Method for efficiently expressing and purifying Taq DNA polymerase by using HEK293T

Abstract

The invention provides a method for efficiently expressing and purifying Taq DNA polymerase by using HEK293T, and belongs to the technical field of molecular biology. According to an amino acid sequence of Taq DNA polymerase, thrombin recognition and cleavage sites, six connected His amino acid tags and IFNalpha-2 signal peptides are sequentially designed at the N end of the Taq DNA polymerase, reverse translation is performed to obtain a nucleotide sequence, codons are optimized according to the environment of HEK293T cells, and then an eukaryotic expression vector is constructed. And transfecting the eukaryotic expression vector to an HEK293T cell to obtain the target protein. The purity of the obtained Taq DNA polymerase can reach 100%, the protein yield is high, about 2 mg of Taq DNA polymerase can be obtained by 20 mL of a culture medium, and the method is economical and efficient.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a method for efficiently expressing and purifying Taq DNA polymerase using human embryonic kidney cells HEK293T. Background Technology

[0002] Taq DNA polymerase is derived from thermophilic bacillus ( ) Thermus aquaticus The thermostable DNA polymerase isolated from [the organism] is the core enzyme preparation for polymerase chain reaction (PCR) technology. Currently, commercially available Taq enzymes are mainly derived from prokaryotic expression systems (such as *E. coli*). Although the *E. coli* system has advantages such as low culture cost, rapid reproduction, and high expression levels, it also has several inherent drawbacks: (1) Inclusion body problem: Exogenous proteins easily form insoluble inclusion bodies, requiring a complex renaturation process to obtain active proteins, which is cumbersome and has a low yield. (2) Residual host nucleic acid: Escherichia coli itself contains abundant genomic DNA and plasmid DNA, which are easily mixed into the final product during purification. These residual nucleic acids will become non-specific templates in PCR amplification, leading to false positive results, especially in high-sensitivity detection (such as pathogen detection, trace DNA analysis) where they cause serious interference. (3) Endotoxin contamination: Lipopolysaccharide (LPS, i.e., endotoxin), a component of the E. coli cell wall, is difficult to completely remove, which may affect downstream cell experiments and even trigger human immune responses, limiting its application in clinical treatment. (4) Complex purification process: In order to remove nucleic acids and endotoxins, multiple purification steps are required, such as nucleic acid precipitation, ion exchange, heparin affinity chromatography, etc., which is cumbersome, leading to enzyme activity loss and increased costs. (5) Although some technologies claim to produce “nuclease-free” or “endotoxin-free” Taq enzymes, their detection standards are lenient (e.g., only detectable up to 35 cycles) or the purification process is extremely complex, resulting in high product prices.

[0003] Eukaryotic expression systems (such as mammalian cells and yeast cells) possess complete post-translational protein modification capabilities and low levels of endogenous endotoxins, making them ideal for producing high-quality recombinant proteins. HEK293T cells are widely used due to their high transfection efficiency, ease of culture, and strong protein expression capabilities. However, to date, no successful reports have been made of utilizing HEK293T cells to efficiently secrete and express prokaryotic Taq DNA polymerase and establishing a corresponding simple and efficient purification process. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned deficiencies of the prior art and provide a method for the efficient expression and purification of Taq DNA polymerase using HEK293T cells. This method aims to achieve efficient secretory expression of Taq DNA polymerase in eukaryotic systems, avoid inclusion body formation, simplify the purification process, obtain high-purity products through a single affinity chromatography step, fundamentally solve the problems of bacterial DNA residue and endotoxin contamination, obtain ultrapure enzyme preparations suitable for high-end molecular diagnostics, and establish a stable, economical, and scalable production process.

[0005] This invention is achieved through the following technical solution: 1. Design of fusion proteins based on the amino acid sequence of Taq DNA polymerase First, the amino acid sequence of Taq DNA polymerase was retrieved using NCBI. To facilitate subsequent purification of Taq DNA polymerase, a six-linked His amino acid tag was designed at the N-terminus of the enzyme, forming a 6×His-Taq structure. Considering that redundant sequences may need to be removed for large-scale commercialization of Taq DNA polymerase, a thrombin recognition and cleavage site was inserted between the 6×His and Taq DNA polymerase sequences to facilitate subsequent His tag removal. It should be noted that not removing the 6×His tag does not affect the activity of the Taq DNA polymerase itself; therefore, the thrombin recognition and cleavage site can be omitted in the preparation of Taq DNA polymerase.

[0006] Traditional Taq DNA polymerase expression typically involves intracellular expression, followed by cell lysis to release the Taq DNA polymerase and subsequent purification. This purification method is relatively cumbersome, and the lysis process inevitably leads to the degradation of some proteins or the binding of Taq DNA polymerase to some host DNA. Furthermore, due to the limited space within the cell, excessive foreign proteins can be toxic to the host and even cause cell death. To simplify protein purification, a signal peptide is specifically added to the N-terminus of the 6×His protein. This specific signal peptide recognizes the cell-expressed fusion Taq DNA polymerase protein and secretes it extracellularly. Before secretion, the signal peptide is cleaved, reducing the impact of excess polypeptide sequences on the activity of the fusion protein. Afterward, cell culture medium is collected, and Ni-NTA affinity purification is used to purify and collect the desired fusion Taq DNA polymerase protein in one step. However, different signal peptides have different secretion and cleavage efficiencies for different proteins, and their adaptability varies in different cells. This invention preferably uses the IFNα-2 signal peptide, which has the characteristics of high-efficiency secretion and high-efficiency cleavage in HEK293 cells, thereby obtaining the framework of the IFNα-2-6×His-Taq DNA polymerase protein. The amino acid sequence of the IFNα-2-6×His-Taq DNA polymerase protein, starting from the N-terminus, includes the amino acid sequence of the IFNα-2 signal peptide, the amino acid sequence of 6×His, the amino acid sequence of the thrombin recognition and cleavage site, and the amino acid sequence of the Taq DNA polymerase.

[0007] 2. Codon optimization and gene synthesis of IFNα-2-6×His-Taq DNA polymerase protein The nucleotide sequence of IFNα-2-6×His-Taq DNA polymerase protein was obtained by reverse translation of the amino acid sequence. Codons were optimized according to the expression environment of HEK293T cells to adapt to the expression environment of HEK293T cells. Finally, the expression sequence of IFNα-2-6×His-Taq DNA polymerase protein was obtained by gene synthesis based on the nucleotide sequence.

[0008] 3. Construction of expression carrier The expression sequence of IFNα-2-6×His-Taq DNA polymerase protein was constructed into a eukaryotic expression vector (such as pIRES2-EGFP), thus completing the construction of the Taq DNA polymerase expression vector pIFNα-2-6×His-Taq-IRES2-EGFP.

[0009] In addition, to enhance the translation efficiency of the expression sequence of IFNα-2-6×His-Taq DNA polymerase protein, a Kozak expression sequence that enhances translation efficiency was added to the 5' end of the nucleotide sequence corresponding to the IFNα-2 signal peptide, resulting in the Kozak-IFNα-2-6×His-Taq DNA polymerase expression sequence. The addition of this Kozak expression sequence can be performed before codon optimization, so that the resulting Kozak-IFNα-2-6×His-Taq DNA polymerase expression sequence can be optimized together.

[0010] 4. Cell transfection and purification The constructed recombinant expression vector pIFNα-2-6×His-Taq-IRES2-EGFP was transformed into competent cells (e.g., TOP10) and cultured to expand. The plasmid was extracted using a low-endotoxin plasmid extraction kit (e.g., plasmid extraction kit with catalog number CR-NAE-001) and then transfected into healthy HEK293T cells using a transfection reagent (e.g., Beyotime Lipo8000™). After transfection, the cells were cultured in DMEM complete medium (DMEM medium + 10wt% FBS: DMEM medium + 10% fetal bovine serum) for 24 hours. The DMEM complete medium was replaced the next day, and the expression of the fluorescent protein was observed to determine the transfection efficiency and the expression of the target protein. Culture was continued, and the supernatant was collected the following day, with fresh DMEM complete medium added.

[0011] Add Tris-HCl (pH 8.0) and NaCl to the collected cell culture supernatant to final concentrations of 20 mM and 300 mM, respectively. Equilibrate the Ni-NTA purification medium with binding buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl). Incubate the Ni-NTA purification medium treated with binding buffer with the treated supernatant to allow the target protein to bind to the purification medium. After incubation, transfer the supernatant to a gravity purification column. Filter all supernatant and purification medium. Wash the purification column sequentially with elution buffers containing low-concentration imidazole and high-concentration imidazole (Tris-HCl 20 mM, pH 8.0, NaCl 300 mM). The low-concentration and high-concentration imidazole elution buffers are used to remove impurity proteins and elute the target protein, respectively. Finally, collect the eluent containing the target protein.

[0012] Thus, Ni-NTA purification medium can purify any His-tagged protein. However, since the bovine serum in DMEM complete medium contains His-tagged proteins, the eluted proteins will contain impurities. To address this issue, this invention later replaced the HEK293T cell culture medium with a serum-free dedicated medium. The specific type of medium is not limited. The optional media used in the experiment were OPMA 293F Hi-exp medium (catalog number AC601501) and OPMA 293F Hi-exp feed (catalog number AC601502). The feed was added at a ratio of 5%, which means 4.75 ml of medium per plate and 0.25 ml of feed per plate.

[0013] This invention utilizes the eukaryotic cell HEK293T as a host and introduces the IFNα-2 signal peptide into the expression plasmid of Taq DNA polymerase. This allows the signal peptide to be recognized and transported extracellularly as soon as transcription and translation begin. During this process, the IFNα-2 signal peptide is recognized and cleaved by the intracellular cleavage system. The Taq DNA polymerase subsequently secreted extracellularly does not contain the signal peptide or other redundant sequences. The Taq DNA polymerase released into the culture medium contains a 6×His tag, and after using serum-free medium, it can be purified in one step using Ni-NTA purification medium, achieving a purity of almost 100%. SDS-PAGE electrophoresis shows no impurity proteins. Furthermore, since it is purified directly from cell culture medium, the endotoxin and bacterial DNA content is extremely low, and no bacterial DNA was detected using bacterial detection kits. The entire process, from collecting the culture medium to eluting the Taq DNA polymerase, can be completed within 1 hour. No protease inhibitors need to be added during the purification process, and up to about 2 mg of Taq DNA polymerase can be obtained from 20 mL of culture medium. This is economical and efficient, laying the foundation for the future development of high-end Taq DNA polymerases and other high-end molecular enzymes.

[0014] In summary, compared with the prior art, the present invention has the following significant advantages: Source control of contamination: Using HEK293T cells as the host fundamentally avoids contamination by prokaryotic host DNA and endotoxins.

[0015] Advantages of secretory expression: By introducing a highly efficient specific signal peptide (IFNα-2), the protein can be recognized and transported extracellularly when transcription and translation begin. During this process, the signal peptide is recognized and cleaved by the intracellular cleavage system, thus enabling the direct secretion of the target protein into serum-free culture medium. This simplifies the downstream purification process and avoids protein degradation, enzyme activity loss, and additional impurities caused by cell lysis.

[0016] Extremely high purity: Due to the use of serum-free culture medium and Ni-NTA affinity purification, the purity of the obtained Taq DNA polymerase can reach almost 100%; at the same time, since it is purified from cell culture medium, the content of endotoxin and bacterial DNA is extremely low.

[0017] Guaranteed activity: The eukaryotic system is more conducive to the correct folding of complex proteins, and the obtained Taq DNA polymerase has complete biological activity.

[0018] The process is simple and economical: from collecting the culture supernatant to eluting Taq DNA polymerase, the entire process can be completed within 1 hour. No protease inhibitors need to be added during the entire purification process. Up to about 2 mg of Taq DNA polymerase can be obtained from 20 mL of culture medium. It is economical and efficient, which lays the foundation for the future development of high-end Taq DNA polymerase and other high-end molecular enzymes. In addition, the supernatant of HEK293T cells can be collected continuously, which greatly reduces the production cost. Attached Figure Description

[0019] Figure 1 This image shows the plasmid map of the expression vector pIFNα-2-6×His-Taq-IRES2-EGFP constructed in this invention.

[0020] Figure 2 This image shows the fluorescence of HEK293T cells transfected with the expression vector pIFNα-2-6×His-Taq-IRES2-EGFP after culture in DMEM complete medium.

[0021] Figure 3 The image shows an SDS-PAGE electrophoresis image of the protein obtained by eluting HEK293T cells in Ni-NTA purification after culturing them in DMEM complete medium for 1-3 times (the blank control is HEK293T cells without Taq DNA polymerase expression vector).

[0022] Figure 4 This shows the SDS-PAGE electrophoresis images of proteins obtained by washing 1-3 times and eluting 1-3 times during the Ni-NTA purification process.

[0023] Figure 5 This image shows the fluorescence of HEK293T cells after being cultured in serum-free DMEM medium on the third day.

[0024] Figure 6 This image shows an SDS-PAGE electrophoresis image of the protein obtained by eluting HEK293T cells 1-3 times in Ni-NTA purification after culturing in serum-free medium. (The blank control is HEK293T cells without Taq DNA polymerase expression vector).

[0025] Figure 7 The image shows SDS-PAGE electrophoresis of proteins obtained by eluting the supernatant of the culture medium collected from the third and fourth batches of HEK293T cells in serum-free medium after 1-3 times in Ni-NTA purification, indicating that Taq DNA polymerase can be stably expressed.

[0026] Figure 8 The graph shows the results of amplification of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa using a bacterial quadrivalent detection kit with purified Taq DNA polymerase. Detailed Implementation

[0027] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0028] Example 1: Design of fusion proteins based on the amino acid sequence of Taq DNA polymerase 1. Obtain the amino acid sequence of Taq DNA polymerase from the NCBI database.

[0029] 2. At the N-terminus of the Taq DNA polymerase amino acid sequence, the encoding of the thrombin cleavage site, the 6×His tag, and the IFNα-2 signal peptide were sequentially added to obtain the framework of the IFNα-2-6×His-Taq DNA polymerase protein. The amino acid sequence of the IFNα-2-6×His-Taq DNA polymerase protein is as follows: MALTFALLVALLVLSCKSSCSVGMHHHHHHSSGLVPRGSHMRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEDGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE* (SEQ ID NO.1).

[0030] Example 2: Codon Optimization and Gene Synthesis of IFNα-2-6×His-Taq DNA Polymerase Protein The amino acid sequence of the IFNα-2-6×His-Taq DNA polymerase protein from Example 1 was reverse-translated to obtain the corresponding nucleotide sequence (as shown in SEQ ID NO. 2; in fact, the reverse-translated nucleotide sequence can have other sequences, as long as it can completely express the protein peptide chain). Codon optimization was performed according to the expression environment of HEK293T cells to adapt to the HEK293T cell expression environment. Finally, the gene was synthesized by Beijing Qingke Biotechnology Co., Ltd. to obtain the expression sequence of the IFNα-2-6×His-Taq DNA polymerase protein. The codon-optimized expression sequence of the IFNα-2-6×His-Taq DNA polymerase protein is shown in SEQ ID NO. 3.

[0031]

[0032]

[0033] Example 3: Construction of expression vector The expression sequence of IFNα-2-6×His-Taq DNA polymerase protein obtained in Example 2 was cloned into the multiple cloning site of the eukaryotic expression vector pIRES2-EGFP by enzyme digestion and ligation.

[0034] Furthermore, to enhance the translation efficiency of the IFNα-2-6×His-Taq DNA polymerase protein expression sequence, a Kozak expression sequence was added to the 5' end of the nucleotide sequence corresponding to the IFNα-2 signal peptide, resulting in the Kozak-IFNα-2-6×His-Taq DNA polymerase expression sequence. This Kozak expression sequence can be added before codon optimization, allowing for simultaneous codon optimization of the resulting Kozak-IFNα-2-6×His-Taq DNA polymerase expression sequence. The specific Kozak sequence is shown below: GCCACC ATGG (SEQ ID NO.4) The Kozak sequence is special. It is not a standalone element. Its tail needs to contain a portion of the start codon sequence of the fusion protein in order to guide the transcribed mRNA into the ribosome and improve the translation level. Therefore, it overlaps with the ATGG portion of the most proximal part of the sequence in SEQ ID NO.3.

[0035] The expression vector pIFNα-2-6×His-Taq-IRES2-EGFP was constructed (plasmid map shown). Figure 1 The nucleotide sequence of the complete Taq DNA polymerase expression vector pIFNα-2-6×His-Taq-IRES2-EGFP is shown in SEQ ID NO.5.

[0036]

[0037] Example 4: Cell transfection and purification The constructed expression vector pIFNα-2-6×His-Taq-IRES2-EGFP was transformed into competent TOP10 cells, and then cultured to scale up. The plasmid was extracted using a low endotoxin plasmid extraction kit (catalog number CR-NAE-001) and then transfected into well-growing HEK293T cells using a transfection reagent (such as Beyotime Lipo8000™). The culture medium was changed the next day, and the expression of fluorescent protein was observed to determine the transfection efficiency and the expression of the target protein. The cells were cultured for another day, and the supernatant was collected every other day, while fresh DMEM complete medium was added.

[0038] Tris-HCl (pH=8.0) and NaCl were added to the collected supernatant to achieve final concentrations of 20 mM and 300 mM, respectively. 1 mL of Ni-NTA purification medium was taken and equilibrated with 5 mL of binding buffer (Tris-HCl 20 mM, pH=8.0, NaCl 300 mM). The Ni-NTA purification medium was then transferred to the supernatant and incubated vertically at 4°C for 0.5 h to allow the target protein to bind to the purification medium.

[0039] After incubation, the supernatant was transferred to a gravity purification column. This process was repeated until all supernatant and purification media were filtered. Five volumes of elution buffer containing low-concentration imidazole (Tris-HCl 20mM, pH=8.0, NaCl 300mM, 2mM imidazole) were added to elute impurity proteins. The elution buffer was collected, and the elution status was checked to see if the target protein was eluted. Finally, 1 mL of elution buffer containing high-concentration imidazole (Tris-HCl 20mM, pH=8.0, NaCl 300mM, 50mM imidazole) was added for the first elution of the target protein. After standing at room temperature for 10 min, the eluent was collected into a sterile centrifuge tube. Then, 1 mL of elution buffer containing high-concentration imidazole (Tris-HCl 20mM, pH=8.0, NaCl 300mM, 50mM imidazole) was added to the purification column. The target protein was eluted a second time with 300mM and 50mM imidazole. The eluent was collected into a new sterile centrifuge tube. Finally, 1 mL of elution buffer containing a high concentration of imidazole (Tris-HCl 20mM, pH=8.0, NaCl 300mM, 50mM imidazole) was added to the purification column for a third elution of the target protein. The eluent was collected into a new sterile centrifuge tube to obtain the target protein obtained from three elutions.

[0040] Example 5: Detection of the obtained target protein sample SDS-PAGE electrophoresis: Take 10 μL of the eluted protein sample into a new centrifuge tube, add 2 μL of 5×SDS Loading Buffer (containing 5% β-mercaptoethanol), and separately add 2 μL of commercially available Taq DNA polymerase to a new tube. Add 8 μL of 1% SDS solution and 2 μL of 5×SDS Loading Buffer (containing 5% β-mercaptoethanol) to each tube, mix well, and denature at 95℃ for 5 min. Prepare a 16% Tris-SDS-PAGE gel, add both samples to the wells, and incubate at 100V for 2 h. Remove the gel and stain with Coomassie Brilliant Blue rapid staining solution for 15 min, then rinse with tap water overnight, changing the water several times during this period.

[0041] qPCR detection: Prepare Taq DNA polymerase dialysis buffer (Tris-HCl 50mM, pH=8.0, KCl 500mM, EDTA 1mM, DTT 3mM, glycerol 50%) with sterile water. Transfer the eluent containing the target protein to a 14KD dialysis bag and dialyze at 4℃ for 24h, changing the dialysis buffer twice during the process. Finally, transfer the dialyzed Taq DNA polymerase to a new sterile centrifuge tube and store at -20℃.

[0042] To detect microbial nucleic acid contamination in Taq DNA polymerase, purified Taq DNA polymerase and four specific primers and probes from the bacterial quadruple detection kit (catalog number CR-MDK-003) were used to detect residual DNA in Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa. The qPCR reaction system was prepared according to Table 1, and PCR amplification was performed according to the qPCR reaction procedure in Table 2. Table 1 qPCR reaction system

[0043] Table 2 qPCR reaction procedure

[0044] Results Analysis from Figure 2 It can be seen that strong fluorescent protein expression appeared on the day after HEK293T cells were transfected with the pIFNα-2-6×His-Taq-IRES2-EGFP expression vector, indicating that the expression vector has effectively expressed the target protein.

[0045] The collected culture medium supernatant was then enriched and purified using fusion Taq DNA polymerase. The fusion Taq DNA polymerase was detected by SDS-PAGE electrophoresis. Theoretically, the molecular weight of the produced fusion Taq DNA polymerase is approximately 95 kDa. Figure 3The electrophoresis results showed that the 95KD target protein did not appear in the untransfected blank control medium, but a higher expression level of the target protein appeared in the transfected medium, and its size was consistent with that of commercially available Taq DNA polymerase. To determine whether the fusion Taq DNA polymerase was washed away during washing with a low-concentration imidazole elution buffer, wash buffers from washing 1-3 times were collected and analyzed by SDS-PAGE electrophoresis. The results showed that the wash buffer from washing 3 times eluted many other impurity proteins from serum, but no target protein was found to be eluted. However, the eluent obtained using a high-concentration imidazole elution buffer, analyzed by SDS-PAGE electrophoresis, showed that the eluent from washing 3 times contained both impurity proteins and the target protein. This indicates that Ni-NTA purification can be extremely effective in enriching and purifying the target protein. Figure 4 As shown.

[0046] Because fetal bovine serum contains extremely high levels of various proteins, especially those rich in His amino acids, the purification of fusion Taq DNA polymerase using a Ni-NTA purification column results in the non-specific adsorption of other impurity proteins. This leads to the acquisition of fusion Taq DNA polymerase containing a high amount of these impurity proteins, which are difficult to remove completely regardless of the amount of washing solution used. While increasing the concentration of imidazole in the washing solution enhances the washing effect, it also leads to the loss of fusion Taq DNA polymerase and does not completely solve the problem of non-specific adsorption. Therefore, this invention employs an alternative method: after transfecting HEK293T cells with the expression vector pIFNα-2-6×His-Taq-IRES2-EGFP, the cells are cultured in DMEM complete medium to maximize transfection of the expression vector and promote its expression within the cells. The next day, the medium is changed back to DMEM complete medium to promote cell proliferation and the expression of the target protein. On the third day, the medium is changed to serum-free medium specifically for HEK293 cells (in this experiment, OPMA 293F Hi-exp medium was used: catalog number AC601501 and 293F). Hi-exp feed additive: Catalog number AC601502. The feed additive was added at a ratio of 5% (4.75 ml of culture medium per plate, 0.25 ml of feed additive per plate). After incubation, the supernatant was collected on the fourth day for purification with Taq DNA polymerase and SDS-PAGE electrophoresis. Results are as follows: Figure 5 As shown, the results indicated that after switching to serum-free medium, the cells in the culture dish exhibited higher fluorescence density. Furthermore, due to the absence of serum, cell adhesion decreased slightly, and the cells began to round out, indicating that they were starting to grow into a suspension cell state. Figure 6The results of SDS-PAGE electrophoresis showed that no protein bands were produced in the eluents obtained from three elutions during the purification process of HEK293T cells without Taq DNA polymerase expression vector (blank control). However, only one protein band of about 95KD was produced in the culture supernatant of HEK293T cells transfected with Taq DNA polymerase expression vector. This indicates that this method can not only effectively produce fusion Taq DNA polymerase, but also that the purity of the purified fusion Taq DNA polymerase is almost 100%. Moreover, according to UV spectrophotometry, the highest expression level can be purified to about 2 mg of fusion Taq DNA polymerase in 20 mL of culture medium, that is, about 100 mg of fusion Taq DNA polymerase can be obtained per liter of culture medium. Furthermore, continuous culture of transfected HEK293T cells revealed that they continued to produce fusion Taq DNA polymerase, eliminating the need for retransfection and significantly reducing the purification cost of Taq DNA polymerase. Subsequent SDS-PAGE electrophoresis results of the culture supernatants from the third and fourth batches of HEK293T cells are shown below. Figure 7 As shown.

[0047] Although this invention yielded a large quantity of highly pure fusion Taq DNA polymerase using the above method, we were unsure about the activity of the purified Taq DNA polymerase and whether it was contaminated with common microbial DNA. Therefore, we directly verified this using the purified Taq DNA polymerase. Specific primers and probes from a bacterial quadruple assay kit were selected to determine whether the Taq DNA polymerase contained DNA contamination from *Escherichia coli*, *Bacillus subtilis*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. The results are as follows: Figure 8 As shown, the purified fusion Taq DNA polymerase can effectively amplify the positive sample template, indicating that the purified Taq DNA polymerase is active. The negative control, however, produced no bands, indicating that the enzyme does not contain any DNA from the aforementioned bacteria. Therefore, this method can produce large quantities of biologically active fusion Taq DNA polymerase in HEK293T cells in a short time. The negative control, which did not contain any DNA template, serves two purposes: firstly, it acts as a negative control for amplifying the positive sample template; secondly, it serves as an experimental group to verify whether the Taq DNA polymerase is contaminated with microbial DNA. The absence of amplification in the negative control proves that the Taq DNA polymerase protein is active and that the Taq DNA polymerase is free from common microbial contamination.

[0048] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nucleotide sequence expressing a fused Taq DNA polymerase, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

3.

2. A Taq DNA polymerase fused to a polymerase, characterized in that, The N-terminus of the amino acid sequence of Taq DNA polymerase includes, in sequence, a thrombin recognition and cleavage site, six linked His amino acid tags, and an IFNα-2 signal peptide. The amino acid sequence of the fused Taq DNA polymerase is shown in SEQ ID NO.

1.

3. An expression vector for expressing a fused Taq DNA polymerase, characterized in that, The expression sequence of the fusion Taq DNA polymerase comprises, in sequence, an IFNα-2 signal peptide coding sequence, a 6×His tag coding sequence, a thrombin cleavage site coding sequence and a Taq DNA polymerase coding sequence; The nucleotide sequence expressing the fusion Taq DNA polymerase is shown in SEQ ID NO.3; The backbone of the expression vector expressing Taq DNA polymerase fusion is a eukaryotic expression vector.

4. The expression vector for expressing Taq DNA polymerase as described in claim 3, characterized in that, The backbone of this eukaryotic expression vector is pIRES2-EGFP, and the constructed expression vector is pIFNα-2-6×His-Taq-IRES2-EGFP.

5. The expression vector for expressing Taq DNA polymerase as described in claim 4, characterized in that, The 5' end of the fusion Taq DNA polymerase also includes a Kozak sequence, the nucleotide sequence of which is shown in SEQ ID NO.4; The nucleotide sequence of the expression vector expressing the fused Taq DNA polymerase is shown in SEQ ID NO.

5.

6. A method for expressing Taq DNA polymerase using HEK293T cells, characterized in that, Includes the following steps: (a) Constructing an expression vector for expressing Taq DNA polymerase as described in claim 3, 4, or 5; (b) The expression vector constructed in step (a) was transformed and cultured, and the plasmid was extracted and transfected into HEK293T cells; (c) First, the transfected HEK293T cells were cultured in HEK293T cell culture medium containing fetal bovine serum, and then the transfected HEK293T cells were cultured in HEK293T cell culture medium with serum removed, so that Taq DNA polymerase was released into the cell culture supernatant.

7. The method of claim 6 further includes the step of: (d) collecting cell culture supernatant and purifying it using the Ni-NTA purification method to obtain Taq DNA polymerase.

8. The method as described in claim 7, characterized in that, The Ni-NTA purification process includes: mixing cell culture supernatant with equilibrated Ni-NTA medium and incubating; using an elution buffer containing a low concentration of imidazole to remove impurity proteins; and finally eluting the target protein using an elution buffer containing a high concentration of imidazole. The elution buffer containing low concentration of imidazole contains 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 2 mM imidazole; the elution buffer containing high concentration of imidazole contains 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 50 mM imidazole.

9. Use of the Taq DNA polymerase expressed in the expression vector as described in claim 3, 4 or 5 in a kit for preparing a polymerase chain reaction.

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