Method for efficiently preparing high-purity NudCD3 protein in in-vitro eukaryotic cells

Abstract

The invention provides a method for efficiently preparing high-purity NudCD3 protein in an in-vitro eukaryotic cell. The method comprises the following steps: synthesizing a target gene; carrying out enzyme digestion on the target gene and the pLEXmMBP vector, and connecting to obtain a recombinant vector; transforming the recombinant vector into competent cells, culturing, and extracting plasmids; transfecting an HEK 293F cell, culturing, and collecting the cell; resuspending the cells in a lysis buffer solution, carrying out ultrasonication and centrifugation, and taking a supernatant; and carrying out affinity chromatography column purification, Prescission protease digestion and molecular sieve purification to finally obtain the NudCD3 protein with the purity of more than 90%. The problems that in the prior art, NudCD3 protein is difficult to express in quantity and insufficient in purity are solved, high-purity protein is provided for structural biology research, drug screening and antibody preparation, and the method has the advantages of being easy to operate, high in yield, high in protein stability and the like.

Description

Technical Field

[0001] This invention relates to the field of protein engineering technology, and in particular to a method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro. Background Technology

[0002] NudCD3 (NudC domain-containing protein 3) is a protein highly expressed in vertebrates, belonging to the NudC family, which contains a characteristic p23 domain. Proteins in this family play a crucial role in a range of responses, including cell division, protein transport, and nuclear localization. Studies have shown that aberrant expression of NudCD3 severely interferes with V(D)J recombination, leading to severe combined immunodeficiency (SCID) and Omenn syndrome (OS). Furthermore, comparisons of cells from thymic carcinoma patients and normal cells revealed that NudCD3 was almost undetectable in the patient's cells. These studies indicate that NudCD3, like other proteins in this family, is closely related to major diseases such as cancer.

[0003] With the increasing application of artificial intelligence in cancer drug development, high-precision protein target structure is crucial for increasing the affinity of small molecule ligands. However, there is currently no way to express NudCD3 in large quantities in eukaryotic cells in vitro, which has kept its full-length protein structure unsolved.

[0004] Therefore, developing a rapid, simple, highly expressive, and highly pure in vitro expression system has significant application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a rapid, simple and efficient method for preparing high-purity NudCD3 protein in eukaryotic cells in vitro.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro includes the following steps: (1) Synthesize the Homo sapiens NudCD3 target gene with Age I restriction site and Xho I restriction site, the sequence of which is shown in SEQ ID NO.1; (2) Insert an 8×His tag, an MBP tag, a Prescission protease site, an AgeI restriction site and an Xho I restriction site into the pLEXm vector to construct the pLEXm_MBP vector; (3) Digest the target gene and the pLEXm_MBP vector with enzymes respectively; (4) The target gene fragment and vector fragment after enzyme digestion were recovered and ligated to obtain the pLEXm_MBP_Homo sapiens NudCD3 recombinant vector; (5) Transform the recombinant vector into DH5α competent cells, select positive transformants after culture, expand the culture and extract plasmids; (6) Culture HEK 293F cells. When the cell density reaches a suitable concentration, mix the plasmid and PolyethylenimineLiner and add it to the HEK 293F cells; collect the cells after culture. (7) Resuspend the collected cells in lysis buffer, sonicate them, centrifuge, and collect the supernatant; (8) The supernatant was purified using an affinity chromatography column, and the eluent was collected; (9) Mix the protein in the elution buffer with Prescission protease and perform enzymatic digestion; (10) The protein after enzyme digestion was purified by molecular sieve to obtain high-purity NudCD3 protein.

[0007] As one of the preferred embodiments of the present invention, in step (2), the 8×His tag, MBP tag, Prescission protease site, Age I restriction site and Xho I restriction site are sequentially inserted at 1767bp of the pLEXm vector.

[0008] As one of the preferred embodiments of the present invention, in step (3), the enzyme digestion system is as follows: substrate up to 1 μg, 10×CutOne Buffer 2 μL, Lightning Xho I 1 μL, Lightning Age I 1 μL, and deionized water to make up to 20 μL.

[0009] As one of the preferred embodiments of the present invention, in step (4), the ligation system is as follows: 20 ng of vector, the molar ratio of target gene to vector is 3:1, 2 μL of 10×T4 Dna Ligest Buffer, 1 μL of T4 Dna ligest (fast), and 20 μL of deionized water; incubate at 22℃ for 30 min.

[0010] As one of the preferred embodiments of the present invention, in step (6), when the cell density reaches 1.5 × 10⁻⁶, 6 ~2.0×10 6 When the cell / mL ratio is 1, the plasmid is mixed with Polyethylenimine Liner at a mass ratio of 1:4, and then allowed to stand for 15-25 min, more preferably 20 min. The cell culture conditions are 37℃, 5% CO2, and the culture time is 72 h.

[0011] As one of the preferred embodiments of the present invention, in step (7), the lysis buffer is a buffer containing 20mM HEPES pH7.5, 500mM KCl, 2mM DTT, 0.5mM EDTA and 1mM PMSF; the ultrasonic disruption power is 10% and the duration is 30min; the centrifugation conditions are 4℃, 16900rpm and the centrifugation time is 1h.

[0012] As one of the preferred embodiments of the present invention, in step (8), the packing material of the affinity chromatography column is Agarose Resin resin, and the elution buffer is a buffer containing 300mM maltose. The buffer composition is: 20mM HEPES pH7.5, 500mM KCl, 2mM DTT, 0.5mM EDTA, and 1mM PMSF.

[0013] As one of the preferred embodiments of the present invention, in step (9), the mass ratio of protein to Prescission protease is 10:1, and the digestion condition is overnight at 4°C.

[0014] As one of the preferred embodiments of the present invention, in step (10), the molecular sieve purification is performed using a Superdex 200Increase 10 / 300 GL chromatography column, and the mobile phase is: 20mM HEPES pH 7.5, 100mM KCl, 2mM DTT, 0.5mM EDTA, and 1% glycerol.

[0015] As one of the preferred embodiments of the present invention, in step (10), before loading the sample, a buffer containing 20 mM HEPES pH7.5, 2 mM DTT, 0.5 mM EDTA and 1% glycerol is added to the protein sample to reduce the salt concentration; then, the sample is centrifuged, and the supernatant is collected for loading at a flow rate of 0.3 mL / min.

[0016] As one of the preferred embodiments of the present invention, in step (10), the purity of the high-purity NudCD3 protein reaches more than 90%.

[0017] The advantages of this invention compared to the prior art are: (1) Achieve efficient preparation and high-purity acquisition of NudCD3 protein: This invention establishes for the first time a complete system for the large-scale expression of NudCD3 protein in eukaryotic cells in vitro. By modifying the pLEXm vector, optimizing transfection and culture conditions, and combining affinity chromatography, Prescission protease cleavage tag, and molecular sieve purification steps, the final NudCD3 protein obtained has a purity of over 90%, and the protein is highly stable and not easily degraded, effectively solving the problems of insufficient purity and difficulty in the large-scale expression of endogenous NudCD3 protein in the prior art.

[0018] (2) Supporting multi-dimensional research and application needs: Laying the foundation for structural biology research, the high-purity and stable NudCD3 protein can be directly used to resolve its full-length protein structure and reveal its functional mechanism; it can also assist in drug development and antibody preparation, meeting the demand for high-precision target structures in AI-assisted drug screening, serving as a targeted screening tool for small molecule ligand screening, and providing high-purity antigens for the development of therapeutic antibodies; and it can support the study of interaction mechanisms, as the constructed recombinant vector can express the NudCD3 protein alone in mammalian cells, and can also be co-transfected with other interacting proteins that depend on eukaryotic cell expression, providing a reliable tool for studying the formation and interaction mechanisms of macromolecular complexes involved by NudCD3.

[0019] (3) It has practicality and expandability: The method of this invention is rapid and easy to operate, and can stably and repeatedly obtain large amounts of high-purity protein. Moreover, the system is adapted to the eukaryotic cell environment, providing a molecular basis for subsequent research on the synergistic effects of NudCD3 with other eukaryotic interacting proteins and the structural analysis of related complexes. It has broad scientific research and application value. Attached Figure Description

[0020] Figure 1 This is the structural spectrum of the pLEXm_MBP vector in Example 1; Figure 2 This is the structural spectrum of the pLEXm_MBP_Homo sapiens NudCD3 recombinant vector in Example 1; Figure 3 These are SDS-PAGE results of the cell culture supernatant, precipitate, and affinity chromatography purified extracts from Example 1; Figure 4 These are SDS-PAGE results before and after enzyme digestion in Example 1; Figure 5 This is a molecular sieve diagram after enzyme digestion in Example 1; Figure 6 This is an SDS-PAGE result diagram of each peak of the molecular sieve in Example 1; Figure 7 This is a graph showing the results of the purification condition optimization in Experiment Example 1. Detailed Implementation

[0021] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, all reagents used in the present invention are commercially available products; and all methods and equipment used are conventional methods and equipment in this technical field, and will not be described in detail further.

[0022] Example 1 This embodiment describes a method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro, comprising the following steps: (1) Obtain the original full gene sequence of Homo sapiens NudCD3 (Gene id: 23386), add Age I at its N end and Xho I restriction site at its C end, and synthesize the corresponding target gene (sequence as shown in SEQ ID NO.1).

[0023] (2) Using the vector pLEXm (Addgene: 99844) as the original vector, an 8×His tag, an MBP tag, a Prescission protease site, an Age I restriction site, and an Xho I restriction site were sequentially inserted at 1767bp to construct the pLEXm_MBP vector ( Figure 1 (As shown).

[0024] (3) The target gene and pLEXm_MBP vector were digested with enzymes respectively. The enzyme digestion reaction system is shown in Table 1.

[0025] Table 1 Enzyme digestion reaction system

[0026] Run the gel using 1% agarose gel, cut out the target fragment, and recover the target fragment using Trans's Easypure QuickGel Extraction Kit.

[0027] (4) The target gene fragment and vector fragment after enzyme digestion were recovered, and ligation reaction was performed according to the ligation system shown in Table 2 (mix thoroughly and centrifuge briefly, incubate at 22℃ for 30 min) to obtain the pLEXm_MBP_Homo sapiens NudCD3 recombinant vector ( Figure 2 (As shown).

[0028] Table 2 Connection System

[0029] (5) Take 10 μL of recombinant vector and add it to 100 μL of DH5α competent cells. Incubate at 4℃ for 25 min, heat shock at 42℃ for 90 s, incubate at 4℃ for 2 min, add 200 μL of antibiotic-free LB (LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L), recover at 37℃ for 1 h, spread on LB plates with 100 μg / mL ampicillin resistance, incubate at 37℃ for 12 h, and select positive transformants.

[0030] Single colonies were picked and placed into 5 mL of LB medium containing ampicillin. After culturing for 12 h, the colonies were added to 300 mL of LB medium containing ampicillin at a ratio of 1:60. The colonies were then incubated at 37 °C for 24 h and collected by centrifugation at 5000 rpm for 10 min.

[0031] Plasmids were extracted using TIANGEN's endotoxin-free plasmid large-scale extraction kit and stored at -20 °C.

[0032] (6) Mammalian HEK 293F cells were cultured using SMM293-TII medium. Before transfection, the cell density was increased to 1.5 × 10⁻⁶ cells / year. 6 ~2.0×10 6 cells / mL. The NudCD3 plasmid obtained in the above steps was filtered through a 0.22 μm filter. 1 mg of NudCD3 plasmid was added to 30 mL of serum-free culture medium, followed immediately by 4 mg of Polyethylenimine Liner (PEI). The mixture was shaken well and incubated at room temperature for 20 min. The mixture was then added to 1 L of HEK 293F cells and cultured at 37 °C with 5% CO2 for 72 h. Cell bodies were collected by centrifugation at 500 g.

[0033] (7) Resuspend the cell bodies from 1L culture in 50mL Buffer C (20mM HEPES pH 7.5, 500mM KCl, 2mM DTT, 0.5mM EDTA, 1mM PMSF) lysis buffer, and sonicate the cell suspension using an ultrasonic homogenizer at 10% power for 30min. Centrifuge the mixture at 4℃ and 16900rpm for 1h, and collect the supernatant for subsequent affinity chromatography purification (SDS-PAGE results of supernatant and precipitate are shown in [reference]). Figure 3 ).

[0034] (8) The chromatography column is 30 mL, and the packing material is Agarose Resin. Specifically, first wet the chromatography column with deionized water, then add 10 mL of packing material to the chromatography column. After the alcohol flows out, add three column volumes of deionized water to wash away the residual alcohol. Equilibrate the column with three column volumes of Buffer C (20 mM HEPES pH 7.5, 500 mM KCl, 2 mM DTT, 0.5 mM EDTA, 1 mM PMSF) for later use.

[0035] Mix the supernatant from step (7) centrifugation with the resin and incubate by rotation for 2 hours. Pour the mixture back into the column, let it stand for 20 minutes to allow the gel to settle, and collect the flow-through sample. Wash the resin with 20 column volumes of Buffer C (20 mM HEPES pH 7.5, 500 mM KCl, 2 mM DTT, 0.5 mM EDTA, 1 mM PMSF). Add 100 μL of Coomassie Brilliant Blue G250 protein detection solution to 10 μL of the flow-through, mix well, and observe whether it turns blue compared to the negative control. If it turns blue, it indicates that some protein has still been washed down, indicating that there are still impurities. Continue washing with Buffer C (20 mM HEPES pH 7.5, 500 mM KCl, 2 mM DTT, 0.5 mM EDTA, 1 mM PMSF) until the detection solution no longer turns blue. Collect the washing sample. The target protein was eluted using elution buffer containing 300 mM maltose (Buffer C + 300 mM maltose). Coomassie Brilliant Blue G250 protein assay was used to check for complete elution. The eluted samples were then collected. All samples were analyzed by SDS-PAGE. The results are shown below. Figure 3 As shown. Combined with Figure 3 As a result, the eluent contained a large amount of the target protein, achieving preliminary protein purification.

[0036] The protein in the eluent was concentrated to 5 mg / mL using a 10000 M WCO concentration tube and stored at -80 °C.

[0037] (9) In order to further improve the purity of the protein, the obtained protein was purified by enzyme digestion and molecular sieve.

[0038] The protein was mixed with Prescission protease at a mass ratio of 1:10 and digested overnight at 4°C (SDS-PAGE analysis before and after digestion is as follows). Figure 4 (as shown), to remove the MBP tag.

[0039] The digested protein was purified by molecular sieving using a Superdex 200 Increase 10 / 300 GL. First, two column volumes were equilibrated with Buffer C (20 mM HEPES pH 7.5, 100 mM KCl, 2 mM DTT, 0.5 mM EDTA, 1% glycerol) at a flow rate of 0.3 mL / min. Next, four volumes of Buffer D (20 mM HEPES pH 7.5, 2 mM DTT, 0.5 mM EDTA, 1% glycerol) were added to the protein sample to reduce the salt concentration. The sample was centrifuged at 12,000 rpm for 20 min at 4°C to remove the precipitate. The sample was loaded using a 2 mL loop at a flow rate of 0.3 mL / min. The buffer was continued as the mobile phase, and peaks P1, P2, and P3 were collected at 0.5 mL / tube and analyzed by SDS-PAGE.

[0040] The results are as follows Figure 5 , 6 As shown in the figure. The results show that P1 is a miscellaneous protein, P3 is an MBP tag, and P2 is the target protein, with a purity of over 90%.

[0041] Experimental Example 1 This experimental example illustrates the optimization process of the buffer formulation used in affinity chromatography and pre-chromatographic cell lysis according to the present invention.

[0042] To improve the yield of NudCD3 protein, the buffer formulations used for affinity chromatography and pre-chromatographic cell lysis were optimized in the early stages of this invention, as follows: 1. Experimental Design: Using the lysis procedure of step (7) and affinity chromatography of step (8) in Example 1, only the lysis buffer and the formulations of the equilibration column, washing resin, and elution buffer were changed to set up three sets of buffers: Buffer A: 20mM Tris-HCl pH 7.5, 500mM KCl; Buffer B: 20mM HEPES pH 7.5, 500mM KCl, 1mM PMSF; Buffer C: 20mM HEPES pH 7.5, 500mM KCl, 2mM DTT, 0.5mM EDTA, 1mM PMSF (same as Example 1).

[0043] 2. Experimental procedure: Cell bodies were resuspended in different lysis buffers (Buffer A / Buffer B / Buffer C), and the cell suspensions were disrupted using an ultrasonic disruptor at 10% power for 30 min. The mixtures were then centrifuged at 16900 rpm for 1 h at 4 °C, and the supernatant was collected for subsequent affinity chromatography purification. After centrifuging 1L of HEK 293F cell culture for each group of experiments (selecting the appropriate buffer according to the corresponding group), affinity chromatography was performed to purify the cells according to the method in step (8) of Example 1. After collecting the eluent, the protein concentration was determined by Coomassie Brilliant Blue method and detected by SDS-PAGE.

[0044] 3. Experimental Results: The results are as follows Figure 7 As shown in the figure. The results showed that the highest concentration of NudCD3 protein, approximately 21 mg / mL, was obtained by cleavage and purification using Buffer C; followed by Buffer B, and the lowest concentration was obtained by Buffer A. SDS-PAGE analysis showed that the target protein band was clear in the Buffer C group, with fewer contaminating proteins, suggesting that Buffer C can significantly improve protein yield and initial purity.

[0045] 4. Conclusion: Adding DTT (reducing agent), EDTA (metal ion chelator), and PMSF (protease inhibitor) to the buffer can reduce protein oxidation, degradation, and non-specific binding. The optimized Buffer C is the best choice for affinity chromatography and pre-chromatographic cell lysis.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro, characterized in that, Includes the following steps: (1) Synthesize the Homo sapiens NudCD3 target gene with Age I restriction site and Xho I restriction site, the sequence of which is shown in SEQ ID NO.1; (2) Insert an 8×His tag, an MBP tag, a Prescission protease site, an Age I restriction site and an Xho I restriction site into the pLEXm vector to construct the pLEXm_MBP vector; (3) Digest the target gene and the pLEXm_MBP vector with enzymes respectively; (4) The target gene fragment and vector fragment after enzyme digestion were recovered and ligated to obtain the pLEXm_MBP_Homosapiens NudCD3 recombinant vector; (5) Transform the recombinant vector into DH5α competent cells, select positive transformants after culture, expand the culture and extract plasmids; (6) Culture HEK 293F cells. When the cell density reaches a suitable concentration, mix the plasmid and PolyethylenimineLiner and add it to the HEK 293F cells; collect the cells after culture. (7) Resuspend the collected cells in lysis buffer, sonicate them, centrifuge, and collect the supernatant; (8) The supernatant was purified using an affinity chromatography column, and the eluent was collected; (9) Mix the protein in the elution buffer with Prescission protease and perform enzymatic digestion; (10) The protein after enzyme digestion was purified by molecular sieve to obtain high-purity NudCD3 protein.

2. The method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro according to claim 1, characterized in that, In step (2), the 8×His tag, MBP tag, Prescission protease site, Age I restriction site and Xho I restriction site are inserted sequentially at 1767bp of the pLEXm vector.

3. The method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro according to claim 1, characterized in that, In step (3), the enzyme digestion system is as follows: substrate up to 1 μg, 10×CutOne Buffer 2 μL, Lightning Xho I 1 μL, Lightning Age I 1 μL, and deionized water to make up to 20 μL.

4. The method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro according to claim 1, characterized in that, In step (4), the ligation system is as follows: 20 ng of vector, the molar ratio of target gene to vector is 3:1, 2 μL of 10×T4Dna Ligest Buffer, 1 μL of T4 Dna ligest (fast), and 20 μL of deionized water; incubate at 22℃ for 30 min.

5. The method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro according to claim 1, characterized in that, In step (6), when the cell density reaches 1.5 × 10⁻⁶, 6 ~2.0×10 6 When the cell / mL ratio is 1, mix the plasmid with Polyethylenimine Liner at a mass ratio of 1:4 and let it stand for 15-25 min. The cell culture conditions are 37℃, 5% CO2, and culture time is 72 h.

6. The method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro according to claim 1, characterized in that, In step (7), the lysis buffer is a buffer containing 20mM HEPES pH 7.5, 500mM KCl, 2mM DTT, 0.5mM EDTA, and 1mM PMSF; the ultrasonic disruption power is 10% and the duration is 30min; the centrifugation conditions are 4℃, 16900rpm, and the centrifugation time is 1h.

7. The method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro according to claim 1, characterized in that, In step (8), the affinity chromatography column is filled with Agarose Resin resin, and the elution buffer is a buffer containing 300mM maltose. The buffer composition is: 20mM HEPES pH 7.5, 500mM KCl, 2mM DTT, 0.5mM EDTA, and 1mM PMSF.

8. The method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro according to claim 1, characterized in that, In step (9), the mass ratio of protein to Prescission protease is 10:1, and the digestion condition is overnight at 4°C.

9. The method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro according to claim 1, characterized in that, In step (10), molecular sieve purification is performed using a Superdex 200 Increase 10 / 300 GL chromatography column. The mobile phase is: 20mM HEPES pH 7.5, 100mM KCl, 2mM DTT, 0.5mM EDTA, and 1% glycerol. Before loading, a buffer solution containing 20mM HEPES pH 7.5, 2mM DTT, 0.5mM EDTA, and 1% glycerol is added to the protein sample. After centrifugation, the supernatant is collected for loading at a flow rate of 0.3mL / min.

10. The method for efficiently preparing high-purity NudCD3 protein in eukaryotic cells in vitro according to claim 1, characterized in that, In step (10), the purity of the high-purity NudCD3 protein reaches over 90%.

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