Eukaryotic protein modification system as well as preparation method and application thereof

Abstract

The invention discloses an eukaryotic protein modification system and a preparation method and application thereof, the eukaryotic protein modification system comprises a cell extract and a supplement comprising a modification donor and an energy donor, and a to-be-modified protein is added into the cell extract to incubate to obtain a functional protein. The eukaryotic protein modification system provided by the invention makes up for the defect of insufficient post-translational modification of prokaryotic cell expression protein, can quickly modify target protein in vitro, effectively improves the functionality of protein, realizes efficient production of functionalized protein, is beneficial to reducing the production cost of bioactive protein, improves the production efficiency, and is suitable for industrial production. Wide application prospects are realized in the fields of biological medicines and the like.

Description

Technical Field

[0001] The present invention relates to a protein modification system, in particular to a eukaryotic protein modification system and application thereof in preparing functionalized proteins, and belongs to the technical field of biomedicine. Background Art

[0002] Post-translational modification is an important part of protein function realization, involving various modification forms such as spatial folding, glycosylation, phosphorylation, acylation, methylation, ubiquitination, hydroxylation, etc., which can significantly affect the stability, activity and interaction of proteins with other molecules. Although prokaryotic cells have been widely used in large-scale production of proteins, they have limitations in protein post-translational modification, resulting in incomplete functions of the produced proteins, which cannot meet the application needs in the fields of drug development, biopharmaceuticals, biosensors, etc. With the development of proteomics research and the biopharmaceutical industry, the demand for functional proteins with high activity and high stability after post-translational modification is increasing day by day, but the eukaryotic expression system and cell-free system technology that can prepare such proteins currently have obvious defects.

[0003] The cell culture cost of the eukaryotic expression system is high, the cycle is long, and the operation of introducing the target gene into eukaryotic cells is complicated, so there are problems such as low yield and high cost of functionalized proteins. In addition, the purification of proteins by this technology requires cell wall breaking, which may change the properties of the protein and then affect its function. The cell-free system uses the lysate of prokaryotic cells or eukaryotic cells to synthesize functionalized proteins from the target gene in vitro. However, the protein yield of this technology depends on the number of cells used to construct the system. In addition, since this technology uses genes as the starting point to prepare functionalized proteins, it needs to go through the two processes of translation and modification, so the raw materials, process steps and process conditions used are very complicated. In summary, the existing technology cannot efficiently produce functionalized proteins. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a eukaryotic protein modification system capable of efficiently producing functionalized proteins.

[0005] Technical solution: The eukaryotic protein modification system described in the present invention includes a cell extract and a supplement, wherein the cell extract is an extract of eukaryotic cells, and the supplement includes a modification donor and an energy donor.

[0006] The eukaryotic cells include animal cells (such as human cells, CHO cells, HEK293 cells), plant cells (such as tobacco cells, rice cells), fungal cells (such as yeast, Aspergillus niger) and protist cells (such as amoeba), etc. The modification donors include UDP-glucose, ATP, S-adenosylmethionine, etc. The energy donors include ATP and GTP, etc.

[0007] Eukaryotic cells naturally contain a variety of enzyme systems such as tyrosine kinases and serine kinases, which can catalyze protein modification to form functionalized proteins. Lysing eukaryotic cells can obtain cell extracts containing the enzyme system, and adding supplements including modification donors and energy donors thereto can construct the eukaryotic protein modification system. Different modification donors and energy donors can construct eukaryotic protein modification systems that can produce different functionalized proteins, such as spatial folding, hydroxylation, glycosylation, phosphorylation, acylation, methylation, ubiquitination, etc. of proteins.

[0008] Preferably, the eukaryotic cell has been subjected to mutagenesis to enhance protein modification function.

[0009] The eukaryotic protein modification system constructed by using the mutagenized eukaryotic cells can produce functional proteins more efficiently.

[0010] Preferably, the mutagenesis is carried out using cisplatin, fluorouracil, ultraviolet light, colchicine, cyclophosphamide or sodium hydroxide.

[0011] Cisplatin can enhance the protein modification function of eukaryotic cells by inducing DNA damage and activating cell stress response. Other mutagens such as 5-FU, ultraviolet rays, colchicine, cyclophosphamide and sodium hydroxide can also be used to enhance the modification ability of cells, such as 5-FU treatment, which increases the phosphorylation ability of cells; ultraviolet (UV) irradiation treatment, which enhances the glycosylation ability of cells; colchicine treatment, which increases the ubiquitination ability of cells.

[0012] Preferably, the eukaryotic cell is at least one of an animal cell, a plant cell, a fungal cell, and a protist cell.

[0013] Preferably, the modification donor is at least one of UDP-glucose, ATP, and S-adenosylmethionine, and the energy donor is ATP or GTP.

[0014] ATP hydrolysis can release phosphate and energy. When constructing a eukaryotic protein modification system with protein phosphorylation function, ATP is used as a supplement and can serve as both the modification donor and the energy donor.

[0015] Preferably, the volume ratio of the cell extract to the supplement is 100-1000:1-10.

[0016] The present invention also provides a method for preparing the eukaryotic protein modification system. The first step is to lyse eukaryotic cells to obtain cell extracts; the second step is to add supplements to the cell extracts.

[0017] Preferably, the lysed eukaryotic cells can be lysed by chemical lysis, enzymatic lysis or mechanical lysis, such as grinding.

[0018] Preferably, in the first step, eukaryotic cells are first mutated, and then cells with enhanced protein modification function are screened for lysis.

[0019] The present invention also provides application of the eukaryotic protein modification system in preparing functionalized proteins.

[0020] The protein to be modified is added into the eukaryotic protein modification system and incubated to obtain the functionalized protein.

[0021] Preferably, the functionalization includes spatial folding, glycosylation, phosphorylation, acylation, methylation, ubiquitination, and hydroxylation of proteins.

[0022] Preferably, the eukaryotic protein modification system is used to modify proteins expressed by prokaryotic cells to obtain functionalized proteins, thereby effectively integrating the advantages of different systems to achieve efficient production of functionalized proteins.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The eukaryotic protein modification system of the present invention can effectively improve the functionality of proteins and realize the efficient production of functionalized proteins, which is beneficial to their application in the fields of drug development and biopharmaceuticals; 2. The present invention integrates the high protein yield advantage of the prokaryotic cell system with the high-efficiency modification advantage of the eukaryotic protein modification system, and can quickly modify the target protein in vitro, which is beneficial to reducing the production cost of biologically active proteins and improving production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the eukaryotic protein modification system of the present invention and its preparation and application; Figure 2 This is a diagram showing the enzyme activity detection results of GST produced by different modification systems after mutagenesis. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0026] The examples take protein phosphorylation modification as an example to specifically illustrate the eukaryotic protein modification system and its preparation method and application according to the present invention.

[0027] Main materials and sources: 1. Eukaryotic cells HEK293T: purchased from Shanghai Kisdno Biotechnology Co., Ltd. and preserved by our research group.

[0028] 2. Cisplatin: purchased from Anhui Zesheng Technology Co., Ltd.

[0029] 3. ATP: purchased from Macklin.

[0030] 4. Glutathione S-transferase (GST): Human GST expressed by prokaryotic cell Escherichia coli (E. coli), the amino acid sequence is shown in SEQ ID NO. 1. The GST protein sequence has multiple phosphorylation sites and needs to be phosphorylated to have enzyme activity.

[0031] 5. GST activity detection kit: purchased from Beijing Solebow Technology Co., Ltd. The detection principle is that the substrate labeled with chemiluminescent substances produces chemiluminescence under the action of GST, and the enzyme activity of GST is calculated by detecting the chemiluminescence intensity.

[0032] Example 1: A eukaryotic protein modification system and its preparation method Preparation method of eukaryotic protein modification system Figure 1 shown.

[0033] 1. Eukaryotic cell culture: Select an appropriate culture medium (such as DMEM or RPMI-1640) and inoculate HEK293T cells in a culture flask under sterile conditions. Cultivate the cells to the logarithmic growth phase, which usually takes 48-72 hours, and the cell density should reach 70%-80%. During this stage, regularly replace the culture medium to maintain the cell growth environment, and monitor the cell morphology and growth status to ensure cell health.

[0034] 2. Mutagenesis and screening: 2.1 Mutagenesis treatment. The cells were divided into five groups, each group was inoculated in the same culture medium, and 0 µM, 1 µM, 5 µM, 10 µM and 20 µM cisplatin solutions were added for 24 hours. During the treatment, the cell culture environment was kept at a constant temperature and humidity to ensure the effectiveness of cisplatin.

[0035] 2.2 Cell viability detection. After treatment, the cell viability was assessed using the MTT method or the cell counting method. For the MTT method, MTT solution (0.5 mg / mL) was added to each well and cultured for 4 hours, followed by the addition of DMSO solution to dissolve the purple crystals formed. The absorbance value (OD) was measured at a wavelength of 570 nm using a microplate reader to calculate the survival rate of each group of cells. For the cell counting method, a cell counting plate or an automatic cell counter was used to count the number of live cells in each group of cells.

[0036] 2.3 Selective culture. The treated cells are inoculated in a selective culture medium containing cisplatin at the same concentration as during treatment, and the culture time is 7-14 days. During this period, the growth of cells is regularly observed, and the morphological changes and growth rate of the cells are recorded. Surviving cells that are tolerant to cisplatin are screened out.

[0037] 2.4 Screening of surviving cells. After the selective culture is completed, select the surviving cells for subsequent analysis. The morphology of the cells can be observed under a microscope to ensure that the selected cells are healthy and growing well. Record the number and growth status of surviving cells for subsequent single cell isolation and cloning culture.

[0038] 2.5 Single cell isolation. Use dilution method or flow cytometry to separate surviving mutant cells into single cells. For dilution method, dilute surviving cells to an appropriate concentration (e.g., 1 cell / well) and then inoculate them into a 96-well plate. For flow cytometry, sort the cells using a flow cytometer to ensure that there is only one cell in each well.

[0039] 2.6 Clonal culture. Cultivate the single cells in the 96-well plate until clones are formed. The culture medium should be a suitable complete culture medium, and the culture time is usually 7-14 days. During this period, regularly observe the growth of cells to ensure the formation and growth of clones.

[0040] 2.7 Clone expansion. Select the formed clones for expansion and transfer to larger culture flasks. Each clone (named mHEK293T1, mHEK293T2, mHEK293T3, etc.) continues to be cultured in appropriate culture medium until the required number of cells is reached. Record the growth of each clone and use the expanded cell lines for subsequent analysis and application.

[0041] 3. Lyse cells: The cells were lysed by grinding. Precool the homogenizer and HEPES buffer in ice water for 15 min. Wash the cells twice with HEPES buffer at a concentration of 10 mM and a pH of 7.5. Suspend the cells in 1 ml HEPES buffer by blowing and incubate on ice for 10 min. Put the cell suspension into the homogenizer and push it about 10-15 times to lyse the cells. Incubate the cell lysate in ice water for 20 min and centrifuge at 10,000 xg and 4°C for 10 min. Take the supernatant and transfer it to a precooled Eppendorf tube. This is the cell extract.

[0042] 4. Add supplements: ATP was used as a supplement and added to the aforementioned cell extracts. The ratio of the cell extract to the supplement was 1000:1. A eukaryotic protein modification system (named E-HEK293T, E-mHEK293T1, E-mHEK293T2, E-mHEK293T3...) was obtained.

[0043] Example 2: Preparation of functionalized proteins using eukaryotic protein modification system like Figure 1As shown, to 1-10 ml of the eukaryotic protein modification system constructed in Example 1, equal volumes of GST protein and 0.02-0.2 ml ATP were added and incubated (incubated at 4°C for 12-24 h) to obtain 50-400 μM of phosphorylated GST protein.

[0044] Enzyme activity detection The enzyme activity of the functionalized protein prepared in Example 2 was detected using a GST activity detection kit, and compared with the enzyme activity of the GST protein expressed by prokaryotic cell Escherichia coli (E. coli) that was not treated with the eukaryotic protein modification system, thereby evaluating the modification effect of the eukaryotic protein modification system.

[0045] Test results such as Figure 2 HEK293T, mHEK293T1, mHEK293T2, and mHEK293T3 represent eukaryotic protein modification systems without GST protein, and none of them have GST enzyme activity. Compared with GST (E. coli) protein not treated with the eukaryotic protein modification system, the enzyme activities of GST (E-HEK293T), GST (E-mHEK293T1), GST (E-mHEK293T2), and GST (E-mHEK293T3) treated with the eukaryotic protein modification system are all improved, indicating that the eukaryotic protein modification system can effectively improve the functionality of proteins. Among them, GST (E-mHEK293T1) treated with the mutagenic eukaryotic protein modification system showed a significant improvement in enzyme activity: compared with GST (E-HEK293T) without mutagenesis, its activity increased by 127%; compared with GST (E. coli) expressed from prokaryotic cells, it increased by 502%. This indicates that the cell line with the best phosphorylation modification function in Example 1 is mHEK293T1.

[0046] The above examples demonstrate that the eukaryotic protein modification system of the present invention can combine the rapid protein expression of prokaryotic cells with the efficient protein modification ability of eukaryotic systems, thereby significantly improving the production efficiency of functionalized proteins.

Claims

1. A eukaryotic protein modification system, characterized in that: The invention comprises a cell extract and a supplement, wherein the cell extract is an extract of eukaryotic cells, and the supplement comprises a modification donor and an energy donor.

2. The eukaryotic protein modification system according to claim 1, characterized in that: The eukaryotic cell has been subjected to mutagenesis that enhances protein modification function.

3. The eukaryotic protein modification system according to claim 2, characterized in that: The mutagenesis was performed using cisplatin, fluorouracil, ultraviolet light, colchicine, cyclophosphamide or sodium hydroxide.

4. The eukaryotic protein modification system according to claim 1, characterized in that The eukaryotic cell is at least one of an animal cell, a plant cell, a fungal cell, and a protist cell.

5. The eukaryotic protein modification system according to claim 1, characterized in that: The modification donor is at least one of UDP-glucose, ATP, and S-adenosylmethionine, and the energy donor is ATP or GTP.

6. A method for preparing the eukaryotic protein modification system as claimed in claim 1, characterized in that: In the first step, eukaryotic cells are lysed to obtain cell extracts; in the second step, supplements are added to the cell extracts.

7. The preparation method according to claim 6, characterized in that: In the first step, eukaryotic cells are mutagenized and cells with enhanced protein modification functions are selected for lysis.

8. Use of the eukaryotic protein modification system according to claim 1 in the preparation of functionalized proteins.

9. The use according to claim 8, characterized in that: The functionalization includes spatial folding, glycosylation, phosphorylation, acylation, methylation, ubiquitination, and hydroxylation of proteins.

10. The use according to claim 8, characterized in that: The eukaryotic protein modification system is used to modify the protein expressed by prokaryotic cells to obtain functionalized protein.

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