Fusion enzymes for producing pseudouridine monophosphate and uses thereof

Abstract

The application provides a fusion enzyme for producing pseudouridine monophosphate, characterized by being composed of a ribokinase gene rbsK-rigid connection peptide-pseudouridine 5' phosphate glycosidase gene psuG. Its expression gene, expression plasmid and prokaryotic expression vector are also disclosed. The fusion enzyme of the application can catalyze D-ribose and uracil to synthesize pseudouridine monophosphate in vitro, greatly simplifying the synthesis steps of pseudouridine monophosphate and related compounds, and has a wide application prospect.

Description

Technical Field

[0001] This invention patent relates to a fusion enzyme for the production of pseudouridine monophosphate and its application, belonging to the field of bioengineering technology. Background Technology

[0002] mRNA-mediated biotherapy has become a major focus of biomedical research in recent years, primarily encompassing mRNA prophylactic vaccines, mRNA therapeutic vaccines, and mRNA drugs. Compared to traditional vaccines, mRNA vaccines offer advantages such as shorter production cycles and longer durations of action. Therefore, in development, mRNA vaccines can respond rapidly to outbreaks and are more advantageous in the face of viral strain mutations. In 2005, Katalin Karikó et al. discovered numerous pseudouridine modifications on mRNA. These modifications significantly reduce the immunogenicity of mRNA, preventing the induction of extensive inflammatory responses and reducing the side effects of mRNA vaccines and drugs on the human body. Pseudouridine, N1-methylpseudouridine, and 5-methoxyuridine are the main modification forms that replace natural uridine. Furthermore, pseudouridine monophosphate, as a primary synthetic raw material, has significant research value in its synthesis and production.

[0003] Currently, there are few pseudouridine monophosphate products on the market. Literature records two main methods for synthesizing pseudouridine monophosphate: chemical and enzymatic. The chemical method uses pseudouridine as a substrate, involves fewer steps, but is more expensive and requires flammable and explosive hazardous materials. The enzymatic method uses D-ribose as a substrate, is less expensive, but requires constructing vectors for multiple enzymes in the synthetic pathway, expressing and purifying them to obtain the enzyme protein, which is more complex. Therefore, this invention simplifies the protein expression and purification steps in enzymatic synthesis by constructing and preparing a fusion enzyme, enabling rapid and safe production of pseudouridine monophosphate. Summary of the Invention

[0004] The purpose of this invention is to provide a fusion enzyme for the production of pseudouridine monophosphate.

[0005] This invention discloses a fusion enzyme for producing pseudouridine monophosphate, which consists of a ribokinase gene rbsK, a rigid linker peptide, and a pseudouridine 5' phosphoglycosidase gene psuG. The sequence of rbsK is shown in SEQ ID No. 1, and the sequence of psuG is shown in SEQ ID No. 2.

[0006] Preferably, the sequence of the rigid linker peptide is EAAAKEAAAK.

[0007] Preferably, the C-terminus of the fusion enzyme also has a His tag.

[0008] The present invention also discloses the expression gene of the fusion enzyme for producing pseudouridine monophosphate, the sequence of which is shown in SEQ ID No. 10.

[0009] The present invention also discloses an expression plasmid for producing a fusion enzyme of pseudouridine monophosphate, expressing the above-mentioned fusion enzyme.

[0010] Preferably, the plasmid vector is PET-28a.

[0011] The present invention also discloses a prokaryotic expression vector for producing a fusion enzyme of pseudouridine monophosphate, expressing the above-mentioned fusion enzyme.

[0012] Preferably, the expression vector is Escherichia coli BL21.

[0013] This invention also discloses the preparation method of the above-mentioned fusion enzyme for producing pseudouridine monophosphate, characterized in that its steps include:

[0014] (1) Primers for ribokinase gene rbsK and pseudouridine 5' phosphate glycosidase gene psuG were designed respectively. Homologous fragments of rigid linking peptides and vectors were added during the primer design process. Using Escherichia coli BL21 cell lysate as template, psuG gene fragments and rbsK gene fragments were cloned.

[0015] (2) The gene fragment cloned by the primers in step (1) is ligated into a plasmid vector to construct the PET-rbsK-psuG expression plasmid;

[0016] (3) The expression plasmid was transformed into Escherichia coli BL21, and positive clones were selected to express the fusion enzyme protein;

[0017] (4) The positive clone strains were cultured, the bacterial cells were collected, and the total protein was obtained by ultrasonic disruption and centrifugation. Then, the fusion enzyme protein was purified by Ni-NTA agarose gel chromatography column.

[0018] Preferably, the primer sequence described in step (1) is:

[0019] rbsK upstream primer: 5'-TAAGAAGGAGATATACCATGATGCAAAACGCAGGCAGCC-3';

[0020] rbsK downstream primer: 5'-GGAAATTTTTAATTCAGACGCTTTCGCCGCCGCTTCTTTCGCCGCCGCTTCCCTCTGCCTGTCTAAAAATGCGTC-3';

[0021] psuG upstream primer: 5'-TCTGAATTAAAAATTTCCCCTGAATTATTACA-3';

[0022] psuG downstream primer: 5'-GTGGTGGTGGTGCTCGAGACCCGCGAGACGC-3'.

[0023] Preferably, the plasmid vector is PET-28a. Step (2) specifically involves digesting PET-28a with NcoⅠ and XhoⅠ, recovering the digestion products, and ligating the digestion products of the plasmid with the cloned gene fragment to obtain the rbsK-rigid linker peptide-psuG expression plasmid.

[0024] The present invention also discloses the application of the above-mentioned fusion enzyme in catalyzing the conversion of D-ribose to pseudouridine monophosphate.

[0025] Preferably, the reaction is carried out by mixing D-ribose with the fusion enzyme. The reaction system includes: ATP 1mM, uracil 1mM, D-ribose 1mM, PEP 2mM, fusion enzyme 2ug / mL, PK enzyme 2ug / mL, with a total volume of 1mL and a reaction temperature of 37℃.

[0026] The beneficial effects of this invention are as follows: The fusion enzyme preparation method of this invention is simple, low-cost, and can be used for large-scale production. The fusion enzyme of this invention can catalyze the synthesis of pseudouridine monophosphate from D-ribose and uracil in vitro, greatly simplifying the synthesis steps of pseudouridine monophosphate and its related compounds, and has broad application prospects. Attached Figure Description

[0027] Figure 1 Electrophoresis results of PCR products of rbsK and psuG gene clones. Lanes 1 and 2 are rbsK; lanes 4 and 5 are psuG; lane 3 is a 2000bp DNA marker, with values ​​of 2000, 1000, 750, 500, 250, and 100bp from top to bottom.

[0028] Figure 2 The synthesis of pseudouridine monophosphate using the fusion enzyme described in this invention was performed, and the reaction solution was analyzed by HPLC after 4 hours of reaction. The peak at approximately 6.3 min was pseudouridine monophosphate.

[0029] Figure 3 The synthesis of pseudouridine monophosphate using the fusion enzyme described in this invention was performed, and the reaction solution was analyzed by HPLC after 20 hours of reaction. The peak at approximately 6.3 min was pseudouridine monophosphate.

[0030] Figure 4 Liquid phase results detected by LC-MS of the reaction solution. Figure 4 As a result, the peak around 1.5 min is pseudouridine monophosphate.

[0031] Figure 5MS results of LC-MS detection of the reaction solution are shown. The left graph shows the real-time result at 1.11 min, and the right graph shows the real-time result at 1.5 min. The peak around 1.5 min is pseudouridine monophosphate, which is related to... Figure 4 The results correspond. Detailed Implementation

[0032] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The provided embodiments are merely illustrative of the method of the present invention and do not limit the rest of the content disclosed herein in any way.

[0033] Example 1

[0034] In this embodiment, an expression element (SEQ ID No. 10) consisting of the ribokinase gene (SEQ ID No. 3), the linker peptide, and the pseudouridine 5' phosphoglycosidase gene (SEQ ID No. 4) was recombined into the E. coli expression plasmid PET-28a.

[0035] In this embodiment, the Escherichia coli DH5α competent cells, PET-28a plasmid, multi-fragment one-step cloning kit, high-fidelity enzyme premix, and gel recovery kit were all from Yisheng Biotechnology Co., Ltd.

[0036] Using a multi-fragment one-step cloning kit, primers were designed to clone the rbsK and psuG genes using E. coli BL21 as a template. To ligate the two gene fragments to the NcoⅠ and XhoⅠ sites of PET-28a, homologous plasmid sequences were added to the 5' ends of the upstream primer of rbsK and the downstream primer of psuG. The linker peptide was designed at the 5' end of the downstream primer of rbsK.

[0037] The rbsK primer sequences are as follows:

[0038] Upstream 5'-TAAGAAGGAGATATACCATGATGCAAAACGCAGGCAGCC-3' (SEQ ID No. 5)

[0039] Downstream

[0040] 5'-GGAAATTTTTAATTCAGACGCTTTCGCCGCCGCTTCTTTCGCCGCCGCTTCCCTCTGCCTGTCTAAAAATGCGTC-3' (SEQ ID No. 6)

[0041] The psuG primer sequence is as follows:

[0042] Upstream 5'-TCTGAATTAAAAATTTCCCCTGAATTATTACA-3'(SEQ ID No. 7)

[0043] Downstream 5'-GTGGTGGTGGTGCTCGAGACCCGCGAGACGC-3' (SEQ ID No. 8)

[0044] The system was supplemented with 25 μL of 2× high-fidelity enzyme premix, 10 μM each of forward and reverse primers, and 1 μL of pre-cooked E. coli BL21.

[0045] The bacterial culture was used as a template; ddH2O was added to a total volume of 50 μL. The PCR reaction program was: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min 10 s, 35 cycles; 72℃ for 5 min. The PCR products were separated by agarose gel electrophoresis. The electrophoresis results are shown below. Figure 1 As shown.

[0046] The PET-28a plasmid was double-digested with restriction endonucleases NcoⅠ and XhoⅠ. The digestion system consisted of 1 μL each of TaKaRa restriction enzymes, 10 μL of TaKaRa2*Universal buffer, 1 μg of plasmid, and ddH2O added to a total volume of 20 μL. The reaction was carried out at 37°C for 20 minutes, followed by electrophoresis. The linearized plasmid fragment was then recovered by gel extraction. The PCR product and the linearized plasmid were ligated using a cloning kit. The ligated recombinant plasmid was transformed into DH5α competent cells, plated on LB agar containing 50 mg / mL kanamycin sulfate, and the correct clones were screened and sequenced for verification.

[0047] The sequence of the linker peptide is EAAAKEAAAK (SEQ ID No. 9).

[0048] Example 2

[0049] This example aims to overexpress and purify the fusion enzyme protein.

[0050] The correct recombinant plasmid was screened and transformed into Escherichia coli strain BL21. The strain was cultured in shake flasks with LB liquid medium. When the OD600 of the bacterial culture reached 0.8-1, IPTG inducer at a final concentration of 0.1 mM was added and the culture was induced overnight at 16°C.

[0051] The target protein was isolated and purified using a HisSep Ni-NTA 6FF pre-packed column (Yisheng Biotechnology).

[0052] First, prepare the purification buffer:

[0053] Lysis buffer: 50mM NaH2PO4, 300mM NaCl, pH 7.9

[0054] Elution buffer: 50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH7.9

[0055] The bacterial cells were collected by centrifugation at 8000 rpm for 10 minutes and resuspended in one-tenth of a volume of lysis buffer. The cells were then sonicated for 25 minutes at 300 W. A pre-packed column was installed on an AKTA protein purification system at a flow rate of 1 mL / min. The purification steps were as follows: equilibration with 4 column volumes of Lysis buffer; sample loading; washing with 3 column volumes of Lysis buffer; washing with 3 column volumes of 92% Lysis buffer and 8% Elution buffer; elution with 80% Lysis buffer and 20% Elution buffer. After elution, the solution was dialyzed through a 7 kDa dialysis bag to 20 mM Tris and 200 mM NaCl, and 50% glycerol was added. The solution was then stored at -20°C.

[0056] Example 3

[0057] This embodiment relates to the synthesis of pseudouridine monophosphate using the fusion enzyme prepared according to the present invention.

[0058] Prepare a 50 mM Tris buffer solution and adjust the pH to 7.8 with HCl. The reaction system includes: 1 mM ATP, 1 mM uracil, 1 mM D-ribose, 2 mM PEP, 2 μg / mL RK-psuG fusion enzyme, and 2 μg / mL pyruvate kinase, with a total volume of 1 mL. The reaction temperature is 37 °C, and the reaction solution is detected by HPLC. Before detection, the sample solution needs to be heated at 95 °C for 10 minutes and centrifuged at 12,000 rpm for 10 minutes to remove proteins.

[0059] HPLC was performed using a C18 column. Phase A consisted of 20 mM potassium phosphate and 100 mM triethylamine, adjusted to pH 6.0 with glacial acetic acid, filtered through a 0.22 μm filter and degassed by sonication before use. Phase B consisted of methanol (analytical grade). Phase C consisted of ultrapure water. The analytical methods were as follows: 95% Phase A, 5% Phase B, 5 min; 95%-80% Phase A, 5%-20% Phase B, 15 min; 80% Phase A, 20% Phase B, 5 min; 95% Phase A, 5% Phase B, 5 min. HPLC results are attached. Figure 2 , 3 As shown in the attached figure. The LC-MS detection and analysis results are as follows. Figure 4 , 5 As shown.

[0060] Appendix Figure 2 , 3The reaction solution analysis results are shown for 4 hours and 20 hours, respectively. The peak at 6.303 min is pseudouridine monophosphate, and at 7.177 min it is uracil. These results indicate that the conversion rate of the substrate uracil is approximately 50% after 4 hours and approximately 99% after 20 hours.

[0061] Figure 4 It is the LC spectrum in LC-MS. Figure 5 The MS spectrum shows that component B at 1.5 min matches the molecular weight of pseudouridine monophosphate. This indicates that the reaction did indeed produce pseudouridine monophosphate.

Claims

1. A fusion enzyme for the production of pseudouridine monophosphate, characterized in that: It consists of the ribokinase gene rbsK, the rigid linker peptide, and the pseudouridine 5' phosphoglycosidase gene psuG. The sequence of rbsK is shown in SEQ ID No. 1, the sequence of psuG is shown in SEQ ID No. 2, and the amino acid sequence of the rigid linker peptide is EAAAKEAAAK.

2. A fusion enzyme for the production of pseudouridine monophosphate, characterized in that: The C-terminus of the fusion enzyme according to claim 1 also has a His tag.

3. The expression gene for the fusion enzyme used to produce pseudouridine monophosphate according to claim 1, the sequence of which is shown in SEQ ID No.

10.

4. An expression plasmid for producing a fusion enzyme of pseudouridine monophosphate, characterized in that... The fusion enzyme of any one of claims 1-2 is expressed using a plasmid vector of PET-28a.

5. A prokaryotic expression host bacterium for producing a fusion enzyme of pseudouridine monophosphate, characterized in that... The expression of the fusion enzyme according to any one of claims 1-2, wherein the expression host bacterium is Escherichia coli BL21.

6. The method for preparing the fusion enzyme according to claim 2, characterized in that... The steps include: (1) Primers for ribokinase gene rbsK and pseudouridine 5' phosphate glycosidase gene psuG were designed respectively. Homologous fragments of rigid linking peptides and vectors were added during the primer design process. Using Escherichia coli BL21 cell lysate as template, the full-length gene fragments of psuG and rbsK were cloned. (2) The gene fragment cloned by the primers in step (1) is ligated into a plasmid vector to construct the rbsK-rigid linker peptide-psuG expression plasmid; (3) Transform the expression plasmid into Escherichia coli BL21 and select positive clones to express the fusion enzyme protein; (4) The positive clone strains were cultured, the bacterial cells were collected, and the total protein was obtained by ultrasonic disruption and centrifugation. Then, the fusion enzyme protein was purified by Ni-NTA agarose gel chromatography column.

7. The method for preparing the fusion enzyme according to claim 6, characterized in that... The primer sequence mentioned in step (1) is as follows: rbsK upstream primer: 5'-TAAGAAGGAGATATACCATGATGCAAAACGCAGGCAGCC-3'; rbsK downstream primer: 5'-GGAAATTTTTAATTCAGACGCTTTCGCCGCCGCTTCTTTCGCCGCCGCTTCCCTCTGCCTGTCTAAAAATGCGTC-3'; psuG upstream primer: 5'-TCTGAATTAAAAATTTCCCCTGAATTATTACA-3'; psuG downstream primer: 5'-GTGGTGGTGGTGCTCGAGACCCGCGAGACGC-3'.

8. The method for preparing the fusion enzyme according to claim 6, characterized in that: The plasmid vector is PET-28a. Step (2) specifically involves digesting PET-28a with NcoⅠ and XhoⅠ, recovering the digestion products, and linking the digestion products of the plasmid with the cloned gene fragment to obtain the rbsK-rigid linking peptide-psuG expression plasmid.

9. The use of the fusion enzyme according to any one of claims 1-2 in the in vitro catalytic conversion of D-ribose to pseudouridine monophosphate.

10. The application according to claim 9, characterized in that... The D-ribose was mixed with the fusion enzyme and reacted. The reaction system included: ATP 1mM, uracil 1mM, D-ribose 1mM, PEP 2mM, fusion enzyme 2ug / mL, PK enzyme 2ug / mL, with a total volume of 1mL and a reaction temperature of 37℃.

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