Method for separating and purifying recombinant conopeptide through one-step chromatography and application thereof

Abstract

The invention provides a method for rapidly preparing recombinant conopeptide through one-step chromatography and application. According to the method disclosed by the invention, after fermentation liquor of conopeptide subjected to recombinant expression of a pichia pastoris system is subjected to crude purification, the refined purification of the conopeptide subjected to enzyme digestion is completed only through a one-step cation chromatography method, and the high-purity conopeptide with the purity of 98% or above is obtained. The invention further finds that the conopeptide biosynthesized by using a pichia pastoris expression system has the characteristics of high yield and easiness in large-scale production, the TrxA tag protein is screened and adopted, so that folding and expression of the recombinant conopeptide are efficiently promoted, and the TrxA tag protein has oxidation-reduction activity, so that a suitable environment is provided for formation of disulfide bonds of the conopeptide, and the conopeptide has a good application prospect. The problem of disulfide bond mismatching is obviously reduced. The invention provides an effective solution for biosynthesis and large-scale production of conopeptide.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and pharmaceutical engineering, specifically to a method for separating and purifying recombinant conospirin, and particularly to a one-step chromatographic separation and purification method for recombinant conospirin and its application. Background Technology

[0002] Conopeptides, also known as conotoxins, are small-molecule polypeptide neurotoxins secreted by the venom glands of marine gastropods (Conodontidae family). They hold broad application prospects in the pharmaceutical and cosmetic fields. They have shown great potential in treating neurological disorders, depression, and diabetes, with related drugs (such as ziconopeptide) already successfully marketed and several others in clinical trials. μ-conopeptides, in particular, interfere with nerve signal transmission by blocking the influx of sodium ions into voltage-gated sodium channels, thereby producing potent paralyzing and analgesic effects. Furthermore, μ-conopeptides are often referred to as "topical botox," especially in the cosmetic field, demonstrating significant anti-wrinkle efficacy. However, the biggest limitation to the application of conopeptides is their sourcing.

[0003] Currently, there are two main sources of conotinamide: one is bio-extraction, which involves directly isolating and extracting conotinamide from natural conotinoids. However, this method faces challenges such as resource scarcity, complex processes, extraction difficulties, and high costs, severely limiting yield. The other method is chemical synthesis, which is currently the primary method for obtaining conotinamide. However, large-scale production using chemical synthesis faces numerous technical challenges, such as cumbersome steps, numerous byproducts, low efficiency in achieving correct disulfide bond pairing, and environmental unfriendliness. Furthermore, there are reports of obtaining conotinamide via in vitro enzymatic semi-biosynthesis (CN120591222A) and coliform expression system biosynthesis (CN116355932A). These methods suffer from low yields, disulfide bond mispairing, and the inability to achieve large-scale production.

[0004] For the isolation and purification of conospirin, HPLC is generally required in the art to obtain conospirin with high purity. For example, patent application WO2024114331A1 discloses the purification of conospirin peptides by HPLC reversed-phase column chromatography desalting. However, the HPLC method is expensive and has low throughput. Therefore, there is an urgent need in the art for a simple, high-yield, low-cost, and environmentally friendly method for the isolation and purification of recombinant conospirin. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to overcome the difficulties in scaling up high-purity conospiropeptides using current techniques, and provides a rapid and efficient method for preparing recombinant conospiropeptides. This invention creatively discovers that after crude purification of the fermentation broth from recombinant conospiropeptide expression in a Pichia pastoris system, the enzymatically digested conospiropeptides can be purified using a single-step cation exchange chromatography method, yielding high-purity conospiropeptides with a purity exceeding 98%. This invention further discovers that the biosynthesis of conospiropeptides using the Pichia pastoris expression system has the advantages of high yield and ease of large-scale production. By screening for tag proteins, specifically the TrxA tag protein, not only is the folding and expression of recombinant conospiropeptides efficiently promoted, but the TrxA tag protein itself also possesses redox activity, providing a suitable environment for the formation of disulfide bonds in conospiropeptides, significantly reducing disulfide bond mismatch problems, and providing an effective solution for the biosynthesis and large-scale production of conospiropeptides.

[0006] To achieve the above objectives, the present invention provides a recombinant conotoxin nucleic acid expression cassette, characterized in that it comprises, from the 5' end to the 3' end, the following elements in sequence: a nucleic acid sequence encoding a HIS tag, a nucleic acid sequence encoding a TrxA tag, a nucleic acid sequence encoding an enterokinase cleavage site, and a Conotoxin nucleic acid sequence; wherein, the amino acid sequence of the HIS tag is as shown in SEQ ID NO. 2, the amino acid sequence of the TrxA tag is as shown in SEQ ID NO. 4, the amino acid sequence of the enterokinase cleavage site is as shown in SEQ ID NO. 6, and the amino acid sequence of the Conotoxin is as shown in SEQ ID NO. 8.

[0007] Furthermore, the nucleic acid sequence of the HIS tag is shown in SEQ ID NO. 1, the nucleic acid sequence of the TrxA tag is shown in SEQ ID NO. 3, the nucleic acid sequence of the enterokinase cleavage site is shown in SEQ ID NO. 5, and the nucleic acid sequence of the Conotoxin is shown in SEQ ID NO. 7.

[0008] Furthermore, the nucleic acid sequence of the nucleic acid expression cassette is shown in SEQ ID NO.9; the amino acid sequence encoded by the nucleic acid expression cassette is shown in SEQ ID NO.10.

[0009] Another aspect of the present invention provides a method for constructing a recombinant expression bacterium, characterized in that the nucleic acid expression cassette described in any one of the present invention is used to construct a recombinant expression plasmid by enzyme digestion and ligation or one-step cloning, and then linearized plasmid is obtained by enzyme digestion, and then the linearized plasmid is transformed into competent host cells by electroporation; preferably, the competent host cells are Pichia pastoris competent cells.

[0010] Another aspect of the present invention provides a method for preparing recombinant conotoxin expressed by yeast, characterized in that it includes fermentation using the recombinant expression bacteria constructed by the method of the present invention, digestion with enterokinase to cleave the tag protein and conotoxin, and purification to obtain the recombinant conotoxin; preferably, NTA affinity chromatography is further used for preliminary purification before enterokinase digestion; more preferably, ion exchange chromatography is further used for purification after enterokinase digestion.

[0011] Another aspect of the present invention provides a method for one-step chromatographic separation and purification of recombinant conotinoids, characterized in that the fermentation broth containing the recombinant conotinoid with an amino acid sequence as shown in SEQ ID NO.10 is subjected to crude purification and enterokinase digestion, and then subjected to one-step cation chromatography to obtain a purified sample of the recombinant conotinoid; preferably, the recombinant conotinoid is obtained by expression in a Pichia pastoris system; more preferably, the Pichia pastoris system is a P. astoris X-33 system.

[0012] Further, the crude purity is selected from affinity chromatography; preferably, the affinity chromatography is NTA affinity chromatography; the cation chromatography is selected from NanoGel-50SP-HP cation chromatography; the enterokinase digestion conditions are 5 Units of enterokinase added to each 1 mg of recombinant conospirin, and incubated at 10°C for 30 h.

[0013] Further, the one-step cation chromatography includes the following steps: rinsing the chromatography column with pure water, equilibrating the chromatography column with cation balancing buffer, loading the enterokinase digestion reaction solution, reequilibrating the chromatography column with cation balancing buffer, isocratic elution with cation elution buffer to remove impurities, and then eluting the target protein conospiropeptide with cation elution buffer; preferably, the cation balancing buffer is 20 mM Tris, pH 8.0, and the cation elution buffer is 20 mM Tris + 1 M NaCl, pH 8.0; more preferably, the isocratic elution buffer for removing impurities is a 15% cation elution buffer, and the cation elution buffer for eluting the target protein conospiropeptide is a 35% cation elution buffer.

[0014] Further, the one-step cation chromatography includes the following steps: rinsing the chromatography column with 2-3 column volumes of purified water, then equilibrating the column with 2-3 column volumes of cation equilibration buffer (20 mM Tris, pH 8.0); loading the enterokinase digestion reaction solution at a certain flow rate to ensure sufficient binding of conopeptide with the chromatography packing material; after loading, reequilibrating the column with cation equilibration buffer until the baseline is stable; then isocratic elution with 15% cation elution buffer (20 mM Tris + 1 M NaCl, pH 8.0) to remove impurities, approximately 20 CV; and finally eluting with 35% cation elution buffer to obtain a purified conopeptide sample.

[0015] In another aspect, this invention provides the application of the recombinant conotoxin nucleic acid expression cassette according to any one of the present invention, or the product obtained by the construction method of the recombinant expression bacteria according to the present invention, or the product obtained by the yeast expression method of the present invention, or the product obtained by the one-step chromatographic separation and purification method of the present invention, in the preparation of medical aesthetic products or clinical drugs; preferably, the medical aesthetic product is selected from anti-wrinkle medical aesthetic products and repair medical aesthetic products; preferably, the clinical drug is selected from pain treatment drugs, muscle relaxants, neuroprotective and repair drugs, and hormone regulation and metabolic disease drugs.

[0016] Through the above technical solutions, the expression cassette and separation and purification method of the present invention can achieve the following beneficial effects:

[0017] (1) The recombinant vector provided by the present invention can encode a fusion protein of TrxA-tagged protein and conopodyte, which has the characteristics of high yield and promotes the correct folding of conopodyte disulfide bonds. On the other hand, the recombinant vector provided by the present invention inserts an HIS tag and an enterokinase recognition site, wherein the HIS tag is used for rapid capture of the fusion protein and the enterokinase site is used for cleavage and separation to obtain conopodyte.

[0018] (2) The present invention uses the Pichia pastoris expression system, which significantly improves the yield of conotoxin. Compared with the Escherichia coli expression system, the yield of conotoxin fusion protein is about 1 g / L, while the expression level of Pichia pastoris can be increased to about 5 g / L, which greatly reduces the production cost.

[0019] (3) The present invention completes the purification of enzymatically digested conospirin by one-step cation chromatography, and obtains high-purity conospirin with a purity of over 98%, avoiding the traditional RP-HPLC purification method and significantly reducing the preparation cost.

[0020] (4) LC-MS analysis showed that the molecular weight of the conospirol obtained in this invention was 2391.901 Da (consistent with the theoretical molecular weight of 2.39 kDa) and contained 3 pairs of disulfide bonds. This indicates that the conospirol with high purity and correct structure can be obtained through the technology of this invention.

[0021] (5) The method for rapid preparation of recombinant conotoxin provided by the present invention achieves efficient preparation of conotoxin, with the characteristics of high yield, low cost and green environmental protection, and is suitable for large-scale production of conotoxin. Attached Figure Description

[0022] Figure 1 Map of recombinant conopeptide expression plasmids;

[0023] Figure 2Agarose gel electrophoresis image of the linearized recombinant conospirin expression plasmid;

[0024] Figure 3 Recombinant conopeptide Pichia pastoris expression engineered strain resistance screening plate;

[0025] Figure 4 SDS-PAGE results of SGC screening of engineered strains expressing recombinant conopeptide in Pichia pastoris;

[0026] Figure 5 Ni-NTA chromatography capture of conopeptide fusion protein SDS-PAGE detection results;

[0027] Figure 6 Results of conopeptide fusion protease digestion and conopeptide isolation SDS-PAGE assay;

[0028] Figure 7 Molecular weight determination of conopeptide (LC-MS). Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the content described. The preparation, separation, purification, and detection methods of recombinant conotoxin provided by the present invention, etc., all use commercially available raw materials and reagents. Based on the basic knowledge of conventional genetic engineering, protein engineering, and genetic modification methods in the art, those skilled in the art can implement the methods and embodiments of the present invention.

[0030] The main bottlenecks in the preparation of recombinant conotinoids lie in the correct folding and efficient expression of conotinoids, as well as the cumbersome and time-consuming isolation and purification process. The inventors of this invention discovered in their research that direct expression of mature conotinoids using conventional microbial expression systems (such as *E. coli* and *Pichia pastoris*) is difficult. On the one hand, conotinoids themselves have a small molecular weight (typically containing only 20-30 amino acid residues), making them unsuitable for microbial expression; on the other hand, the unique molecular conformation of conotinoids (rich in polydisulfide bonds) makes correct expression difficult. Although some researchers have achieved the expression of recombinant conotinoids in *E. coli* through fusion expression, it is well known that intracellular expression in *E. coli* has limited yield, making large-scale production difficult. In contrast, secretory expression through *Pichia pastoris* strains offers significant potential for increased yield and is more easily scalable. Furthermore, the inventors of this invention previously learned through experiments that recombinant conotinoids obtained through the *E. coli* expression system exhibit severe isomerism, primarily due to disulfide bond mismatches. Therefore, increasing the yield of recombinant conospirin and reducing the isomerism problem of recombinant conospirin are the main challenges to achieving large-scale production of recombinant conospirin.

[0031] The inventors of this invention discovered that expressing recombinant conotinib using Pichia pastoris strain significantly improves the yield compared to expressing conotinib using Escherichia coli. Furthermore, through screening fusion-promoting tag proteins, the inventors compared different tags, such as the NusA tag, MBP tag, GST tag, SUMO tag, and TrxA tag, and ultimately determined the TrxA tag as the preferred protein tag. This tag protein can effectively solve the expression challenges posed by the small molecular weight and disulfide-rich structure of conotinib, enabling recombinant expression and large-scale production of conotinib.

[0032] Furthermore, this invention directly purifies conospirin using one-step cation chromatography. Compared with traditional purification methods, such as RP-HPLC, the one-step purification of this invention is direct, rapid, more economical and environmentally friendly, and suitable for large-scale production and application.

[0033] Example 1: Design and construction of the modified recombinant conotoxin of the present invention.

[0034] The recombinant conotoxin of this invention is designed to sequentially comprise, starting from the 5' end: a. a nucleotide sequence encoding tag protein 1 (histidine tag, i.e., HIS tag) as shown in SEQ ID NO. 1, and its encoded amino acid sequence as shown in SEQ ID NO. 2; b. a nucleotide sequence encoding tag protein 2 (thioredoxin, i.e., TrxA tag) as shown in SEQ ID NO. 3, and its encoded amino acid sequence as shown in SEQ ID NO. 4; c. a nucleotide sequence encoding a protease recognition site (i.e., enterokinase recognition site) as shown in SEQ ID NO. 5, and its encoded amino acid sequence as shown in SEQ ID NO. 6; d. a nucleotide sequence encoding mature conotoxin as shown in SEQ ID NO. 7, and its encoded amino acid sequence as shown in SEQ ID NO. 8. The nucleotide sequence of the ReConotoxin constructed in this invention is shown in SEQ ID NO. 9, and the amino acid sequence of the ReConotoxin is shown in SEQ ID NO. 10. The codons of the ReConotoxin gene sequence were optimized to the codons preferred by Pichia pastoris. The optimized sequence must avoid the presence of Sac I restriction sites. The obtained optimized sequence is the ReConotoxin gene of SEQ ID NO. 9.

[0035] Table 1 Sequence of the present invention

[0036] name Sequence and number HIS tag (nucleic acid sequence) ATGAGAGGTTCTCACCATCACCATCACCATCAC (SEQ ID NO.1) HIS tag (amino acid sequence) MRGSHHHHHHH (SEQ ID NO.2) TrxA tag (nucleic acid sequence) ATGTCTGATAAAATTATTCACTTGACTGATGATTCTTTTGATACTGATGTTTTGAAAGCTGATGGTGCTATTTTGGTTGATTTTTGGGCTGAATGGTGTGGTCCATGTAAAATGATTGCTCCAATTTTGGATGAAATTGCTGATGAATATCAAGGTAAATTGACT GTTGCTAAATTGAATATTGATCAAAATCCTGGTACTGCTCCAAAATATGGTATTAGAGGTATTCCAACTTTGTTATTGTTTAAAAATGGTGAAGTTGCTGCTACTAAAGTTGGTGCTTTGTCTAAAGGTCAATTGAAAGAATTTTTGGATGCTAATTTGGCT (SEQ ID NO.3) TrxA tag (amino acid sequence) MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLA (SEQ ID NO.4) Enterokinase recognition site (nucleic acid sequence) GATTATAAAGATGACGATGATAAA (SEQ ID NO.5) Enterokinase recognition site (amino acid sequence) DYKDDDDK (SEQ ID NO.6) Mature conospirin (nucleic acid sequence) CAAGGTTGTTGTAATGGTCCAAAAGGTTGTTCTTCTAAATGGTGTAGAGATCATGCTAGATGTTGTTAA (SEQ ID NO.7) Mature conospirin (amino acid sequence) QGCCNGPKGCSSKWCRDHARCC (SEQ ID NO.8) ReConotoxin (nucleic acid sequence) ATGAGAGGTTCTCACCATCACCATCACCATCACATGTCTGATAAAATTATTCACTTGACTGATGATTCTTTTGATACTGATGTTTTGAAAGCTGATGGTGCTATTTTGGTTGATTTTTGGGCTGAATGGTGTGGTCCATGTAAAATGATTGCTCCAATTTTGGATGAAATTGCTGATGAATATCAAGGTAAATTGACTGTTGCTAAATTGAATATTGATCAAAATCCTGGTACTGCTCCAAAATATGGTATTAGAGGTATTCCAACTTTGTTATTGTTTAAAAATGGTGAAGTTGCTGCTACTAAAGTTGGTGCTTTGTCTAAAGGTCAATTGAAAGAATTTTTGGATGCTAATTTGGCTGATTATAAAGATGACGATGATAAACAAGGTTGTTGTAATGGTCCAAAAGGTTGTTCTTCTAAATGGTGTAGAGATCATGCTAGATGTTGTTAA (SEQ ID NO.9) ReConotoxin (Amino Acid Sequence) MRGSHHHHHHHMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLADYKDDDDKQGCCNGPKGCSSKWCRDHARCC (SEQ ID NO.10)

[0037] Example 2: Construction of a recombinant expression plasmid containing the sequence from step 1

[0038] The ReConotoxin gene was synthesized by Genewiz Biotechnology Co., Ltd., and then inserted between 5'-CTCGAGAAAAGA and 3'-GCTGGCGGCCGC in the pPICZα A plasmid vector via enzyme digestion and ligation or one-step cloning. This resulted in the construction of the pPICZα A-ReConotoxin expression plasmid (i.e., ReConotoxin in pPICZα A), as shown in the plasmid map below. ​ As shown.

[0039] Example 3 Construction of recombinant expression strain

[0040] The recombinant plasmid ReConotoxin in pPICZα A was linearized by digestion with Sac I (Quick Cut Sac I, TaKaRa). The linearized recombinant plasmid ReConotoxin in pPICZα A was purified and recovered using a SanPrep column PCR product kit (Sangon Biotech (Shanghai) Co., Ltd.). ​ Agarose gel electrophoresis image of the linearized recombinant conotoxin expression plasmid shows that the linearized recombinant plasmid ReConotoxin in pPICZαA was successfully extracted. 5–20 μg of pre-chilled linearized ReConotoxin in pPICZαA recombinant plasmid (≤10 μL) was added to 80 μL of P. astoris X-33 competent cells, mixed well, and incubated on ice for 5 minutes. The mixture was transferred to a pre-chilled electroporation cuvette, which was then placed in an electroporator (Bio-Rad). The electroporator parameters were set to the Pichia preset, and after one electroporation, the mixture was immediately cooled on ice for 30 seconds, followed by ≥2–3 electroporations. 1 mL of YPD liquid medium was added, and the contents were transferred to a sterile centrifuge tube and incubated at 30°C for 0.5–1.0 h. Spread an appropriate amount of bacterial cell suspension onto a YPD plate containing 200 μg / mL Zeocin resistance. Incubate at room temperature until no liquid flows on the plate, then transfer to a 30°C incubator and incubate upside down for 48–72 h until distinct single-clone colonies appear. Number the single-clone colonies on the transformation plates and transfer them to different Zeocin-resistant YPD plates containing 500 μg / mL, 1000 μg / mL, and 2000 μg / mL, labeling each plate. Incubate the transferred plates upside down at 30°C for 24–48 h until distinct single-clone colonies appear. ​ This is a plate for screening the resistance of engineered bacteria expressing recombinant conopeptide in Pichia pastoris.

[0041] Example 4 Expression of the target protein

[0042] Single colonies were picked sequentially from high-resistance plates to low-resistance plates and inoculated into 5 mL of BMGY liquid medium. The plates were then incubated overnight (~16 h) at 30°C and 250 rpm on a shaker. The OD of the overnight culture was measured. 600 Collect 10 OD 600 Centrifuge at 1500×g for 5 minutes, discard the supernatant, and resuspend the cells in 10 mL of BMMY medium. Incubate on a shaker at 30°C and 250 rpm for 72 hours. Add methanol to a final concentration of 0.5% every 24 hours. After incubation, centrifuge at 12000×g for 5 minutes and collect the supernatant. ​ The SDS-PAGE results for screening the recombinant conopodiopeptide expression in Pichia pastoris X-33 show that the recombinant conopodiopeptide is well expressed in Pichia pastoris. This embodiment demonstrates that the Pichia pastoris expression system used in this invention significantly improves the yield of conopodiopeptide. Yield testing showed that compared to approximately 1 g / L of conopodiopeptide fusion protein expressed by the E. coli expression system, the expression level in Pichia pastoris of this invention can be increased to approximately 5 g / L, greatly reducing production costs.

[0043] Example 5 Purification of the expression product

[0044] The fermentation supernatant was clarified by filtration through a 0.45µm filter or a 0.5µm Nennis filter. The ReConotoxin fusion protein in the clarified filtrate was captured using an AKTApure150 protein purifier and a Nanomicro NW Rose Ni FF chromatography column. The method was as follows: the column was washed with 2-3 column volumes of purified water, and then equilibrated with 2-3 column volumes of Lysis Buffer (20 mM phosphate buffer + 300 mM sodium chloride + 20 mM imidazole + 10% glycerol, pH 8.0). The clarified filtrate was then loaded at a certain flow rate to ensure sufficient binding of the fusion protein to the chromatographic packing material. After loading, the column was reequilibrated with Lysis Buffer until the baseline stabilized. Finally, isocratic elution with Elution Buffer (20 mM phosphate buffer + 300 mM sodium chloride + 300 mM imidazole, pH 8.0) was used to obtain the ReConotoxin fusion protein. The results were analyzed by SDS-PAGE. ​ As shown, lane 5 is the ReConotoxin conotoxin fusion protein, which has a distinct target band at approximately 16 kDa, representing the captured ReConotoxin conotoxin fusion protein of this invention.

[0045] Example 6 Isolation of conopeptide

[0046] Using Cobetter 5 KD, 0.11 m 2 The eluent of the captured conopodipeptide fusion protein was concentrated and replaced with enzyme digestion buffer (20 mM Tris, pH 8.0). An appropriate amount of enterokinase (Shanghai Yaxin, catalog number REK08) was added to the replaced conopodipeptide fusion protein. The preferred digestion conditions were 5 Units of recombinant enterokinase per 1 mg of fusion protein, incubated at 10°C for 30 h. SDS-PAGE analysis showed the following results: ​ As shown in lanes 4 and 5, this invention obtains tag-removed conotoxin after enzymatic digestion.

[0047] Conopeptides in the above-mentioned enzymatic digestion reaction solution were captured using an AKTA pure150 protein purification system and a NanoGel-50SP-HP cationic chromatography column. The method was as follows: the chromatography column was washed with 2-3 column volumes of purified water, and then equilibrated with 2-3 column volumes of cation equilibration buffer (20 mM Tris, pH 8.0). The above-mentioned enzymatic digestion reaction solution was then loaded at a certain flow rate to ensure sufficient binding of the conopeptides to the chromatographic packing material. After loading, the chromatography column was reequilibrated with cation equilibration buffer until the baseline stabilized. Then, isocratic elution was performed with 15% cation elution buffer (20 mM Tris + 1 M NaCl, pH 8.0) to remove impurities until the baseline stabilized (~20 CV). Finally, isocratic elution was performed with 35% cation elution buffer (20 mM Tris + 1 M NaCl, pH 8.0) to obtain high-purity conopeptides. The results were analyzed by SDS-PAGE. ​ As shown, lanes 11-13 contain the isolated conospirin. Samples from lanes 11-13 were combined and analyzed by HPLC, showing a purity of 98.68%. Further analysis by LC-MS... ​ The molecular weight of conospiropeptide was determined by LC-MS. The target molecular weight was 2391.901 Da (consistent with the theoretical molecular weight of 2.39 kDa) and it contained 3 pairs of disulfide bonds. This indicates that the technique of this invention can obtain conospiropeptide with high purity and correct structure.

[0048] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A recombinant conotoxin nucleic acid expression cassette, characterized in that, The sequence from the 5' end to the 3' end includes the following elements in sequence: a nucleic acid sequence encoding an HIS tag, a nucleic acid sequence encoding a TrxA tag, a nucleic acid sequence encoding an enterokinase cleavage site, and a Conotoxin nucleic acid sequence; wherein, the amino acid sequence of the HIS tag is shown in SEQ ID NO. 2, the amino acid sequence of the TrxA tag is shown in SEQ ID NO. 4, the amino acid sequence of the enterokinase cleavage site is shown in SEQ ID NO. 6, and the amino acid sequence of the Conotoxin is shown in SEQ ID NO.

8.

2. The nucleic acid expression cassette according to claim 1, characterized in that, The nucleic acid sequence of the HIS tag is shown in SEQ ID NO. 1, the nucleic acid sequence of the TrxA tag is shown in SEQ ID NO. 3, the nucleic acid sequence of the enterokinase cleavage site is shown in SEQ ID NO. 5, and the nucleic acid sequence of the Conotoxin is shown in SEQ ID NO.

7.

3. The nucleic acid expression cassette according to claim 1 or 2, characterized in that, The nucleic acid sequence of the nucleic acid expression cassette is shown in SEQ ID NO.9; the amino acid sequence encoded by the nucleic acid expression cassette is shown in SEQ ID NO.

10.

4. A method for constructing a recombinant expression bacterium, characterized in that, The nucleic acid expression cassette according to any one of claims 1-3 is used to construct a recombinant expression plasmid by enzyme digestion and ligation or one-step cloning, and then linearized plasmid is obtained by enzyme digestion. The linearized plasmid is then transformed into competent host cells by electroporation. Preferably, the competent host cells are Pichia pastoris competent cells.

5. A method for preparing recombinant conotoxin expressed in yeast, characterized in that, The method includes fermentation using the recombinant expression bacteria constructed according to claim 4, followed by enterokinase digestion to cleave the tag protein and conospirin, and purification to obtain the recombinant conospirin; preferably, NTA affinity chromatography is used for preliminary purification before enterokinase digestion; more preferably, ion exchange chromatography is used for purification after enterokinase digestion.

6. A method for one-step chromatographic separation and purification of recombinant conospirin, characterized in that, Fermentation broth containing recombinant conospirin with an amino acid sequence as shown in SEQ ID NO. 10 is purified crudely and digested with enterokinase, followed by one-step cation exchange chromatography to obtain a purified sample of recombinant conospirin; preferably, the recombinant conospirin is obtained by expression using a Pichia pastoris system; more preferably, the Pichia pastoris system is... P astoris X-33 system.

7. The method according to claim 6, characterized in that, The crude purity is selected from affinity chromatography; preferably, the affinity chromatography is NTA affinity chromatography; the cation chromatography is selected from NanoGel-50SP-HP cation chromatography; the enterokinase digestion conditions are: 5 Units of enterokinase are added to every 1 mg of recombinant conospirin, and incubated at 10°C for 30 h.

8. The method according to claim 6 or 7, characterized in that, The one-step cation chromatography comprises the following steps: rinsing the chromatography column with pure water, equilibrating the chromatography column with cation balancing buffer, loading the enterokinase digestion reaction solution, reequilibrating the chromatography column with cation balancing buffer, isocratic elution with cation elution buffer to remove impurities, and then eluting the target protein conospiropeptide with cation elution buffer; preferably, the cation balancing buffer is 20 mM Tris, pH 8.0, and the cation elution buffer is 20 mM Tris + 1 M NaCl, pH 8.0; more preferably, the isocratic elution buffer for removing impurities is 15% cation elution buffer, and the cation elution buffer for eluting the target protein conospiropeptide is 35% cation elution buffer.

9. The method according to any one of claims 6-8, characterized in that, The one-step cation chromatography includes the following steps: rinsing the chromatography column with 2-3 column volumes of purified water, then equilibrating the column with 2-3 column volumes of cation equilibration buffer (20 mM Tris, pH 8.0); loading the enterokinase digestion solution at a certain flow rate to ensure sufficient binding of conopeptide with the chromatography packing material; after loading, reequilibrating the column with cation equilibration buffer until the baseline is stable; then isocratic elution with 15% cation elution buffer (20 mM Tris + 1 M NaCl, pH 8.0) to remove impurities (approximately 20 CV); and finally eluting with 35% cation elution buffer to obtain a purified conopeptide sample.

10. The application of the recombinant conotoxin nucleic acid expression cassette according to any one of claims 1-3, or the product obtained by the construction method of the recombinant expression bacteria according to claim 4, or the product obtained by the yeast expression method for recombinant conotoxin according to claim 5, or the product obtained by the one-step chromatographic separation and purification method for recombinant conotoxin according to any one of claims 6-9, in the preparation of medical aesthetic products or clinical pharmaceuticals; preferably, the medical aesthetic product is selected from anti-wrinkle medical aesthetic products and repair medical aesthetic products; preferably, the clinical pharmaceuticals are selected from pain treatment drugs, muscle relaxants, neuroprotective and repair drugs, and hormone regulation and metabolic disease drugs.

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