.

[0052] In a fourth aspect, the present invention provides a kit comprising: expression cells according to the third aspect of the present invention, and instructions on how to use the kit to produce uracil glycosylase (UDG).

[0053] In summary, this invention provides a method for producing uracil glycosylation enzyme (UDG), which can significantly improve the solubility of UDG, thereby increasing the expression level and yield of UDG.

[0054] The present invention will now be described in more detail with reference to the embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional chemical reagent stores. It should be noted that the above summary of the invention and the following detailed description are for illustrative purposes only and are not intended to limit the invention in any way.

[0055] Example

[0056] 1. Evolutionary analysis and gene design of UDG

[0057] Through comprehensive analysis of UDG / UNG products from different companies, the inventors discovered that current UDGs mainly originate from *E. coli* and the psychrophilic bacterium BMTU3346 (an Antarctic thermosensitive UDG). Currently, there is no sequence information for BMTU3346 UDG in databases. Based on the protein sequence provided in the literature (Jaeger et al., 2000), BLAST analysis in NCBI and UniProt revealed a protein with 98% homology to *Micrococcus terreus* UDG, thus raising questions about the information for this protein. Since thermosensitive UDGs from psychrophilic bacteria have a significant advantage in controlling PCR residual contamination, a search for psychrophilic UDGs was conducted in the NCBI database, resulting in 12 possible thermosensitive UDGs. A phylogenetic tree was constructed using psychrophilic UDGs and commonly used UDGs as the basis, with three main branches (see [link to phylogenetic tree]). Figure 1Branch 2 includes human UDG (18), Escherichia coli UDG (17), and previously synthesized Pseudomonas UDG (9). It is preliminarily speculated that the main drawback of this branch is that it cannot be completely inactivated at high temperatures, which may affect the yield of subsequent amplification products in practical applications. Branch 1 consists almost entirely of UDGs from psychrobacterial bacteria, and includes the previously reported HJ147-UDG and HJ171-UDG. Therefore, this branch has become the focus of development. Using UDG stability data from the BRENDA database, the inventors finally selected four UDG enzymes with thermal stability less than 50°C, namely, psychrobacter sp. HJ147 UDG (HJ147-UDG), Bacillus sp. HJ171 UDG (HJ171-UDG), Photobacterium aplysiae GMD509UDG (Pap-UDG), and Atlantic cod (Gadus morhus) (Cod-UDG).

[0058] 2. UDG sequence analysis and plasmid construction

[0059] Due to the lack of three-dimensional structural information of the thermosensitive UDG protein, the inventors selected two expression vectors, pET21a and pHUE, to construct the protein, which respectively produced UDG protein with a 6×His tag fused to the carboxyl terminus and UDG protein with a 6×His tag fused to the amino terminus + ubiquitin.

[0060] Different UDG protein sequences were obtained using the NCBI and UniProt databases. The amino acid sequences were then imported into the ProtParam (https: / / web.expasy.org / protparam / ) online tool to obtain the amino acid number, molecular weight, isoelectric point, extinction coefficient, etc. of different UDG proteins, as shown in Table 1 below.

[0061] Table 1. UDG sequence analysis

[0062]

[0063] In addition, the inventors selected the pBAD33 plasmid to express the GroEL-GroES protein. By comparing the restriction endonuclease cleavage sites in the nucleic acid sequence of the plasmid target protein with the multiple cloning sites of the corresponding expression plasmid, the restriction enzyme sites used for gene cloning were determined. The GroEL-GroES expression plasmid shares a single promoter and terminator, and a ribosome bind site (RBS) sequence needs to be added to either the GroEL or GroES protein during construction. Finally, the plasmid and gene information were submitted to the Nanjing Qingke Gene Synthesis Department to complete the construction of all UDG and pBAD33 gene expression plasmids.

[0064] 3. Construction of the dual-plasmid expression system

[0065] To construct a dual-plasmid expression system of UDG protein and molecular chaperone system GroEL-GroES in Escherichia coli expression strain BL21(DE3), the first plasmid is first used to transform E. coli strain BL21 with the first plasmid by heat shock, and then the first plasmid is used to transform the E. coli strain BL21 that was transformed with the first plasmid by electroporation.

[0066] 3.1 Preparation of heat-competent BL21 cells

[0067] 1. Activate the original bacterial strain stored at -80°C by streaking on antibiotic-free plates, and incubate the strain at 37°C overnight.

[0068] 2. The following day, pick single-clone BL21 cells and seed them into test tubes containing 50 mL of LB liquid medium. Incubate at 37°C for 8-12 hours at 200 rpm. Transfer the cells to 1 L of LB liquid medium at a dilution ratio of 1:50-1:100 and incubate at 37°C for 200 rpm until the OD600 is approximately 0.3-0.4.

[0069] 3. Place the bacterial cells on ice for 15 to 30 minutes to cool them down, then centrifuge at 2500 rpm at 4°C for 15 minutes. Discard the supernatant and resuspend the bacterial cells thoroughly in 800 mL of pre-cooled 0.1 M CaCl2. Then place the bacterial cells on ice for 15 minutes.

[0070] 4. Centrifuge at 2500 rpm at 4℃ for 15 minutes, discard the supernatant, and fully resuspend the bacterial cells in 500 mL of pre-cooled 0.1 M CaCl2. Then place the bacterial cells on ice for 15 minutes.

[0071] 5. Add 40 mL of pre-chilled 0.1 M CaCl2 solution (containing 15% glycerol) to slowly resuspend the bacterial cells, and place the cells on ice for 15 minutes. Then, aliquot the bacterial cells into 1.5 mL centrifuge tubes at doses of 50 μL to 100 μL, flash-freeze in liquid nitrogen, and immediately store at -80°C or use directly for subsequent transformation experiments.

[0072] 3.2 Thermal shock conversion

[0073] 1. First, thaw competent BL21 cells on ice, then add 1 μL of the first plasmid, i.e., UDG-pET21a plasmid, or 10 μL of ligation product, and incubate on ice for 30 minutes.

[0074] 2. Heat shock in a 42℃ water bath for 90 seconds, then immediately place on ice for 2 minutes.

[0075] 3. In a clean bench, add 800 μL of antibiotic-free LB medium and incubate at 37°C at 200 rpm for 40 to 60 minutes.

[0076] 4. Centrifuge the bacterial cells at low speed, then discard part of the supernatant in a laminar flow hood and resuspend the bacterial cells in the remaining supernatant.

[0077] 5. Aspirate the bacterial cells and spread them evenly onto LB agar plates containing the resistant bacteria using a sterile pipette tip. Incubate overnight at 37°C to obtain E. coli BL21 cells transformed with the first plasmid.

[0078] 3.3 Preparation of electrocompetent cells

[0079] 1. Select BL21 cells that have undergone heat shock transformation into monoclonal cells, seed them into 5 mL of liquid culture medium, and culture them at 37°C and 200 rpm for 8 to 12 hours.

[0080] 2. Transfer the cells to 5 mL of LB liquid medium at a dilution ratio of 1:50 to 1:100 and incubate at 37°C at 200 rpm until the OD600 is approximately 0.3-0.4.

[0081] 3. Place the test tube on ice for 30 minutes to cool it. Dispense the bacterial solution into 2 mL centrifuge tubes and centrifuge at 1500 g for 10 minutes at 4°C to collect the bacterial cells. The bacterial volume in each centrifuge tube should be 2 mL to 4 mL.

[0082] 4. In a clean bench, slowly discard the supernatant, resuspend the bacterial cells in 1 mL of pre-cooled sterile ddH2O, invert several times, and mix thoroughly.

[0083] 5. Centrifuge at 1500g for 10 minutes at 4℃, then discard the supernatant in a clean bench and resuspend the cells in 800μL of pre-cooled sterile ddH2O. Invert the container several times to mix thoroughly.

[0084] 6. Centrifuge at 1500g for 10 minutes at 4℃, then discard the supernatant in a clean bench and resuspend the bacterial cells in 1mL of pre-cooled sterile 10% glycerol, inverting the container several times to mix thoroughly.

[0085] 7. Centrifuge at 1500g for 10 minutes at 4℃, then discard the supernatant in a clean bench and resuspend the bacterial cells in 800μL of pre-cooled sterile 10% glycerol, inverting the container several times to mix thoroughly.

[0086] 8. Centrifuge at 1500g for 10 minutes at 4℃, then discard the supernatant in a clean bench, add 100μL of pre-cooled sterile 10% glycerol to resuspend the cells, flash freeze in liquid nitrogen, and store in a -80℃ freezer or use directly for subsequent transformation experiments.

[0087] 3.4 Electroporation of Electrocompetent Cells

[0088] 1. Remove BL21 electrotransfer competent cells from the -80℃ freezer, add 1μL-2μL of the second plasmid, namely GroEL-GroES-pBAD33 plasmid, and place on ice for 30 minutes.

[0089] 2. Transfer the mixture of competent cells and plasmids to a pre-chilled 0.2 cm electroporation vessel and incubate on ice for 5 minutes.

[0090] 3. Subsequently, Bio-Rad MicroPulser was used. TM The electric converter is set to program Ec2 (2.5kV, pulse 1) for electric shock conversion.

[0091] 4. In a clean bench, transfer the electroporated bacterial culture to a 1 mL centrifuge tube and quickly add 800 μL of antibiotic-free LB liquid medium.

[0092] 5. Incubate at 37°C with shaking at 200 rpm for 1 hour.

[0093] 6. Spread 100 μL to 200 μL of bacterial culture onto LB agar plates containing antibiotics and incubate overnight at 37°C.

[0094] For multi-plasmid expression systems, antibiotic resistance can increase accordingly with the increase in the number of plasmids.

[0095] 4. Induction and detection of UDG expression

[0096] 4.1 Induction and Detection of Protein Expression in Single Plasmid System

[0097] 1. In a clean bench, streak the UDG expression strain taken from the -80℃ freezer onto LB solid medium containing the corresponding antibiotic resistance and incubate overnight at 37℃.

[0098] 2. Pick a single colony and inoculate it into 5 mL of LB liquid medium containing antibiotics, and incubate at 37°C with shaking at 200 rpm for 8 to 10 hours.

[0099] 3. Dilute the bacterial culture at a ratio of 1:50 to 1:100. Transfer the diluted culture to eight test tubes containing 5 mL of LB liquid medium and incubate at 37°C and 200 rpm until OD (dose retardation) is reached. 600 It is approximately 0.6 to 0.8.

[0100] 4. Divide the 8 test tubes into 2 groups, take 1 mL of bacterial solution from each test tube, and store 1 mL of bacterial solution from each group in a 4℃ refrigerator.

[0101] 5. Set the inducer to different concentration gradients (taking IPTG as an example, the final concentrations are 0.1mM, 0.2mM, 0.5mM, and 1.0mM), with 4 test tubes in each group, and add different concentrations of inducer to each tube.

[0102] 6. The first group was placed at 37℃ and induced at 200 rpm for 4 hours; the second group was placed at 16℃ and induced at 200 rpm for 16 to 20 hours.

[0103] 7. After induction, take 1 mL of bacterial culture from each test tube, centrifuge at 13000 rpm for 10 minutes, and collect the bacterial cells.

[0104] 8. Add the two samples before induction to 150 μL of ultrasonic buffer (50 mM Tris-HCl pH 7.5, 25 mM NaCl), and add the eight samples after induction to 400 μL of the same buffer.

[0105] 9. Perform small-volume ultrasound using a Biosafer 2100F batch ultrasound machine. The small-volume ultrasound program is: 30% intensity, continuous ultrasound for 10 minutes.

[0106] 10. Take 40 μL of bacterial culture from each sample.

[0107] 11. Centrifuge the induced ultrasonic sample at 13,000 rpm for 10 minutes at 4℃.

[0108] 12. Take 40 μL of supernatant from each sample.

[0109] 13. Finally, SDS-PAGE was used to detect UDG and compare the expression of UDG protein in whole cells and supernatant before and after induction to confirm the most suitable induction conditions for each protein.

[0110] 4.2 Induction and Detection of Protein Expression in a Dual Plasmid System

[0111] A. Step-by-step induction

[0112] 1. Remove the BL21 strain, which has been successfully transformed with two plasmids, from the -80℃ freezer, streak it on LB plates (ampicillin (Amp) and chloramphenicol (Chl)), and incubate it overnight in a 37℃ incubator.

[0113] 2. Pick a single colony and inoculate it into 5 mL of LB liquid medium containing antibiotics, and incubate at 37°C with shaking at 200 rpm for 8 to 10 hours.

[0114] 3. Dilute the bacterial suspension at a ratio of 1:50 to 1:100, and transfer the diluted suspension to multiple 5 mL LB liquid medium containing antibiotics. Incubate at 37°C and 200 rpm until OD500 is reached. 600 It is approximately 0.2 to 0.4.

[0115] 4. Take 1 mL of bacterial culture as a negative control (before induction), then add 0.4% arabinose and incubate at 25°C for 3 hours.

[0116] 5. Then centrifuge at 1500g for 15 minutes at 4℃ and collect the bacterial cells.

[0117] 6. Discard the supernatant in a clean bench, add 4 mL of fresh LB medium (resistance to Amp and Chl), then add 0.4% arabinose and IPTG at different concentration gradients (0.1 mM, 0.2 mM, 0.5 mM, 1 mM), and induce at 16°C for 16 to 20 hours.

[0118] 7. After induction, take 1 mL of bacterial culture from each test tube, centrifuge at 13000 rpm for 10 minutes, and collect the bacterial cells.

[0119] 8. Following the recombinant protein small-scale expression procedure, sonication and sampling were performed to obtain samples. Finally, SDS-PAGE was used to detect UDG, and the amount of UDG in whole cells and supernatant before and after induction was compared to confirm the solubility of UDG.

[0120] B. Simultaneous induction

[0121] 1. Remove the BL21 strain, which has been successfully transformed with two plasmids, from the -80℃ freezer, streak it on LB plates (Amp, Chl), and incubate it overnight in a 37℃ incubator.

[0122] 2. Pick a single colony and inoculate it into 5 mL of LB liquid medium containing antibiotics, and incubate at 37°C with shaking at 200 rpm for 8 to 10 hours.

[0123] 3. Dilute the bacterial suspension at a ratio of 1:50 to 1:100, and transfer the diluted suspension to multiple 5 mL LB liquid medium containing antibiotics. Incubate at 37°C and 200 rpm until OD500 is reached. 600 It is approximately 0.6 to 0.8.

[0124] 4. Take 1 mL of bacterial culture as a negative control (before induction), then add 0.4% arabinose and IPTG at different concentration gradients (0.1 mM, 0.2 mM, 0.5 mM, 1 mM), and incubate at 16℃ for 16 to 20 hours.

[0125] 7. After induction, take 1 mL of bacterial culture from each test tube, centrifuge at 13000 rpm for 10 minutes, and collect the bacterial cells.

[0126] 8. Following the recombinant protein small-scale expression procedure, sonication and sampling were performed to obtain samples. Finally, SDS-PAGE was used to detect UDG, and the amount of UDG in whole cells and supernatant before and after induction was compared to confirm the solubility of UDG.

[0127] 5. Purification of UDG

[0128] To obtain high-purity UDG, the expressed UDG can be further purified by affinity chromatography. Additionally, because this UDG is thermosensitive, the entire purification process needs to be carried out at 4°C to 10°C to maintain its activity.

[0129] 5.1 Pretreatment before purification – Cell disruption

[0130] 1. Dispense the induced bacterial cells into 50mL centrifuge tubes, with each tube containing 0.5L to 1L of bacterial cells.

[0131] 2. Resuspend the bacterial cells using lysis buffer (containing 2% totipotent nuclease at a final concentration), adding 40 mL of the lysis buffer per liter of bacterial cells.

[0132] 3. Perform a large-scale ultrasound using an ultrasonic instrument. The large-scale ultrasound procedure is as follows: intensity 30%, ultrasound time 4 seconds, dwell time 10 seconds, and total ultrasound time 20 minutes.

[0133] 4. Centrifuge at 10,000 rpm for 40 minutes at 4°C using a benchtop centrifuge 5810R (Eppendorf).

[0134] 5. Transfer the supernatant to a pre-cooled beaker, filter the sample through a 0.2 μm filter membrane, and obtain the supernatant.

[0135] 5.2 Affinity Chromatography

[0136] 1. Connect the affinity chromatography column to the Insys Autopure100 HPLC system and set the pressure limit (HisTrap HP 5mL: 0.3MPa; HisCap 6FF 5mL: 0.3MPa) and flow rate according to the type of column packing and the instruction manual.

[0137] 2. Rinse the chromatography column with 3-5 column volumes of ddH2O.

[0138] 3. Equilibrate the chromatography column with 5-10 column volumes of wash buffer. After equilibration, the Unique CDSystem software will display the protein UV absorption peak. 280 Located at the baseline.

[0139] 4. Use pump A to directly load the sample until all samples have entered the chromatography column.

[0140] 5. Set up the elution program using the Unique CDSystem software. The first part of the program involves thorough washing with 10-15 column volumes (50 mL to 75 mL) of washing buffer until UV light is applied. 280 Returning to baseline, this process removes non-specifically bound or weakly bound proteins. The second part is a linear gradient elution procedure (elution buffer: 0% to 100%, wash buffer: 100% to 0%). The initial linear gradient elution volume is 20 column volumes. This process can elute all UDG and efficiently separate it from Ni. 2+ Proteins with different affinities.

[0141] 6. Analyze and record protein UV. 280 The position of the absorption peak corresponds to the location of the sample being removed from the collector.

[0142] 7. Use SDS-PAGE to determine the location and purity of UDG.

[0143] 8. Collect and combine UDG, and dialyze overnight at 4°C to 10°C using dialysis eluent-I.

[0144] 9. Wash the chromatography column sequentially with 50 mL of 0.5 M imidazole and 50 mL of ddH2O, and finally pack the chromatography column with 20% ethanol.

[0145] The buffer solutions used in each of the above steps are shown in Table 3.

[0146] Table 2. Buffer systems used for affinity chromatography

[0147] 20mM Tris-HCl 8.0 20 mL 1M Tris-HCl 8.0 25mM NaCl 5mL 5M NaCl 1mM DTT 1mL 1M DTT 10% glycerin 100mL glycerin 10mM imidazole 8.0 5mL 2M imidazole 8.0 Elution buffer 1000mL 20mM Tris-HCl 8.0 20 mL 1M Tris-HCl 8.0 25mM NaCl 5mL 5M NaCl 1mM DTT 1mL 1M DTT 10% glycerin 100mL glycerin 250mM imidazole 8.0 125mL 2M imidazole 8.0 Dialysis eluent-I 4000mL 20mM Tris-HCl 8.0 80 mL 1M Tris-HCl 8.0 25mM NaCl 20mL 5M NaCl 10% glycerin 400mL glycerin

[0148] 6. Results

[0149] 6.1 Detection results of UDG expression in the single plasmid system

[0150] The inventors first studied the effects of induction culture at 37℃, 0.5mM IPTG, and 220rpm for 3-4 hours. UDG expression and dissolution results The detection results of SDS-PAGE are as follows: Figure 2 As shown, whole cells refer to the cell lysate after induction, and the supernatant is the soluble fraction of whole cells after centrifugation. Whole cells can be used to determine the presence or absence of target protein expression, while comparing whole cells and the supernatant can determine the solubility of the target protein. SDS-PAGE analysis showed that all UDGs (U1-U8) exhibited significant protein-inducible expression, but the content of soluble UDGs was very low, accounting for only 5%-10% of the total induced protein.

[0151] Subsequently, the inventors discovered, by adjusting the induction conditions, that under lower temperature and inducer concentrations (i.e., temperatures of 20°C or 16°C and IPTG concentrations of 0.2 mM or 0.1 mM), the solubility of all UDG proteins improved (all by more than 10%), with the solubility of U3 and U4 increasing by 30% to 40%. Figure 3 (and Table 3).

[0152] Table 3. Solubility of UDG protein at 16℃ and 0.1 mM inducer concentration

[0153]

[0154] 6.2 Detection results of UDG expression in the dual plasmid system

[0155] Considering that most proteins in living organisms cannot form the correct three-dimensional structure through self-folding, the inventors introduced the molecular chaperone system GroEL-GroES through a dual-plasmid system to assist and enhance protein folding efficiency, thereby improving the solubility of the target protein, in order to further improve the solubility of the UDG protein. In exploring the induced expression of the dual-plasmid system, the inventors adopted two induction methods: simultaneous induction and stepwise induction.

[0156] Simultaneously induce

[0157] Simultaneous induction refers to the simultaneous addition of GroEL-GroES and UDG inducers to induce protein expression. However, SDS-PAGE analysis showed that simultaneous induction did not significantly improve the solubility of UDG protein. Figure 4 ).

[0158] Step-by-step induction

[0159] Stepwise induction refers to first inducing the molecular chaperone GroEL-GroES to ensure that the molecular chaperone accumulates to a certain protein concentration before inducing the target protein UDG. SDS-PAGE analysis showed that stepwise induction significantly improved the solubility of U3 and U4, raising them to a completely soluble level. Figure 4 However, the expression of other UDG proteins did not change significantly (not shown).

[0160] To more clearly illustrate the changes in the solubility of U3 and U4, the inventors compared the expression results of U3 in *E. coli* containing a single plasmid system with the expression results in *E. coli* containing a dual plasmid system under simultaneous and stepwise induction conditions, where the suffixes 1 and 2 in each group represent different single colonies (…). Figure 5 ).from Figure 5 The results show that, under the same induction conditions (i.e., the same induction temperature, the same induction time, and the same concentration of inducer), simultaneously inducing the expression of the molecular chaperone systems GroEL-GroES and UDG in *E. coli* containing a single plasmid system (containing only the U3 expression gene) did not significantly improve the solubility of U3 compared to *E. coli* containing a single plasmid system (containing only the U3 expression gene). In fact, based on the SDS-PAGE plot, the solubility of U3 in these two production methods is similar, both ranging from 30% to 40%. Figure 5 However, by employing a stepwise induction method on Escherichia coli containing a dual plasmid system, first inducing the expression of the molecular chaperone system GroEL-GroES for a period of time, and then inducing U3 expression, the solubility of U3 can be significantly improved, making U3 completely soluble, thereby greatly increasing the yield of U3.

[0161] 6.3 Results of UDG content detection after affinity chromatography purification

[0162] For the same volume of fermentation broth, after the same affinity chromatography purification, the U3 yield of E. coli expressing a dual-plasmid system, induced simultaneously and stepwise, was significantly higher in the stepwise induction group (the gradient elution fraction represents the total amount of UDG protein) than in the simultaneous induction group. Figure 6 Specifically, according to the UV spectrophotometer... 280 The test results showed that the U3 production in the simultaneous induction group was 45 mg, while the U3 production in the stepwise induction group was 97 mg.

[0163] Sequence information:

[0164] sequence list <110> Beijing Qingke Biotechnology Co., Ltd. Hubei Qingke Biotechnology Co., Ltd. <120> A method for producing uracil glycosylation enzyme and a dual-carrier system for this method. <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 245 <212> PRT <213> Bacillus sp. HJ171 <400> 1 Met Glu Leu Phe Asp Glu Gln Thr Pro Lys Ser Pro Ala Gln Lys Gln 1 5 10 15 Ala Ile Leu Asp Asn Val Arg Leu Pro Glu Asp Trp Lys Lys Ala Leu 20 25 30 Ala Asn Glu Leu Thr Ser Asn Asn Met Asp Asp Leu Arg Ala Phe Leu 35 40 45 Lys Glu Ala Tyr Gln Ser Glu Asn Ser Ile Tyr Pro Pro Ala Pro Leu 50 55 60 Ile Phe Asn Ala Leu Asn Leu Thr Pro Leu Ser Gln Val Lys Val Val 65 70 75 80 Ile Leu Gly Gln Asp Pro Tyr His Gly Pro Gly Gln Ala Met Gly Leu 85 90 95 Ser Phe Ser Val Pro Lys Ala Ile Pro Lys Pro Pro Ser Leu Asn Asn 100 105 110 Leu Leu Lys Glu Met Ala Ser Asp Val Gly Ile Ala Pro Ser Lys His 115 120 125 Gly Asp Leu Thr Tyr Trp Ala Gln Gln Gly Val Leu Leu Leu Asn Ser 130 135 140 Ser Leu Thr Val Arg Glu Ser Glu Pro Asn Ser His Gln Asn Asn Gly 145 150 155 160 Trp Glu Gln Phe Thr Asp Ala Val Ile Asp Val Val Asn Glu Gln Thr 165 170 175 Glu His Thr Val Phe Ile Leu Trp Gly Ser Lys Ala Gln Lys Lys Gly 180 185 190 Lys Tyr Ile Asn Thr Asp Lys His Leu Ile Leu Thr Ala Val His Pro 195 200 205 Ser Pro Leu Ala Ala Asn Arg Gly Gly Phe Phe Gly Ser Lys Pro Phe 210 215 220 Ser Lys Thr Asn Asp Tyr Leu Val Gln Tyr Gly Gln Thr Pro Ile Asp 225 230 235 240 Trp Gln Leu Pro Gln 245 <210> 2 <211> 548 <212> PRT <213> Artificial Sequence <400> 2 Met Ala Ala Lys Asp Val Lys Phe Gly Asn Asp Ala Arg Val Lys Met 1 5 10 15 Leu Arg Gly Val Asn Val Leu Ala Asp Ala Val Lys Val Thr Leu Gly 20 25 30 Pro Lys Gly Arg Asn Val Val Leu Asp Lys Ser Phe Gly Ala Pro Thr 35 40 45 Ile Thr Lys Asp Gly Val Ser Val Ala Arg Glu Ile Glu Leu Glu Asp 50 55 60 Lys Phe Glu Asn Met Gly Ala Gln Met Val Lys Glu Val Ala Ser Lys 65 70 75 80 Ala Asn Asp Ala Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala 85 90 95 Gln Ala Ile Ile Thr Glu Gly Leu Lys Ala Val Ala Ala Gly Met Asn 100 105 110 Pro Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Val Thr Val Ala Val 115 120 125 Glu Glu Leu Lys Ala Leu Ser Val Pro Cys Ser Asp Ser Lys Ala Ile 130 135 140 Ala Gln Val Gly Thr Ile Ser Ala Asn Ser Asp Glu Thr Val Gly Lys 145 150 155 160 Leu Ile Ala Glu Ala Met Asp Lys Val Gly Lys Glu Gly Val Ile Thr 165 170 175 Val Glu Asp Gly Thr Gly Leu Gln Asp Glu Leu Asp Val Val Glu Gly 180 185 190 Met Gln Phe Asp Arg Gly Tyr Leu Ser Pro Tyr Phe Ile Asn Lys Pro 195 200 205 Glu Thr Gly Ala Val Glu Leu Glu Ser Pro Phe Ile Leu Leu Ala Asp 210 215 220 Lys Lys Ile Ser Asn Ile Arg Glu Met Leu Pro Val Leu Glu Ala Val 225 230 235 240 Ala Lys Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly 245 250 255 Glu Ala Leu Ala Thr Leu Val Val Asn Thr Met Arg Gly Ile Val Lys 260 265 270 Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 275 280 285 Leu Gln Asp Ile Ala Thr Leu Thr Gly Gly Thr Val Ile Ser Glu Glu 290 295 300 Ile Gly Met Glu Leu Glu Lys Ala Thr Leu Glu Asp Leu Gly Gln Ala 305 310 315 320 Lys Arg Val Val Ile Asn Lys Asp Thr Thr Thr Ile Ile Asp Gly Val 325 330 335 Gly Glu Glu Ala Ala Ile Gln Gly Arg Val Ala Gln Ile Arg Gln Gln 340 345 350 Ile Glu Glu Ala Thr Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg 355 360 365 Val Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Val Gly Ala Ala 370 375 380 Thr Glu Val Glu Met Lys Glu Lys Lys Ala Arg Val Glu Asp Ala Leu 385 390 395 400 His Ala Thr Arg Ala Ala Val Glu Glu Gly Val Val Ala Gly Gly Gly 405 410 415 Val Ala Leu Ile Arg Val Ala Ser Lys Leu Ala Asp Leu Arg Gly Gln 420 425 430 Asn Glu Asp Gln Asn Val Gly Ile Lys Val Ala Leu Arg Ala Met Glu 435 440 445 Ala Pro Leu Arg Gln Ile Val Leu Asn Cys Gly Glu Glu Pro Ser Val 450 455 460 Val Ala Asn Thr Val Lys Gly Gly Asp Gly Asn Tyr Gly Tyr Asn Ala 465 470 475 480 Ala Thr Glu Glu Tyr Gly Asn Met Ile Asp Met Gly Ile Leu Asp Pro 485 490 495 Thr Lys Val Thr Arg Ser Ala Leu Gln Tyr Ala Ala Ser Val Ala Gly 500 505 510 Leu Met Ile Thr Thr Glu Cys Met Val Thr Asp Leu Pro Lys Asn Asp 515 520 525 Ala Ala Asp Leu Gly Ala Ala Gly Gly Met Gly Gly Met Gly Gly Met 530 535 540 Gly Gly Met Met 545 <210> 3 <211> 97 <212> PRT <213> Artificial Sequence <400> 3 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala <210> 4 <211> 738 <212> DNA <213> Bacillus sp. HJ171 <400> 4 atggaattat tcgatgaaca aacgccaaaa tcgccagcgc aaaagcaggc tattttagac 60 aatgtgcgct tgccagaaga ttggaaaaag gcgttagcaa acgagttaac ttctaacaat 120 atggacgact tgcgtgcgtt tttaaaagaa gcctatcaat cagaaaacag tatctatccg 180 ccagcaccct taatatttaa cgcgttaaac ctgacccctt tatcacaagt taaagtcgtg 240 atactagggc aagatcctta tcatggacca gggcaggcaa tgggcttatc gttttcagtg 300 cccaaagcca ttccaaagcc accctcactc aataatttgt taaaagagat ggcaagtgac 360 gttggtatcg caccttcaaa acatggcgac ctgacttact gggcgcagca aggagtttta 420 ctattaaata gctctctgac cgtgcgagaa agtgagccaa atagtcatca aaataatggc 480 tgggagcagt ttaccgatgc ggtgattgat gtggttaatg aacaaacaga acataccgta 540 tttatattgt ggggcagtaa agcacagaaa aaaggcaaat atatcaatac tgataagcat 600 cttattctga ccgctgtaca tccatcacca cttgctgcca atcgtggtgg attctttggt 660 tccaagccgt tttctaagac caatgattat ctggtacagt atgggcaaac gcctatcgat 720 tggcaattac cgcaatag 738 <210> 5 <211> 244 <212> PRT <213> Psychrobacter sp. HJ147 <400> 5 Met Glu Leu Phe Asp Glu Gln Thr Pro Lys Thr Pro Ala Gln Lys Gln 1 5 10 15 Ala Ile Leu Asp Asn Val Arg Leu Pro Glu Asp Trp Lys Thr Ala Leu 20 25 30 Ala Asp Glu Leu Thr Ser Asn Asn Met Asp Asp Leu Arg Ala Phe Leu 35 40 45 Lys Glu Ala Tyr Gln Ser Glu Asn Ser Ile Tyr Pro Pro Ala Pro Leu 50 55 60 Ile Phe Asn Ala Leu Asn Leu Thr Pro Leu Ser Gln Ile Lys Val Val 65 70 75 80 Ile Leu Gly Gln Asp Pro Tyr His Gly Pro Gly Gln Ala Met Gly Leu 85 90 95 Ser Phe Ser Val Pro Lys Val Ile Pro Lys Pro Pro Ser Leu Asn Asn 100 105 110 Leu Leu Lys Glu Met Ala Ser Asp Val Gly Ile Ala Pro Ser Lys His 115 120 125 Gly Asp Leu Thr Tyr Trp Ala Gln Gln Gly Val Leu Leu Leu Asn Ser 130 135 140 Ser Leu Thr Val Arg Glu Ser Glu Pro Asn Ser His Gln Asn Lys Gly 145 150 155 160 Trp Glu Gln Phe Thr Asp Ala Val Ile Asp Val Val Asn Glu Gln Thr 165 170 175 Glu His Thr Val Phe Ile Leu Trp Gly Ser His Ala Gln Lys Ser Lys 180 185 190 Tyr Ile Asn Thr Asp Lys His Leu Ile Leu Thr Ala Val His Pro Ser 195 200 205 Pro Leu Ala Ala Asn Arg Gly Gly Phe Phe Gly Ser Lys Pro Phe Ser 210 215 220 Lys Thr Asn Asp Tyr Leu Val Gln Tyr Gly Gln Thr Pro Ile Asp Trp 225 230 235 240 Gln Leu Pro Gln <210> 6 <211> 226 <212> PRT <213> Photobacterium aplysiae GMD509 <400> 6 Met Ser Thr Pro Lys Thr Trp Glu Ser Ile Ile Asn Asp Glu Arg Glu 1 5 10 15 Lys Glu Tyr Phe Gln Ser Val Leu Ala Phe Val Glu Gln Gln Arg Asn 20 25 30 Ser Gly Lys Thr Ile Tyr Pro Pro Gln Glu Gln Val Phe Ser Ala Phe 35 40 45 Asp Met Thr Pro Phe Glu Ser Val Arg Val Val Ile Leu Gly Gln Asp 50 55 60 Pro Tyr His Gly Ala Asn Gln Ala His Gly Leu Ala Phe Ser Val Leu 65 70 75 80 Pro Gly Val Lys Ile Pro Pro Ser Leu Arg Asn Met Tyr Lys Glu Leu 85 90 95 Ala Gln Asp Ile Glu Gly Phe Glu Ile Pro Ser His Gly Tyr Leu Asp 100 105 110 Ala Trp Ala Ser Gln Gly Val Leu Met Leu Asn Thr Val Leu Thr Val 115 120 125 Glu Glu Ala Lys Ala His Ser His Ala Lys Cys Gly Trp Glu Thr Phe 130 135 140 Thr Asp Ala Ile Ile Ala Glu Leu Asn Gln Arg Ser Glu Pro Ile Ile 145 150 155 160 Phe Leu Leu Trp Gly Ala His Ala Gln Lys Lys Gly Gln Ala Ile Asp 165 170 175 Thr Gly Met His His Val Leu Ala Ala Pro His Pro Ser Pro Leu Ser 180 185 190 Ala Arg Arg Gly Phe Phe Gly Cys Lys His Phe Ser Thr Thr Asn Lys 195 200 205 Leu Leu Ser Ser Met Asp Gln Gln Pro Ile Asp Trp His Leu Pro Ala 210 215 220 Glu Val 225 <210> 7 <211> 272 <212> PRT <213> Codfish (Gadus morhua) <400> 7 Met Leu Phe Lys Leu Gly Leu Cys Gln Arg Cys Ile Ser Ser Asn Arg 1 5 10 15 Val Leu Pro Gly Leu Leu Ile Pro Gln Thr Leu Cys Phe Ser Lys Leu 20 25 30 Met Lys Ile Thr Pro Lys Lys Leu Arg Ser Ser Asn Val Glu Gln Lys 35 40 45 Thr Ser Ser Pro Gln Leu Ser Val Glu Gln Leu Glu Arg Met Ala Lys 50 55 60 Asn Lys Lys Ala Ala Leu Asp Lys Ile Arg Ala Lys Ala Thr Pro Ala 65 70 75 80 Gly Phe Gly Glu Thr Trp Arg Arg Glu Leu Ala Ala Glu Phe Glu Lys 85 90 95 Pro Tyr Phe Lys Gln Leu Met Ser Phe Val Ala Asp Glu Arg Ser Arg 100 105 110 His Thr Val Tyr Pro Pro Ala Asp Gln Val Tyr Ser Trp Thr Glu Met 115 120 125 Cys Asp Ile Gln Asp Val Lys Val Val Ile Leu Gly Gln Asp Pro Tyr 130 135 140 His Gly Pro Asn Gln Ala His Gly Leu Cys Phe Ser Val Gln Lys Pro 145 150 155 160 Val Pro Pro Pro Pro Ser Leu Val Asn Ile Tyr Lys Glu Leu Cys Thr 165 170 175 Asp Ile Asp Gly Phe Lys His Pro Gly His Gly Asp Leu Ser Gly Trp 180 185 190 Ala Lys Gln Gly Val Leu Leu Leu Asn Ala Val Leu Thr Val Arg Ala 195 200 205 His Gln Ala Asn Ser His Lys Asp Arg Gly Trp Glu Thr Phe Thr Asp 210 215 220 Ala Val Ile Lys Trp Leu Ser Val Asn Arg Glu Gly Val Val Phe Leu 225 230 235 240 Leu Trp Gly Ser Tyr Ala His Lys Lys Gly Ala Thr Ile Asp Arg Lys 245 250 255 Arg His His Val Leu Gln Ala Val His Pro Ser Pro Leu Ser Ala His 260 265 270

Claims

1. A method for producing uracil glycosylation enzyme (UDG), the method comprising the following steps: a) Provides an expression cell comprising a first vector capable of expressing the UDG and a second vector capable of expressing the molecular chaperone system GroEL-GroES, wherein the amino acid sequence of the UDG is shown in SEQ ID NO: 1, and the amino acid sequences of the GroEL and GroES are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively; b) Inducing the expression cells to express the molecular chaperone system GroEL-GroES under a first induction condition, wherein the first induction condition is to induce the expression cells to express the molecular chaperone system GroEL-GroES at 16°C to 37°C for 0.5 to 5 hours using a first inducing agent. c) Inducing the expression cells to simultaneously express the molecular chaperone system GroEL-GroES and the UDG under a second induction condition, wherein the second induction condition is to induce the expression cells to simultaneously express the molecular chaperone system GroEL-GroES and the UDG using a second inducing agent and a third inducing agent at 16°C to 20°C for 16 to 20 hours, wherein the second inducing agent induces the expression cells to express the molecular chaperone system GroEL-GroES, and the third inducing agent induces the expression cells to express the UDG.

2. The method according to claim 1, wherein, The first induction condition is induction at 25°C for 3 hours using arabinose; the second induction condition is induction at 16°C for 16 to 20 hours using arabinose and isopropyl-β-D-thiogalactoside (IPTG), wherein arabinose is the second inducing agent and isopropyl-β-D-thiogalactoside (IPTG) is the third inducing agent.

3. The method according to claim 2, wherein, The concentrations of the first inducer and the second inducer are each independently from 0.001% to 1.0%; the final concentration of the third inducer is from 0.1 mM to 1.0 mM.

4. The method according to claim 3, wherein, The concentrations of the first and second inducers are 0.4%; the final concentration of the third inducer is 0.1 mM.

5. The method according to any one of claims 1-4, wherein, In step a), the expression cells are obtained by transforming the same host cells with the first vector and the second vector, respectively.

6. The method according to claim 5, wherein, The first vector and the second vector are used to transform the host cell through different transformation methods; the first vector and the second vector are plasmids; the host cell is a prokaryotic cell.

7. The method according to claim 6, wherein, The transformation method is heat shock and / or electroporation; the first vector is a plasmid expressing the UDG, the second vector is a plasmid expressing the molecular chaperone system GroEL-GroES; the host cell is a bacterial cell.

8. The method according to claim 7, wherein, The first vector transforms the host cells via heat shock, and the second vector transforms the host cells via electroporation; the first vector is pET21a, pHUE, pET20b, pET-22b, pET-28a, or pET-32a plasmid, and the second vector is pBAD33 plasmid; the host cell is *Escherichia coli* (E. coli). Escherichia coli ).

9. The method according to claim 8, wherein, The first vector is the pET21a plasmid.

10. The method according to any one of claims 1-4, wherein, The method further includes step d): lysing the expression cells obtained in step c) and purifying the UDG by affinity chromatography at a temperature of 4°C to 10°C.

11. The method according to claim 10, wherein, The first carrier also expresses an affinity tag, and the affinity chromatography is performed using the affinity tag.

12. The method according to claim 11, wherein, The affinity tag is His tag, FLAG tag, HAT tag, HPC tag, MBP tag, NusA tag, UB tag, or GST tag.

13. The method according to claim 12, wherein, The affinity tag is 6. His tag.

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