A method for expressing a purified crystallized CD40L protein and the resulting protein

Abstract

The application discloses a method for expressing and purifying CD40L protein of a crystallization level and the obtained protein. The application realizes efficient soluble expression of the CD40L protein by constructing a CD40L recombinant expression vector, transforming the vector into host cells of Escherichia coli, optimizing induced expression conditions and combining a multi-stage purification process. The obtained protein can form a natural functional trimer, the purity reaches a crystallization level, and qualified X-ray diffraction data can be obtained. The method is simple in operation, low in cost and good in repeatability, solves the technical problems that the CD40L protein expressed by the Escherichia coli is difficult to form a trimer and cannot reach a crystallization level of purity, and provides high-quality protein raw materials and an efficient preparation method for crystal structure research, functional mechanism analysis and related drug research of the CD40L protein.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and protein purification technology, and relates to a method for expressing and purifying crystalline CD40L protein and the resulting protein. Background Technology

[0002] CD40 ligand (CD40L, also known as CD154) is a type II transmembrane glycoprotein belonging to the tumor necrosis factor superfamily. It is mainly expressed on the surface of activated T cells, B cells, and monocytes. By binding to the CD40 receptor on the cell surface, it participates in regulating the activation, proliferation, and differentiation of immune cells, playing a crucial role in immune responses, inflammatory reactions, and tumorigenesis and development. The crystal structure determination of CD40L is fundamental to in-depth research into its interaction mechanism with the CD40 receptor and the development of drugs targeting the CD40 / CD40L pathway.

[0003] Currently, the PDB (Protein Data Bank) database contains seven CD40L crystal structures, and the corresponding CD40L proteins are all prepared using insect cell or yeast expression systems. While insect cell and yeast expression systems can achieve soluble expression of CD40L proteins, they suffer from the following technical drawbacks: First, the expression cost is high; insect cell culture requires specialized culture media and equipment, and the fermentation process of yeast expression systems is complex, making large-scale production difficult. Second, the expression cycle is long; the insect cell expression cycle is typically 30-40 days, and the yeast expression cycle is 3-5 days, much longer than the E. coli expression system. Third, the subsequent purification process is cumbersome; proteins expressed by insect cells and yeast often have complex glycosylation modifications, increasing the difficulty and cost of purification and hindering the large-scale preparation of high-quality CD40L proteins.

[0004] E. coli expression systems offer advantages such as ease of operation, low culture costs, rapid growth, and easy scalability, making them the preferred system for recombinant protein expression. However, CD40L protein is highly hydrophobic and exists in its native trimer form. When using conventional E. coli expression methods, the protein easily forms inclusion bodies, making soluble expression difficult. Even if soluble expression is achieved, it is difficult to form functional trimers, let alone achieve crystal-grade purity, which fails to meet the protein quantity requirements for crystal structure analysis. Therefore, there are currently no reports on the preparation of crystalline CD40L protein using E. coli expression systems.

[0005] Therefore, developing a simple, low-cost method for expressing and purifying E. coli that enables soluble expression of CD40L protein and the formation of trimers, can be purified to the crystal level, and can obtain qualified X-ray diffraction data has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a method for expressing and purifying crystalline CD40L protein and the resulting protein. It aims to overcome the technical limitations of existing technologies, such as the reliance on insect cells or yeast for expression of CD40L crystal structures, high expression costs, complex processes, long cycles, and difficulty in achieving crystalline purity through E. coli expression of CD40L protein in forming trimers. This invention achieves efficient and low-cost preparation of CD40L protein, providing support for its crystal structure research and related drug development.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for expressing and purifying crystalline CD40L protein, the method comprising: cloning the CD40L mature peptide encoding gene into a prokaryotic expression vector to construct a recombinant expression vector; transforming the recombinant expression vector into host cells; screening for positive recombinant engineered bacteria; inducing expression in the positive recombinant engineered bacteria to obtain a fermentation broth containing CD40L protein; lysing the bacterial cells; collecting the supernatant containing CD40L protein; performing multi-stage purification of the CD40L protein by nickel affinity chromatography, cation exchange chromatography, and gel filtration chromatography to obtain CD40L trimer protein; and culturing the purified CD40L trimer protein to obtain CD40L protein crystals; wherein the nucleotide sequence of the CD40L mature peptide encoding gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0009] This invention marks the first successful formation of a natural functional trimer from CD40L protein expressed in *E. coli*, with a purity ≥98%, and yields X-ray diffraction data at a resolution of 3.5 Å. Compared to existing insect cell or yeast expression systems, this invention reduces costs by over 60%, shortens the cycle to 1-2 days, and is simple to operate and easily scaled up. It provides high-quality protein raw materials for CD40L crystal structure analysis and drug screening and vaccine development targeting the CD40 / CD40L pathway. A schematic diagram of the construction of the CD40L recombinant expression vector in this invention is shown below. Figure 1 As shown.

[0010] In the prior art, those skilled in the art generally believe that CD40L protein is strongly hydrophobic and depends on the trimer conformation. Its soluble expression and correct folding require post-translational modification and molecular chaperone assistance from eukaryotic expression systems. However, prokaryotic expression systems (such as Escherichia coli) cannot meet the above requirements. Therefore, it has long been excluded from the candidate schemes for the preparation of crystalline CD40L protein. This invention breaks the technical prejudice that Escherichia coli cannot prepare crystalline CD40L protein and for the first time uses an Escherichia coli expression system to obtain CD40L protein crystal structures that are included in the PDB (Protein Data Bank) database.

[0011] Nucleotide sequence of the gene encoding the mature human CD40L peptide (SEQ ID NO.1):

[0012] atgatcgaaacatacaaccaaacttctccccgatctgcggccactggactgcccatcagcatgaaaatttttatgtatttacttactgtttttcttatcacccagatgattgggtcagcactttttgctgtgtatcttcatagaaggttggacaagatagaagatgaaaggaatcttcatgaagattttgtattcatgaaaacgatacagagatgcaacacaggagaaagatccttatccttactgaactgtgaggagattaaaagccagtttgaaggctttgtgaaggatataatgttaaacaaagaggagacgaagaaagaaaacagctttgaaatgcaaaaaggtgatcagaatcctcaaattgcggcacatgtcataagtgaggccagcagtaaaacaacatctgtgttacagtgggctgaaaaaggatactacaccatgagcaacaacttggtaaccctggaaaatgggaaacagctgaccgttaaaagacaaggactctattatatctatgcccaagtcaccttctgttccaatcgggaagcttcgagtcaagctccatttatagccagcctctgcctaaagtcccccggtagattcgagagaatcttactcagagctgcaaatacccacagttccgccaaaccttgcgggcaacaatccattcacttgggaggagtatttgaattgcaaccaggtgcttcggtgtttgtcaatgtgactgatccaagccaagtgagccatggcactggcttcacgtcctttggcttactcaaactctga。

[0013] SEQ ID NO.2

[0014] MGSSHHHHHHSSGLVPRGSHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGDQNPQIAAHVISE ASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL.

[0015] Preferably, the CD40L mature peptide encoding gene is cloned into a prokaryotic expression vector via NcoI and XhoI restriction sites.

[0016] Preferably, the prokaryotic expression vector includes any one of pET-28a(+), pET-30a(+), or pET-43.1a(+).

[0017] Preferably, the host cell includes any one of Escherichia coli host cell BL21(DE3), Escherichia coli host cell BL21(DE3)pLysS, or Escherichia coli host cell Rosetta(DE3).

[0018] Preferably, the induced expression includes: inoculating positive recombinant engineered bacteria into a basal culture medium, shaking at 35-37℃ (e.g., 35℃, 36℃, or 37℃) and 200-220 rpm (e.g., 200 rpm, 210 rpm, or 220 rpm) until the OD is 0.6-0.8 (e.g., 0.6, 0.7, or 0.8), adding isopropyl-β-D-thiogalactoside to a final concentration of 0.1-0.5 mmol / L (e.g., 0.1 mmol / L, 0.3 mmol / L, or 0.5 mmol / L), and inducing expression at 16-25℃ (e.g., 16℃, 18℃, or 25℃) for 8-16 h (e.g., 8 h, 10 h, or 16 h).

[0019] Preferably, the nickel ion affinity chromatography employs a Ni-NTA affinity chromatography column. The equilibration buffer of the nickel ion affinity layer contains 25-75 mmol / L Tris-HCl (e.g., 25 mmol / L, 50 mmol / L, or 75 mmol / L), 100-200 mmol / L NaCl (e.g., 100 mmol / L, 150 mmol / L, or 200 mmol / L), and 5-15 mmol / L imidazole (e.g., 5 mmol / L, 10 mmol / L, or 15 mmol / L). The pH of the equilibration buffer of the nickel ion affinity layer is 7.0-7.6 (e.g., 7.0, 7.4, or 7.6). The elution buffer of the nickel ion affinity layer contains 25-75 mmol / L Tris-HCl (e.g., 25 mmol / L, 50 mmol / L, or 75 mmol / L), 100-200 mmol / L NaCl (e.g., 100 mmol / L, 150 mmol / L, or 200 mmol / L), and 5-15 mmol / L imidazole (e.g., 5 mmol / L, 10 mmol / L, or 15 mmol / L). The elution buffer for the nickel ion affinity layer has a pH of 7.0-7.6 (e.g., 7.0, 7.4, or 7.6) and 200-300 mmol / L imidazole (e.g., 200 mmol / L, 250 mmol / L, or 300 mmol / L).

[0020] Preferably, the cation exchange chromatography uses a Source 15 S column. The equilibration buffer for the cation exchange chromatography contains 10-30 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), for example, 10 mmol / L, 20 mmol / L, or 30 mmol / L, and the pH of the equilibration buffer is 7.8-8.2, for example, 7.8, 8.0, or 8.2. The elution buffer for the cation exchange chromatography contains 10-30 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) (for example, 10 mmol / L, 20 mmol / L, or 30 mmol / L) and 0.5-2 mol / L NaCl (for example, 0.5 mol / L, 1 mol / L, or 2 mol / L), and the pH of the elution buffer is 7.8-8.2 (for example, 7.8, 8.0, or 8.2), using a 0-1 mol / L... NaCl is used for gradient elution (e.g., 0.1, 0.5 mol / L, or 1 mol / L).

[0021] Preferably, the gel filtration chromatography uses a Superdex 75 gel filtration chromatography column, and the equilibration buffer for the gel filtration chromatography contains 25-75 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) (e.g., 25 mmol / L, 50 mmol / L, or 75 mmol / L), 100-200 mmol / L NaCl (e.g., 100 mmol / L, 150 mmol / L, or 200 mmol / L), and 0.5-2 mmol / L dithiothreitol (DTT) (e.g., 0.5 mmol / L, 1 mmol / L, or 2 mmol / L), and the pH of the equilibration buffer for the gel filtration chromatography is 7.2-7.6 (e.g., 7.2, 7.4, or 7.6).

[0022] Preferably, the disruption of bacterial cells includes ultrasonic disruption, wherein the conditions for ultrasonic disruption are: power 200-300 W (e.g., 200 W, 250 W or 300 W), operation time 2-5 s (e.g., 2 s, 4 s or 5 s), interval 3-8 s (e.g., 3 s, 5 s or 8 s), and total disruption time 20-30 min (e.g., 20 min, 25 min or 30 min).

[0023] Preferably, the crystallization culture comprises: mixing and culturing CD40L protein at a concentration of 5-15 mg / mL (e.g., 5 mg / mL, 10 mg / mL, or 15 mg / mL) with a crystallization-promoting solution at 15-20°C (e.g., 15°C, 18°C, or 20°C) for 7-10 days (e.g., 7 days, 8 days, or 10 days); the crystallization-promoting solution contains: 5%-15% polyethylene glycol (e.g., 5%, 10%, or 15%), 0.1-0.5 mol / L diammonium citrate (e.g., 0.1 mol / L, 0.3 mol / L, or 0.5 mol / L), and 0.05-0.2 mol / L Tris-HCl (e.g., 0.05 mol / L, 0.1 mol / L, or 0.2 mol / L), wherein the pH of the Tris-HCl is 8.4-8.6 (e.g., 8.4, 8.5, or 8.6).

[0024] In a second aspect, the present invention provides a crystalline CD40L trimer protein, which is prepared by the method for expressing and purifying crystalline CD40L protein described in the first aspect.

[0025] Thirdly, the present invention provides the application of the crystalline CD40L trimer protein described in the second aspect in drug screening or vaccine development targeting the CD40 pathway and / or the CD40L pathway.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) This invention breaks through the technical bottleneck of expressing CD40L protein in Escherichia coli and achieves for the first time that CD40L protein expressed in Escherichia coli system can form natural functional trimer, solving the problem that conventional Escherichia coli expression of CD40L protein is easy to aggregate and cannot form trimer. The obtained protein has the same biological activity as natural CD40L.

[0028] (2) This invention achieves crystallization-level purification of CD40L protein. After purification, the protein purity is ≥98%, the uniformity is high, stable crystals can be successfully cultured, and X-ray diffraction data with a resolution of 3.5 Å can be obtained. This fills the technical gap in the expression of crystallization-level CD40L protein in Escherichia coli and breaks the current situation where the CD40L crystal structure depends on insect cells or yeast expression.

[0029] (3) Compared with insect cell and yeast expression systems, the present invention uses Escherichia coli expression system, which reduces the culture cost by more than 60%, shortens the expression cycle to 1-2 days, and is easy to operate and scale up, enabling low-cost, large-scale preparation of CD40L protein, laying the foundation for its industrial application;

[0030] (4) The purification process of the present invention uses multi-stage chromatography in combination. The steps are reasonable and reproducible. It can effectively remove impurities and obtain high-purity and highly uniform CD40L trimer protein. Moreover, no complicated modification or refolding steps are required during the purification process, which further reduces the preparation cost and operation difficulty.

[0031] (5) The crystalline CD40L protein obtained by this invention can be directly used for crystal structure analysis, providing high-quality protein raw materials for in-depth research on the CD40 / CD40L interaction mechanism and revealing the functional sites of CD40L. It also provides important support for drug screening and vaccine development targeting the CD40 / CD40L pathway, and has significant economic value and application prospects. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the construction of the CD40L recombinant expression vector;

[0033] Figure 2 SDS-PAGE electrophoresis image of CD40L protein induced expression; where lane 1 is the protein marker, lane 2 is the supernatant after post-induction sonication, lane 3 is the recombinant bacteria after induction, and lanes 4-5 are the recombinant bacteria before induction.

[0034] Figure 3The image shows an SDS-PAGE electrophoresis result of CD40L protein after multi-stage purification according to the present invention; the left image shows the result of nickel ion affinity chromatography, the middle image shows the result of cation exchange chromatography, and the right image shows the result of gel filtration chromatography.

[0035] Figure 4 This is the cation exchange chromatography pattern of the CD40L protein of the present invention; wherein, the peak represents the CD40L trimer protein;

[0036] Figure 5 This is a gel filtration chromatography pattern of the CD40L protein of the present invention, wherein the peak represents the CD40L trimer protein;

[0037] Figure 6 This is a microscope image of the CD40L protein crystal of the present invention;

[0038] Figure 7 The X-ray diffraction pattern of the CD40L protein of this invention is shown. Detailed Implementation

[0039] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0040] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0041] Example 1

[0042] Construction of CD40L recombinant expression vector.

[0043] (1) Obtaining the human CD40L mature peptide encoding gene: The CD40L mature peptide encoding gene as shown in SEQ ID NO.1 was artificially synthesized, and an NcoI restriction site was added to the 5' end of the gene, and a BamHI restriction site and a stop codon were added to the 3' end.

[0044] (2) Enzyme digestion and ligation: The synthesized CD40L mature peptide encoding gene and pET-28a(+) vector were double-digested with NcoI and XhoI restriction endonucleases respectively at 37℃ for 3 h; the digestion products were separated by agarose gel electrophoresis, and the target gene fragment and vector fragment were recovered by a gel recovery kit; the target gene fragment and vector fragment were ligated overnight at 16℃ using T4 DNA ligase to construct the recombinant expression vector pET-CD40L.

[0045] (3) Vector identification: The ligation product was transformed into DH5α competent cells, spread on LB solid medium containing 50 μg / mL kanamycin, and cultured at 37℃ for 12 h; single colonies were picked and inoculated into LB liquid medium containing kanamycin, and cultured at 37℃ with shaking for 8 h; plasmid was extracted, and double enzyme digestion and sequencing were performed for identification. The sequencing results were consistent with the CD40L mature peptide encoding gene sequence, indicating that the recombinant expression vector was successfully constructed.

[0046] Example 2

[0047] Screening and induction expression of positive recombinant engineered bacteria.

[0048] (1) Transformation: The recombinant expression vector pET-CD40L constructed in Example 1 was transformed into Escherichia coli BL21(DE3) competent cells, and the cells were placed on ice for 30 min, heat-shocked at 42℃ for 90 s, and immediately placed on ice for 2 min. 500 μL of antibiotic-free LB liquid medium was added, and the cells were cultured at 37℃ and 200 rpm for 1 h to obtain the transformed bacterial solution.

[0049] (2) Screening: The transformed bacterial culture was spread on LB solid medium containing 50 μg / mL kanamycin and cultured at 37℃ for 16h. Single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ and 220 rpm for 8h. Plasmids were extracted and enzyme digestion was performed for identification. The positive strains were identified as positive recombinant engineered bacteria.

[0050] (3) Induction of expression: The positive recombinant engineered bacteria were inoculated into 1 L of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ with shaking at 220 rpm until OD. 600 The concentration was 0.7; IPTG was added to a final concentration of 0.3 mmol / L, the culture temperature was adjusted to 20℃, and the culture was continued with shaking for 12 h to obtain the fermentation broth. The induced recombinant bacteria were then subjected to SDS-PAGE electrophoresis, and the results are as follows: Figure 2 As shown.

[0051] Example 3

[0052] Disruption and multi-stage purification of CD40L protein.

[0053] (1) Disruption and supernatant collection: The fermentation broth obtained in Example 2 was centrifuged at 8000 rpm for 10 min at 4℃, and the bacterial precipitate was collected. The bacterial cells were resuspended in 50 mL of buffer (50 mmol / L Tris-HCl, 500 mmol / L NaCl, 1 mmol / L DTT, pH 7.4), and sonicated under ice bath conditions at 250 W for 3 s with 5 s intervals, for a total disruption time of 25 min. After disruption, the mixture was centrifuged at 12000 rpm for 25 min at 4℃, and the supernatant was collected and analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown.

[0054] (2) Nickel ion affinity chromatography: A Ni-NTA affinity chromatography column (size: 10 mL) was used. The column was equilibrated with equilibration buffer (50 mmol / L Tris-HCl, 150 mmol / L NaCl, 10 mmol / L imidazole, pH 7.4) until the baseline was stable. The supernatant was loaded onto the column at a flow rate of 1.5 mL / min. The column was washed with equilibration buffer until the baseline was stable. Elution was performed with elution buffer (50 mmol / L Tris-HCl, 150 mmol / L NaCl, 250 mmol / L imidazole, pH 7.4). The elution peak was collected, and the protein purity in the elution peak was detected by SDS-PAGE electrophoresis. The results are shown below. Figure 3 As shown, the protein bands are single, with no obvious contaminating protein bands, and the purity is ≥98%.

[0055] (3) Cation exchange chromatography: The elution peak collected by affinity chromatography was dialyzed into cation exchange equilibration buffer (20 mmol / L HEPES, pH 8.0) using a dialysis bag (molecular weight cutoff 10 kDa) and dialyzed overnight; a Source15S column (specification: 20 mL) was used and equilibrated with equilibration buffer until the baseline was stable; the dialyzed protein sample was loaded at a flow rate of 1 mL / min; elution was performed with elution buffer (20 mmol / L HEPES, 1 mol / L NaCl, pH 8.0) in a 0-1 mol / L NaCl gradient, and the target protein peak was collected. Figure 4 Purity was determined by SDS-PAGE electrophoresis, and the results are as follows: Figure 3 As shown, the protein bands are single, with no obvious contaminating protein bands, and the purity is ≥98%.

[0056] (4) Gel filtration chromatography: The target protein peak collected by affinity chromatography was concentrated to 10 mg / mL using an ultrafiltration centrifuge tube (molecular weight cutoff 10 kDa); a Superdex 75 gel filtration chromatography column (specification: 16 / 600) was used, and the column was equilibrated with equilibration buffer (50 mmol / L HEPES, 150 mmol / L NaCl, 1 mmol / L DTT, pH 7.4); the concentrated protein sample was loaded at a flow rate of 0.8 mL / min; the elution peak corresponding to a molecular weight of approximately 54 kDa was collected ( Figure 5 This refers to the CD40L trimer protein. Purity was determined by SDS-PAGE electrophoresis, and the results are as follows: Figure 3 As shown, the protein purity is ≥98%.

[0057] Example 4

[0058] The only difference between this embodiment and Example 3 is that nickel ion affinity chromatography is performed first, followed by gel filtration chromatography, and finally cation exchange chromatography. The resulting protein purity is similar to that of Example 3, but the time for protein crystal formation increases from 7 days to 1 month.

[0059] Test Example 1

[0060] Identification and crystallization of CD40L protein.

[0061] (1) Purity detection: The purity of CD40L protein obtained after purification in Example 3 was detected by 12% SDS-PAGE electrophoresis. Coomassie Brilliant Blue R-250 staining and gel imaging analysis showed that the protein band was single and there were no obvious contaminating protein bands, with a purity ≥98%.

[0062] (2) Trimer formation detection: Gel filtration chromatography was used; the gel filtration chromatography spectrum showed that the molecular weight of the main peak of the protein was 54 kDa, which was consistent with the molecular weight of CD40L trimer, and the content of impurity peaks was extremely low; indicating that the obtained protein was a stable trimer structure.

[0063] (3) Crystallization culture and X-ray diffraction: The concentration of purified CD40 L protein was adjusted to 10 mg / mL, and crystallization screening was performed using the sitting drop method; the crystallization conditions were: 20% PEG 3350, 0.2 mol / L diammonium citrate, 0.1 mol / L Tris-HCl (pH 8.5), and static culture at 18℃ for 7 days to obtain transparent CD40 L protein crystals. Figure 6 The crystal was cryopreserved in liquid nitrogen and then sent to a synchrotron radiation source for X-ray diffraction data collection, obtaining X-ray diffraction data with a resolution of 3.5 Å, such as... Figure 7 As shown, the diffraction pattern is clear and can be used for crystal structure analysis.

[0064] In summary, the method of this invention is simple to operate, low in cost, and highly reproducible. It solves the technical problem that E. coli expression of CD40L protein is difficult to form trimers and cannot achieve crystallization-level purity. It provides high-quality protein raw materials and efficient preparation methods for the study of the crystal structure, functional mechanism analysis, and related drug development of CD40L protein.

[0065] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for expressing a purified crystallized CD40L protein, characterized in that, The method for expressing and purifying crystalline CD40L protein includes: cloning the CD40L mature peptide encoding gene into a prokaryotic expression vector to construct a recombinant expression vector; transforming the recombinant expression vector into *E. coli*; screening for positive recombinant engineered bacteria; inducing expression in the positive recombinant engineered bacteria to obtain a fermentation broth containing CD40L protein; lysing the bacterial cells; collecting the supernatant containing CD40L protein; performing multi-stage purification of CD40L protein by nickel affinity chromatography, cation exchange chromatography, and gel filtration chromatography to obtain CD40L trimer protein; and culturing the purified CD40L trimer protein to obtain CD40L protein crystals. The nucleotide sequence of the CD40L mature peptide encoding gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2; The nickel ion affinity chromatography used a Ni-NTA affinity chromatography column; the cation exchange chromatography used a Source15 S chromatography column; and the gel filtration chromatography used a Superdex 75 gel filtration chromatography column.

2. The method for expressing and purifying crystalline CD40L protein according to claim 1, characterized in that, The CD40L mature peptide encoding gene was cloned into a prokaryotic expression vector via NcoI and XhoI restriction sites; the prokaryotic expression vector included any one of pET-28a(+), pET-30a(+), or pET-43.1a(+); the Escherichia coli included any one of Escherichia coli host cell BL21(DE3), Escherichia coli host cell BL21(DE3)pLysS, or Escherichia coli host cell Rosetta(DE3).

3. The method for expressing and purifying crystalline CD40L protein according to claim 1, characterized in that, The induction of expression includes: inoculating positive recombinant engineered bacteria into basal culture medium, incubating at 35-37℃ and 200-220 rpm with shaking until the OD is 0.6-0.8, adding isopropyl-β-D-thiogalactoside to a final concentration of 0.1-0.5 mmol / L, and inducing expression at 16-25℃ for 8-16 h.

4. The method for expressing and purifying crystalline CD40L protein according to claim 1, characterized in that, The equilibration buffer of the nickel ion affinity layer contains 25-75 mmol / L Tris-HCl, 100-200 mmol / L NaCl, and 5-15 mmol / L imidazole, and the pH of the equilibration buffer of the nickel ion affinity layer is 7.0-7.6; the elution buffer of the nickel ion affinity layer contains 25-75 mmol / L Tris-HCl, 100-200 mmol / L NaCl, and 200-300 mmol / L imidazole, and the pH of the elution buffer of the nickel ion affinity layer is 7.0-7.

6.

5. The method for expressing and purifying crystalline CD40L protein according to claim 1, characterized in that, The equilibration buffer for the cation exchange chromatography contains 10-30 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid, and the pH of the equilibration buffer for the cation exchange chromatography is 7.8-8.2; the elution buffer for the cation exchange chromatography contains 10-30 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid and 0.5-2 mol / L NaCl, and the pH of the elution buffer for the cation exchange chromatography is 7.8-8.2, and gradient elution is performed using 0-1 mol / L NaCl.

6. The method of claim 1, wherein the CD40L protein is expressed, purified and crystallized. The equilibration buffer for the gel filtration chromatography contains 25-75 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid, 100-200 mmol / L NaCl and 0.5-2 mmol / L dithiothreitol, and the pH of the equilibration buffer for the gel filtration chromatography is 7.2-7.

6.

7. The method of claim 1, wherein the CD40L protein is expressed, purified and crystallized. The disruption of bacterial cells includes ultrasonic disruption, and the conditions for ultrasonic disruption are: power 200-300W, operation time 2-5 s, interval 3-8 s, and total disruption time 20-30 min.

8. The method for expressing and purifying crystalline CD40L protein according to claim 1, characterized in that, The crystallization culture includes: mixing CD40L protein at a concentration of 5-15 mg / mL with a crystallization-promoting solution and culturing at 15-20°C for 7-10 days; the crystallization-promoting solution contains: 5%-15% PEG, 0.1-0.5 mol / L diammonium citrate and 0.05-0.2 mol / L Tris-HCl, wherein the pH of the Tris-HCl is 8.4-8.

6.

9. A crystalline CD40L trimer protein, characterized in that, The crystalline CD40L trimer protein is prepared by the method for expressing and purifying crystalline CD40L protein as described in any one of claims 1-8.

10. The application of the crystalline CD40L trimer protein of claim 9 in drug screening or vaccine development targeting the CD40 pathway and / or the CD40L pathway.

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