A fusion protein NGMet prolonging half-life in vivo, and a preparation method and application thereof

Abstract

The application discloses a fusion protein NGMet with prolonged in-vivo half-life, and a preparation method and application thereof, and belongs to the technical field of medical biological engineering, and particularly relates to a protein NGMet which is formed by fusing methioninase and an albumin binding domain, and a preparation method and application thereof. The fusion protein NGMet is combined with human serum albumin, effectively breaks through the defect that natural methioninase is easily removed by the kidney due to the fact that the molecular weight of the natural methioninase is lower than the molecular weight of a glomerular filtration interception, greatly prolongs the in-vivo half-life of the natural enzyme from less than 8 hours, makes up the technical defects that the natural methioninase has a short in-vivo action time and needs frequent administration, guarantees the stability of the treatment effect, and improves the clinical practical value of the drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical bioengineering technology, specifically relating to NGMet, a protein formed by the fusion of methioninase and an albumin-binding domain (ABD), its preparation method, and its applications. It aims to solve the technical problem of the short in vivo half-life of methioninase. Background Technology

[0002] Methionase is an enzyme that degrades methionine and has attracted much attention in anticancer research in recent years. Methionine is an essential amino acid for the growth of many tumor cells, and methionase inhibits tumor cell proliferation by reducing the intracellular concentration of methionine. Studies have shown that methioninase can effectively inhibit the growth of tumor cells, for example, in gastric cancer (Goseki, N., Yamazaki, S., Shimojyu, K., Kando, F., Maruyama, M., Endo, M., Takahashi, H. Synergistic effect of methionine-depleting total parenteral nutrition with 5-fluorouracil on human gastric cancer-a randomized, prospectiveclinical-trial[J]. Japanese Journal of Cancer Research, 1995, 86(5):484-489.) and cervical cancer (ano, S., Li, S., Han, Q., Tan, Y., Bouvet, M., Fujiwara, T., Hoffman, RM Selective methioninase-induced trap of cancer cells in S / G2 phase visualized by FUCCI imaging confers chemosensitivity[J]. Oncotarget, 2014, 5(18):8729-8736. doi:10.18632 / oncotarget.2369) and prostate cancer (Lu, S., Epner, DE Molecular mechanisms of cell cycle block by methionine restriction in human prostate cancer cells[J]. Nutrition and Cancer - an International Journal, 2000, 38(1):123-130. doi:10.1207 / S15327914NC381_17) showed significant efficacy. In addition, reducing methionine intake in the diet can also effectively inhibit tumor cell growth.However, the molecular weight of natural methioninase is approximately 43 kDa (Lishko, VK, Lishko, OV, Hoffman, RM The preparation of endotoxin-free L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (L-methioninase) from Pseudomonas putida[J]. Protein Expr Purif,1993, 4(6):529-533. doi:10.1006 / prep.1993.1069), which is lower than the glomerular filtration cutoff molecular weight (30-50 kDa), making it easily cleared by the kidneys. Furthermore, the half-life of unengineered methioninase is less than 8 hours (Tan,Y., Sun, X., Xu, M., An, Z., Tan, X., Han, Q., Hoffman, RM Polyethyleneglycol conjugation of recombinant methioninase for cancer therapy[J]. Protein Expr Purif, 1998, 12(1):45-52. doi:10.1006 / prep.1997.0805). Therefore, prolonging the in vivo half-life of methionine enzymes is a technical problem that urgently needs to be solved. Summary of the Invention

[0003] To address the issue of the short in vivo half-life of methionine enzymes, this invention provides a protein called NGMet, which is a fusion of methionine enzyme and an albumin-binding domain (ABD). By binding to human serum albumin (HSA), NGMet significantly prolongs its in vivo half-life.

[0004] The present invention discloses a fusion protein with extended in vivo half-life, specifically a protein formed by the fusion of methioninase and an albumin-binding domain (ABD), named NGMet.

[0005] Furthermore, the fusion protein NGMet disclosed in this invention also includes a peptide linker connecting the C-terminus of the ABD peptide and the N-terminus of the methionine peptide, wherein the peptide linker is a flexible chain peptide having 15 amino acids.

[0006] Furthermore, the fusion protein NGMet disclosed in this invention has gene and protein sequences composed of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively; its complete gene and protein sequences are shown in SEQ ID NO:4 and SEQ ID NO:8.

[0007] Furthermore, the fusion protein NGMet disclosed in this invention has a molecular weight of 49 kDa. It forms a complex with a total molecular weight of 116 kDa by binding its ABD with human serum albumin (HSA, 67 kDa), which is much larger than the molecular weight cutoff for glomerular filtration, thereby significantly prolonging its half-life in vivo.

[0008] Given that the fusion protein NGMet can persistently degrade methionine and inhibit tumor cell proliferation, it can be used as a core active ingredient in the preparation of drugs for treating methionine-dependent tumors such as gastric cancer, cervical cancer, and prostate cancer. By intervening in the metabolism of essential amino acids in tumor cells, it can inhibit tumor growth.

[0009] The fusion protein NGMet described in this invention can also be used to prepare drugs that help improve methionine metabolism in cancer patients. NGMet can be used in synergy with dietary intervention to prepare drugs that help improve methionine metabolism in cancer patients. Through the dual effects of drug degradation and dietary control, it can more efficiently reduce methionine levels in the body and enhance the inhibitory effect on tumor cells. The fusion protein NGMet described in this invention can also be used to prepare formulations that prolong the in vivo action time of methionine enzyme drugs.

[0010] The beneficial effects of this invention are: (1) The fusion protein NGMet effectively overcomes the defect of natural methionine enzymes being easily cleared by the kidneys because their molecular weight is lower than the molecular weight cutoff of the glomerulus by binding to HSA. It significantly extends the half-life in vivo from less than 8 hours for natural enzymes, making up for the technical defects of natural methionine enzymes, such as short in vivo action time and the need for frequent administration.

[0011] (2) The fusion protein NGMet retains the core function of methionine degradation by methionine while prolonging its half-life in vivo, and can continuously reduce the concentration of methionine in the body. Methionine is an essential amino acid for the growth of various tumor cells such as gastric cancer, cervical cancer, and prostate cancer. This makes the fusion protein NGMet able to inhibit tumor cell proliferation more persistently. Compared with natural methionine, its anti-cancer effect is significantly more sustained and effective.

[0012] (3) Due to the extended half-life, the fusion protein NGMet does not need to be administered as frequently as natural methionine enzymes in clinical applications. This not only reduces the burden of administration to patients but also avoids fluctuations in blood drug concentration caused by frequent administration, further ensuring the stability of the therapeutic effect and enhancing the clinical practical value of the drug.

[0013] (4) The fusion protein NGMet has significant application prospects, providing a reference for optimizing the half-life of other methionine enzyme anticancer drugs and expanding the clinical application scenarios of methionine enzyme drugs. Attached Figure Description

[0014] Figure 1 This is an SDS-PAGE analysis of NGMet expression and purification in this invention. Lane 1: Label; Lane 2: Soluble fraction; Lane 3: Nickel column elution; Lane 4: 10% elution buffer; Lane 5: 20% elution buffer; Lane 6: 30% elution buffer; Lane 7: 40% elution buffer; Lane 8: 60% elution buffer; Lane 9: 100% elution buffer.

[0015] Figure 2 This invention provides non-denaturing polyacrylamide gel electrophoresis analysis of NGMet mixed with HSA at different molar ratios. Lane 1: NGMet:HSA = 1:0.25; Lane 2: NGMet:HSA = 1:0.5; Lane 3: NGMet:HSA = 1:0.75; Lane 4: NGMet:HSA = 1:1; Lane 5: NGMet:HSA = 1:1.25; Lane 6: NGMet:HSA = 1:2; Lane 7: NGMet:HSA = 1:0; Lane 8: NGMet:HSA = 0:0.25; Lane 9: NGMet:HSA = 0:1.

[0016] Figure 3 This is the NGMet-HSA complex structure predicted by ColabFold v1.5.5 (AlphaFold2) in this invention.

[0017] Figure 4 This invention demonstrates the inhibitory effects of different concentrations of NGMet on BGC-823 cells.

[0018] Figure 5 The morphology of BGC-823 cells after NGMet treatment and the control group in this invention is shown under a microscope.

[0019] Figure 6 These are the pharmacodynamic results of NGMet in this invention. Detailed Implementation

[0020] All reagents and instruments used in this embodiment are available from commercial sources, and the methods used, unless otherwise specified, are consistent with conventional laboratory methods.

[0021] Example 1

[0022] Expression and purification of NGMet: The NGMet gene was cloned into the pET29a vector and transformed into the BL21(DE3) strain for expression. Single colonies were picked and inoculated into LB medium containing 50 µg / mL kanamycin, and cultured overnight at 30°C and 250 rpm with shaking. The overnight culture was then inoculated 1:100 into 200 mL of LB medium containing 50 µg / mL kanamycin and cultured at 30°C and 250 rpm. When OD... 600 When the expression level reached 0.6-0.8, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce expression. Cells were collected by centrifugation, resuspended in resuspension buffer (20 mM Tris, 1 M NaCl, pH 8.0), and then lysed using an ultrasonic homogenizer. The supernatant was collected and purified using a HiTrap Ni column. NGMet was eluted in 40% elution buffer (20 mM Tris, 1 M NaCl, 500 mM imidazole, pH 8.0). Purity was analyzed by SDS-PAGE. The final product was mixed with 0.3 mM pyridoxal phosphate for subsequent experiments.

[0023] like Figure 1 As shown, NGMet is highly expressed in the soluble fraction (lane 2). Pure NGMet was obtained by nickel column purification in 40% elution buffer with a specific activity of 5.41 ± 0.74 U / mg (lane 7).

[0024] Example 2

[0025] Assay for NGMet binding to human serum albumin (HAS): 2 µg of NGMet was mixed with different molar ratios of HAS (molar ratios: NGMet:HSA = 1:0.25, 1:0.5, 1:0.75, 1:1, 1:1.25, 1:2, 1:0, 0:0.25, 0:1) and incubated at 37 °C for 10 min. The mixture was then analyzed by non-denaturing polyacrylamide gel electrophoresis to evaluate the binding capacity of NGMet to HAS.

[0026] like Figure 2 As shown, NGMet can effectively bind HSA, increasing its molecular weight and preventing it from being cleared by the kidneys. Experimental results show that 1 mole of NGMet can almost completely bind 0.75 moles of HSA (lane 3).

[0027] Predicting NGMet-HSA complexes using ColabFold v1.5.5: AlphaFold2: like Figure 3 As shown, ColabFold v1.5.5 (AlphaFold2) predicts that NGMet (molecular weight 49 kDa) and HSA (molecular weight 67 kDa) form a complex with a total molecular weight of 116 kDa, which is much larger than the molecular weight cutoff for glomerular filtration (30-50 kDa). Figure 3 In the NGMet protein sequence, the red segment represents ABD (sequence shown in SEQ ID NO: 5), the blue segment represents flexible chain polypeptide (sequence shown in SEQ ID NO: 6), the yellow segment represents methionine enzyme (sequence shown in SEQ ID NO: 7), and the green segment represents HAS (protein sequence shown in SEQ ID NO: 9).

[0028] Example 3

[0029] NGMet's inhibitory effect on BGC-823 cells: BGC-823 (human gastric adenocarcinoma cells) were seeded at a density of 3000 cells per well in 96-well plates and cultured overnight in RPMI 1640 medium (containing 10% fetal bovine serum and penicillin / streptomycin) at 37°C with 5% CO2. After 24 hours, different concentrations of NGMet (NGMet concentrations of 30, 15, 7.5, 3.75, 1.875, 0.9375, 0.46875, 0.234375, 0.1171875, and 0 μg / mL) were added, and the cells were cultured for another 72 hours. 10 µL of 5 mg / mL MTT reagent was added to each well, and the cells were incubated at 37°C for 4 hours. After incubation, the culture medium was removed, and 100 µL of DMSO was added to dissolve formazan crystals. Cell viability was determined by absorbance at 540 nm, and IC50 was calculated using Prism software. 50 Value. The experiment was independently repeated three times.

[0030] like Figure 4 As shown, NGMet effectively inhibits the proliferation of BGC-823 gastric cancer cells, with an IC50 value of [missing information]. 50 The value was 4.71 ± 1.04 µg / mL.

[0031] Investigating the effects of NGMet (30 μg / mL concentration) on the morphology and number of BGC-823 cells: BGC-823 cells were uniformly divided into two groups: a control group, cultured in conventional cell culture medium, and an NGMet treatment group, cultured in cell culture medium containing 30 μg / mL NGMet. Both groups of cells were cultured at 37℃ and 5% CO2 for 3 days. Cell morphology was observed under a microscope, with three different fields of view selected from each group. Figure 5 (The corresponding fields of view are 1, 2, and 3 in the middle), and cell images are captured and recorded.

[0032] like Figure 5 As shown, the control group BGC-823 cells were relatively densely distributed and numerous under a microscope; after treatment with 30 μg / mL NGMet, the number of BGC-823 cells was significantly less than that of the control group, and the cell distribution was much sparser. These results indicate that NGMet can significantly inhibit the growth of BGC-823 cells, providing a basis for further investigation into its mechanism of action.

[0033] Example 4

[0034] Pharmacodynamic experiments: To compare the effects of genetically engineered NGMet and unmodified methionine enzyme in mice, and to clarify the half-life of NGMet and its influence on methionine levels in mice.

[0035] Four healthy C57BL / 6 mice of similar weight were selected for this experiment. The mice were randomly divided into two groups: a methionine enzyme group and an NGMet group, with two mice in each group. The mice in each group were intraperitoneally injected with methionine enzyme and NGMet at a dose of 100 mg / kg, respectively.

[0036] Blood samples were collected from two groups of mice at three time points: before drug administration (0 h), 24 h after drug administration, and 48 h after drug administration. The concentration of methionine enzyme in the samples after residual enzyme action was determined using an automated amino acid analyzer (Biochrom 30+) for methionine enzyme concentration detection. Two samples were tested from each group at each time point.

[0037] The concentration data of methionine enzymes in the two groups of mice at different time points were recorded, and the data were then compiled into a bar chart. Figure 6 .

[0038] like Figure 6 As shown, unengineered methionine enzymes acted for less than a day in mice, while NGMet acted for more than a day. However, methionine levels in mice returned to normal on the second day, confirming that the half-life of genetically engineered NGMet was significantly prolonged. This lays the foundation for further investigation into its role in methionine metabolism intervention in the treatment of diseases such as tumors.

Claims

1. A fusion protein NGMet with an extended in vivo half-life, characterized in that, It includes an albumin-binding domain and methionine enzyme.

2. The fusion protein NGMet with extended in vivo half-life according to claim 1, characterized in that, The fusion protein NGMet also includes a peptide linker connecting the C-terminus of an albumin-binding domain polypeptide and the N-terminus of a methionine peptide, wherein the peptide linker is a flexible chain polypeptide having 15 amino acids.

3. The fusion protein NGMet with extended in vivo half-life according to claim 1, characterized in that, The complete gene and protein sequences of the fusion protein NGMet are composed of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

4. The fusion protein NGMet with extended in vivo half-life according to claim 3, characterized in that, The complete gene and protein sequences of the fusion protein NGMet are shown in SEQ ID NO: 4 and SEQ ID NO:

8.

5. The fusion protein NGMet with extended in vivo half-life according to claim 1, characterized in that, The molecular weight of the fusion protein NGMet is 49 kDa.

6. The fusion protein NGMet with extended in vivo half-life according to claim 1, characterized in that, The fusion protein NGMet binds to human serum albumin through its albumin-binding domain to form a complex with a total molecular weight of 116 kDa.

7. The use of the NGMet fusion protein with extended in vivo half-life as described in any one of claims 1-6 in the preparation of a methionine-dependent tumor drug.

8. The use of the NGMet fusion protein with extended in vivo half-life as described in any one of claims 1-6 in a drug for degrading methionine and inhibiting tumor cell proliferation.

9. The use of the fusion protein NGMet with an extended in vivo half-life as described in any one of claims 1-6 in the preparation of a medicament to help improve methionine metabolism in cancer patients.

10. The use of the fusion protein NGMet with an extended in vivo half-life as described in any one of claims 1-6 in the preparation of formulations for prolonging the in vivo action time of methionine enzyme drugs.

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