Linaclotide soluble intermediate and preparation method for linaclotide

Abstract

The present invention provides a linaclotide soluble intermediate and a preparation method for linaclotide. The preparation method comprises: using a host cell containing a recombinant plasmid to express a linaclotide soluble intermediate, wherein the recombinant plasmid contains a gene capable of expressing Sumo tag protein and a gene capable of expressing linaclotide; and the host cell comprises a Shuffle T7-B cell or an Origami B(DE3) cell. The present invention can solve the problem in the prior art of low activity of linaclotide prepared, and is applicable to the field of polypeptide drug biosynthesis.

Description

Linaclotide soluble intermediate and preparation method of linaclotide

[0001] This application is based on the Chinese application with CN application number 202410229264.8 and application date February 29, 2024, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of drug biosynthesis, and in particular to a method for preparing a soluble intermediate of linaclotide and linaclotide. Background Art

[0003] Linaclotide is a GC-C (guanylate cyclase-C) agonist drug that can be used clinically for the treatment of adult patients with constipation-predominant irritable bowel syndrome (IBS-C) and chronic idiopathic constipation (CIC).

[0004] Currently, linaclotide is primarily obtained through chemical conjugation synthesis and inclusion body expression. Chemical conjugation synthesis is a relatively complex process and is prone to impurities during synthesis, resulting in relatively low purity and recovery. While inclusion body expression provides high expression levels, preventing protease degradation, subsequent denaturation and renaturation purification are required to obtain the final product. This process requires the use of large amounts of denaturants such as urea or guanidine hydrochloride, complicates the purification process, and can easily cause internal folding of linaclotide, resulting in low activity.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a soluble intermediate of linaclotide and a method for preparing linaclotide, so as to solve the problem of low activity of linaclotide prepared in the prior art.

[0007] To achieve the above objectives, according to one aspect of the present invention, a method for preparing a soluble intermediate of linaclotide is provided, the preparation method comprising: expressing the soluble intermediate of linaclotide using a host cell containing a recombinant plasmid, wherein the recombinant plasmid contains a gene capable of expressing a Sumo-tagged protein and a gene capable of expressing linaclotide; the host cell comprises a Shuffle T7-B cell or an Origami B (DE3) cell.

[0008] Furthermore, the recombinant plasmid contains a gene capable of expressing Sumo tag protein and a gene capable of expressing linaclotide, which are sequentially connected in the 5'-3' direction.

[0009] Furthermore, the gene capable of expressing the Sumo tag protein includes a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide having more than 70% identity with the nucleotide sequence shown in SEQ ID NO: 1 and containing a disulfide bond structure; the gene capable of expressing linaclotide includes a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2, or a polynucleotide having more than 70% identity with the nucleotide sequence shown in SEQ ID NO: 2.

[0010] Furthermore, the recombinant plasmid contains the nucleotide sequence shown in SEQ ID NO: 3.

[0011] In order to achieve the above-mentioned object, according to a second aspect of the present invention, a method for preparing linaclotide is provided, which comprises: obtaining linaclotide by removing the Sumo tag protein from the soluble intermediate of linaclotide obtained by any of the above-mentioned methods for preparing the soluble intermediate of linaclotide.

[0012] Furthermore, the Sumo tag protein on the soluble intermediate of linaclotide is removed to obtain crude linaclotide; and the crude linaclotide is purified to obtain linaclotide.

[0013] Furthermore, the excision includes: using an enzyme to cut the soluble intermediate of linaclotide to obtain a separated Sumo-tagged protein and a crude linaclotide.

[0014] Furthermore, the purification includes: adjusting the pH of the system containing the crude linaclotide to the isoelectric point of the Sumo-tagged protein to obtain a tagged protein precipitation system; mixing and centrifuging the tagged protein precipitation system with an organic solution, and separating the supernatant to obtain linaclotide.

[0015] Furthermore, the isoelectric point is pH 5.4-5.8, more preferably pH 5.6.

[0016] Furthermore, the organic solution includes one or more of acetonitrile solution, hexafluoroisopropanol solution or acetone solution; preferably, the acetonitrile solution is an acetonitrile aqueous solution with an acetonitrile volume fraction of 50%-70%, more preferably 60% acetonitrile aqueous solution.

[0017] The technical solution of the present invention provides a method for preparing a soluble intermediate of linaclotide, and constructs a recombinant plasmid containing a gene capable of expressing a Sumo-tagged protein and a gene capable of expressing linaclotide. The soluble intermediate of linaclotide prepared by this preparation method has good solubility. In the subsequent preparation of linaclotide, there is no need to denature, renature or reduce linaclotide by adding components such as protein denaturants, renaturing agents or reducing agents, and the final product linaclotide has high activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0019] FIG1 shows the electrophoresis detection results of the precipitate and supernatant of the strain expressing the soluble intermediate of linaclotide according to Example 1 of the present invention after induced centrifugation.

[0020] FIG2 shows the electrophoresis detection results of the purified fusion protein containing the soluble intermediate of linaclotide according to Example 1 of the present invention.

[0021] FIG3 shows the electrophoresis detection results of the purified soluble intermediate of linaclotide according to Example 1 of the present invention.

[0022] FIG4 shows a graph showing the mass spectrum results of LC-MS detection of linaclotide according to Example 1 of the present invention. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0024] As mentioned in the background art, the prior art uses chemical synthesis methods for the preparation of linaclotide. This is not only complex and costly, but also may produce racemates, resulting in a long production cycle and difficult product quality control. When linaclotide is expressed via fusion protein preparation, its expression level is low and inclusion bodies are easily formed. Misfolded inclusion bodies require correction and purification by adding protein denaturants and renaturing agents, resulting in a complex preparation process and low activity of the final product, linaclotide. In this application, the inventors attempted to develop a soluble intermediate of linaclotide and a method for its preparation, thus proposing a series of protection schemes herein.

[0025] In a first typical embodiment of the present application, a method for preparing a soluble intermediate of linaclotide is provided, which comprises: expressing the soluble intermediate of linaclotide using a host cell containing a recombinant plasmid, wherein the recombinant plasmid contains a gene capable of expressing a Sumo tag protein and a gene capable of expressing linaclotide; the host cell comprises a Shuffle T7-B cell or an Origami B (DE3) cell.

[0026] Protein tag refers to the use of gene cloning to fuse polypeptides, protein domains or complete proteins with specific functions with target proteins to achieve application technologies such as expression, purification, detection and tracing of target proteins. Sumo (Small ubiquitin-like modifier) ​​tag protein is a small molecule ubiquitin-like modified protein. Sumo tag protein can be used as a protein tag and molecular chaperone for recombinant protein expression to increase the solubility, stability and purity of the target protein. The present application achieves efficient soluble expression of polypeptides containing three pairs of disulfide bonds by fusing Sumo tag protein with linaclotide, thereby improving the soluble expression of linaclotide.

[0027] Inclusion bodies are high-density, membrane-enclosed, insoluble protein particles that form when exogenous genes are expressed in prokaryotic cells. Their formation is related to the rate of protein production in the cytoplasm. Newly produced polypeptides are concentrated at high concentrations and lack sufficient time to fold, leading to the formation of non-crystalline, amorphous protein aggregates. Therefore, existing methods typically require the addition of protein denaturants and renaturing agents to correct and renature these misfolded inclusion bodies, and purification is required to remove excess reagents before linaclotide can be produced.

[0028] To increase linaclotide production, the prior art typically uses methods to increase the number of linaclotide copies (including but not limited to using a fusion tag to express multiple target proteins in tandem) to prepare fusion proteins. However, there is no mention of achieving soluble expression of fusion proteins obtained by co-expressing multiple copies of linaclotide with a protein tag. Furthermore, prior art methods require that all tandemly expressed fusion proteins be treated with urea, indicating that fusion proteins expressed using this method are insoluble and require treatment with a protein denaturant. Because linaclotide contains multiple pairs of disulfide bonds and is prone to cyclization, this often results in low purification efficiency of the resulting polypeptide using Q Sepharose. To avoid this, prior art methods require linearization of the fusion protein to eliminate the effects of cyclization of the internal disulfide bonds of linaclotide, followed by purification. The purified linearized linaclotide is then subjected to cyclization to obtain the final product. Although the existing technology avoids the internal disulfide cyclization of linaclotide, the reactants undergo multiple denaturation and purification to obtain linaclotide, so the activity of the final product is also low. In addition, this method has many purification times, cumbersome steps, high technical requirements for operators, and relatively high preparation costs.

[0029] In the examples of this application, multiple tags, including Sumo (Small ubiquitin-like modifier), Trx (Thioredoxin), Fh8 (Fasciola hepatica putative calcium-binding protein), Ffu209 (Fluorescent Fusion Ubiquitin 209), and CBM (Carbohydrate-Binding Module), were fused to the target polypeptide sequence and constructed into the pET-28a(+) expression vector. The pET-28a-Sumo, pET-28a-Trx, pET-28a-Fh8, pET-28a-Ffu209, and pET-28a-CBM vector backbones were constructed and linked to genes capable of expressing linaclotide, respectively, and then transformed into Shuffle T7-B, origamiB(DE3), and BL21(DE3) host cells for expression. Experiments have shown that, compared with other tags, Sumo, when linked to the linaclotide gene, can express a more soluble fusion protein in shuffleT7-B cells without forming inclusion bodies, thus avoiding the problem of inclusion body denaturation and renaturation. Furthermore, the expression level is relatively high, which can effectively promote the soluble expression of the protein. Therefore, there is no need to add protein denaturants and renaturing agents, and a soluble linaclotide intermediate can be directly prepared. Then, through a simple purification step, the highly active and high-purity target product, linaclotide, can be obtained.

[0030] In a preferred embodiment, the recombinant plasmid contains a gene capable of expressing a Sumo tag protein and a gene capable of expressing linaclotide, which are sequentially linked in the 5'-3' direction.

[0031] The gene capable of expressing the Sumo-tagged protein is connected to the gene capable of expressing linaclotide, and a soluble linaclotide intermediate formed by the connection of the Sumo protein and linaclotide can be obtained in the subsequent expression. In the above intermediate, the Sumo tag is located at the N-terminus of linaclotide.

[0032] Preferably, on one recombinant plasmid, one gene capable of expressing a Sumo-tagged protein is linked to only one gene capable of expressing linaclotide.

[0033] In a preferred embodiment, the gene capable of expressing the Sumo tag protein includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, or a polynucleotide having greater than 70% identity to the nucleotide sequence of SEQ ID NO: 1 and containing a disulfide bond structure; the gene capable of expressing linaclotide includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, or a polynucleotide having greater than 70% identity to the nucleotide sequence of SEQ ID NO: 2.

[0034] SEQ ID NO: 1:

[0035] SEQ ID NO: 2:

[0036] In a preferred embodiment, the recombinant plasmid contains the nucleotide sequence shown in SEQ ID NO: 3.

[0037] SEQ ID NO: 3:

[0038] Identity in this application refers to the "identity" between amino acid sequences or nucleic acid sequences, that is, the total ratio of amino acid residues or nucleotides of the same type in the amino acid sequence or nucleic acid sequence. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0039] The above proteins have 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity with the amino acid sequence shown in SEQ ID NO: 2 and have the same function, and their active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as those of the proteins provided by the above sequences.

[0040] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0041] Substitution and replacement rules generally state that amino acids with similar properties will have similar effects after substitution. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0042] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0043] The hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0044] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0045] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0046] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0047] By using the above preparation method, a soluble intermediate of linaclotide can be obtained, which includes Sumo-tagged protein and linaclotide.

[0048] The Sumo tag protein comprises the amino acid sequence set forth in SEQ ID NO: 4, or a polypeptide having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 4 and containing a disulfide bond; linaclotide comprises the amino acid sequence set forth in SEQ ID NO: 5, or a polypeptide having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 5. The soluble intermediate of linaclotide comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0049] SEQ ID NO: 4:

[0050] SEQ ID NO: 5:

[0051] SEQ ID NO: 6:

[0052] In a second typical embodiment of the present application, a method for preparing linaclotide is provided, which comprises: obtaining linaclotide by removing the Sumo tag protein from the soluble intermediate of linaclotide prepared by any of the above preparation methods.

[0053] In a preferred embodiment, the Sumo tag protein on the soluble intermediate of linaclotide is removed to obtain crude linaclotide; and the crude linaclotide is purified to obtain linaclotide.

[0054] In a preferred embodiment, the cleavage comprises: using an enzyme to cleave a soluble intermediate of linaclotide to obtain a separated Sumo-tagged protein and a crude linaclotide.

[0055] Because linaclotide contains multiple pairs of cysteines, disulfide bonds easily form within it, which affects the effectiveness of protease cleavage. Therefore, it is difficult to effectively separate the tagged protein from linaclotide when preparing a soluble linaclotide intermediate. In the prior art, crude linaclotide is isolated and purified by adding a reducing agent for decyclization and then enzymatic cleavage. The purified linear polypeptide then needs to be recyclized to form the functional target polypeptide. This method has many steps and requires complex process flows such as decyclization and cyclization, which can easily lead to reduced or even loss of polypeptide activity, and may also result in reduced activity and purity. In the present application, the tagged protein is cleaved without the addition of a reducing agent. The target polypeptide, linaclotide, can be obtained through a single enzymatic cleavage and purification step. Furthermore, the purification method utilizes an organic solvent for precipitation, eliminating the need for column chromatography and other methods to achieve separation and purification of the target polypeptide. The purification steps are simple, the cost is low, and it is conducive to industrial scale-up production. Compared with the prior art, the above preparation method has fewer steps, simpler operation, higher efficiency of enzymatic cleavage and purification, and can prepare highly active and high-purity linaclotide.

[0056] Here, the protease refers to an enzyme that can act on the Sumo protein tag and separate it from the target polypeptide. Such enzymes include but are not limited to Ulp1 enzyme.

[0057] In a preferred embodiment, the purification comprises: adjusting the pH of the system containing the crude linaclotide to the isoelectric point of the Sumo-tagged protein to obtain a tagged protein precipitation system; mixing and centrifuging the tagged protein precipitation system with an organic solution, and separating the supernatant to obtain linaclotide.

[0058] In a preferred embodiment, the isoelectric point is pH 5.4-5.8, including but not limited to 5.4, 5.5 or 5.6; more preferably pH 5.6.

[0059] In a preferred embodiment, the organic solution includes but is not limited to one or more of an acetonitrile solution, a hexafluoroisopropanol solution or an acetone solution; preferably, the acetonitrile solution is an acetonitrile aqueous solution with a volume fraction of 50%-70%, including but not limited to 50%, 55%, 60%, 65% or 70%, more preferably a 60% acetonitrile aqueous solution.

[0060] In the present application, the pH is adjusted to the isoelectric point of the Sumo-tagged protein to precipitate it, and the organic solvent acetonitrile precipitation method is used at the same time. The two methods are combined to separate the Sumo tag from linaclotide to obtain the final product linaclotide. The product prepared by the purification method of the present application has high purity and activity, and the yield is relatively high. The use of organic solvents to precipitate and separate linaclotide makes the preparation process simple and efficient, and reduces costs.

[0061] In the above preparation method, crude linaclotide is treated with an organic solvent such as acetonitrile, and the target product, linaclotide, is isolated by precipitating the tag protein. This avoids the need for purification methods such as chromatography, resulting in a simple purification process, low target product loss, and high activity of the final product, linaclotide.

[0062] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0063] Example 1

[0064] A. Construction of a genetically engineered strain expressing Sumo-tag fusion linaclotide

[0065] The DNA capable of expressing the Sumo protein tag and the DNA sequence capable of expressing the target polypeptide were fused to construct an expression vector in the pET-28a(+) plasmid.

[0066] 1. Connect the Sumo protein tag to the pET-28a(+) plasmid to construct the pET-28a-Sumo vector backbone.

[0067] 2. The target polypeptide sequence is shown in SEQ NO: 1.

[0068] The DNA capable of expressing the target polypeptide fragment was ligated to the pET-28a-Sumo vector backbone by homologous recombination. The ligation product was transformed into Shuffle T7-B (purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., catalog number: zc1229-2). Monoclonal sequencing analysis was performed to screen out the strain containing the correct cloned expression vector pET-28a-Sumo-lina (lina is the abbreviation of Linaclotide).

[0069] 3. Pick three of the above strains and inoculate them in 250mL shake flasks for screening. After the different clones are broken, centrifuged, and the sediment is detected by SDS-PAGE. The groups with better expression are screened by the depth of the electrophoretic bands, and the final expression strains of each type of bacteria are screened. Take 4mL of the strain containing the recombinant plasmid and inoculate it into a 2L Erlenmeyer flask containing 400mL of LB medium. Cultivate at 37℃ and shake at 200rpm until the OD600 reaches 1.0. Add IPTG to a final concentration of 0.2mM and induce at 25℃ overnight. After induction, collect the cells by centrifugation. Disrupt by ultrasound, collect the supernatant, and detect it on 17% separating gel SDS-PAGE. The electrophoresis detection results are shown in Figure 1, where lane M represents the marker band, lane 1 represents the band for electrophoresis detection of the supernatant, and lane 2 represents the band for electrophoresis detection of the precipitate. The band indicated by the arrow represents the position of the target protein Sumo-lina fusion protein in the lane. Sumo-lina in the figure is the soluble intermediate of Sumo tag protein-linaclotide.

[0070] B. Purification of Fusion Protein

[0071] 1. The expressed Sumo-lina strain was resuspended at 20% bacterial concentration and ultrasonically disrupted (5s ultrasonication, 5s interval, 30% power), then centrifuged and the supernatant was filtered through a 0.45 μm filter to obtain crude enzyme solution (crude protein).

[0072] 2. Purify the obtained crude enzyme solution (crude protein) by affinity chromatography (AKTA system equipped with 5 mL HisTrap HP):

[0073] The membrane filtered sample was loaded at a flow rate of 5 mL / min and then rinsed with binding buffer (50 mM Tris-Hcl, 200 mM NaCl, pH 8.0) until the unbound protein was completely eluted. 4 column volumes of miscellaneous proteins were eluted using a buffer solution of 50 mM Tris-Hcl, 200 mM NaCl, 50 mM imidazole at pH 8.0. Finally, the target protein was eluted in an elution buffer of 50 mM Tris-Hcl, 200 mM NaCl, 300 mM imidazole at pH = 8.0. The detection result is shown in FIG2 , wherein lane M represents the Marker band, lane 1 represents the band after electrophoresis detection of the eluted target protein, and the band indicated by the arrow represents that the target protein contains Sumo-lina fusion protein. The Sumo-lina in the figure is a soluble intermediate of Sumo-tagged protein-linaclotide.

[0074] It is calculated that 1g of bacterial mud can produce 40.5mg of purified Sumo-lina fusion protein (which can be further increased after condition optimization).

[0075] C. Purification of Linaclotide

[0076] Purified Sumo-Lina was digested with Ulp1 at a mass ratio of 1:40 (mg / mg) for 16 h at 30°C. The pH was then adjusted to 5.6 with hydrochloric acid, and 60% acetonitrile was added to the reaction system. After mixing, the mixture was shaken at 30°C for 2 h. The supernatant and precipitate were separated by centrifugation at 12,000 rpm. The peptide separation was analyzed by 17% SDS-PAGE. The results are shown in Figure 3 , where lane M represents the marker band, lane 1 represents the band detected by electrophoresis of the supernatant obtained after the purification and separation of the Sumo-lina fusion protein. The band indicated by the arrow represents the presence of linaclotide in the separated supernatant; lane 2 represents the band detected by electrophoresis of the precipitate obtained after the purification and separation of the Sumo-lina fusion protein. The band indicated by the arrow represents the presence of Sumo-tagged protein in the precipitate. In this figure, lina refers to linaclotide, and Sumo refers to Sumo-tagged protein.

[0077] pH 5.6 is the isoelectric point of His-sumo. In the above preparation method, since pET28a(+) contains a His tag, the protein actually expressed is His-Sumo-linaclotide.

[0078] D. Mass spectrometry detection of peptide molecular weight

[0079] The molecular weight of the prepared peptides was analyzed by LC-MS.

[0080] 1. The sample was first separated by HPLC using an Agilent ZORBAX Edipse Plus C18 column, 4.6×100 mm, 3.5 μm. Mobile phase A was 0.1% trifluoroacetic acid, and mobile phase B was 0.1% trifluoroacetic acid in acetonitrile. The gradient elution mode was as follows: 10% B at 0 min, 95% B at 9 min, 100% B at 12 min, 10% B at 12.1 min, and 10% B at 15 min. The column temperature was 50°C, the UV detector was at 210 nm, and the flow rate was 0.3 mL / min.

[0081] 2. The components after HPLC separation were detected using a Q Exactive HF combined quadrupole Orbitrap mass spectrometer using an electrospray ionization source (Dual AJS ESI) in positive ion mode, with a sheath gas flow rate of 35 arb, an auxiliary gas flow rate of 8 arb, a spray voltage of 3800 V, an ion transfer tube temperature of 320 ° C, a scan range of 200-3000 m / z, and the mass spectrometry data were processed by BioPharma Finder software. Finally, the theoretical molecular weight of linaclotide is 1526.8 Da, and the molecular weight of linaclotide analyzed by mass spectrometry is 1526.4. The mass spectrum is shown in Figure 4, where Linaclotide is linaclotide. The expression amount of protein tag-linaclotide in this example is shown in Table 1.

[0082] Example 2

[0083] The difference from Example 1 is that the host cell is Origami B (DE3) (purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number: EC1020), and the remaining steps are the same as Example 1. The expression level of protein tag-linaclotide in this comparative example is shown in Table 1.

[0084] Comparative Example 1

[0085] The difference from Example 1 is that the host cell is Origami B (DE3), the tag protein is Trx, and the remaining steps are consistent with Example 1. The expression level of the protein tag - linaclotide in this comparative example is shown in Table 1.

[0086] Comparative Example 2

[0087] The difference from Example 1 is that the host cell is Origami B (DE3), the tag protein is Ffu209, and the remaining steps are consistent with Example 1. The expression amount of the protein tag - linaclotide in this comparative example is shown in Table 1.

[0088] Comparative Example 3

[0089] The difference from Example 1 is that the host cell is Origami B (DE3), the tag protein is Fh8, and the remaining steps are consistent with Example 1. The expression amount of the protein tag - linaclotide in this comparative example is shown in Table 1.

[0090] Comparative Example 4

[0091] The difference from Example 1 is that the host cell is Origami B (DE3), the tag protein is CBM, and the remaining steps are consistent with Example 1. The expression amount of the protein tag-linaclotide in this comparative example is shown in Table 1.

[0092] Comparative Example 5

[0093] The difference from Example 1 is that the tag protein is Trx, and the remaining steps are the same as Example 1. The expression level of the protein tag - linaclotide in this comparative example is shown in Table 1.

[0094] Comparative Example 6

[0095] The difference from Example 1 is that the tag protein is Ffu209, and the remaining steps are the same as Example 1. The expression level of the protein tag-linaclotide in this comparative example is shown in Table 1.

[0096] Comparative Example 7

[0097] The difference from Example 1 is that the tag protein is Fh8, and the remaining steps are the same as Example 1. The expression level of the protein tag-linaclotide in this comparative example is shown in Table 1.

[0098] Comparative Example 8

[0099] The difference from Example 1 is that the tag protein is CBM, and the remaining steps are the same as Example 1. The expression level of the protein tag-linaclotide in this comparative example is shown in Table 1.

[0100] Comparative Example 9

[0101] The difference from Example 1 is that the host cell is BL21 (DE3) (purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., catalog number: zc121), and the remaining steps are the same as Example 1. The expression level of protein tag-linaclotide in this comparative example is shown in Table 1.

[0102] Comparative Example 10

[0103] The difference from Example 1 is that the host cell is BL21 (DE3), the tag protein is Trx, and the remaining steps are the same as Example 1. The expression amount of the protein tag - linaclotide in this comparative example is shown in Table 1.

[0104] Comparative Example 11

[0105] The difference from Example 1 is that the host cell is BL21 (DE3) and the tag protein is Ffu209; the remaining steps are the same as Example 1. The expression level of the protein tag - linaclotide in this comparative example is shown in Table 1.

[0106] Comparative Example 12

[0107] The difference from Example 1 is that the host cell is BL21 (DE3) and the tag protein is Fh8; the remaining steps are the same as Example 1. The expression level of the protein tag - linaclotide in this comparative example is shown in Table 1.

[0108] Comparative Example 13

[0109] The difference from Example 1 is that the host cell is BL21 (DE3) and the tag protein is CBM; the remaining steps are the same as Example 1. The expression amount of the protein tag - linaclotide in this comparative example is shown in Table 1.

[0110] Table 1

[0111] By comparing Shuffle T7-B, Origami B (DE3) and BL21 (DE3) hosts and different pET-28a-Tag-lina, it was found that pET-28a-Sumo-lina had the highest expression level of lina fusion protein in Shuffle T7-B host cells, reaching 40.5 mg / g wet cells, achieving soluble expression of linaclotide, a peptide containing three pairs of disulfide bonds.

[0112] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the present invention utilizes Sumo-tagged proteins to prepare soluble intermediates of linaclotide in Shuffle T7-B host cells. Compared with other protein tags used to enhance the solubility of target proteins in the prior art, the soluble intermediates of linaclotide containing Sumo-tagged proteins expressed in Shuffle T7-B host cells in the present application are not limited by the problem of inclusion body renaturation, do not require the use of additional reagents to treat the inclusion bodies, have high expression levels, and can more efficiently express linaclotide in a soluble manner. Moreover, when purifying linaclotide, the present application only requires a simple organic solvent precipitation method to purify the target polypeptide, simplifying the product purification steps. Compared with the prior art method of enzymatic cleavage of the protein tag followed by cyclization of the polypeptide and subsequent enzymatic cleavage, the method is relatively low in cost and more efficient, and can further enhance the activity of the final product linaclotide.

[0113] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a soluble intermediate of linaclotide, characterized in that: The preparation method comprises: The soluble intermediate of linaclotide is expressed using a host cell containing a recombinant plasmid, wherein the recombinant plasmid contains a gene capable of expressing a Sumo-tagged protein and a gene capable of expressing linaclotide; The host cells include Shuffle T7-B cells or Origami B (DE3) cells.

2. The preparation method according to claim 1, characterized in that The recombinant plasmid contains the gene capable of expressing the Sumo tag protein and the gene capable of expressing linaclotide, which are sequentially connected in the 5'-3' direction.

3. The preparation method according to claim 1, characterized in that The gene capable of expressing the Sumo tag protein includes a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide having a structure having a homology of more than 70% with the nucleotide sequence shown in SEQ ID NO: 1 and containing a disulfide bond; The gene capable of expressing linaclotide includes a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 2, or a polynucleotide having a homology of more than 70% with the nucleotide sequence shown in SEQ ID NO:

2.

4. The preparation method according to any one of claims 1 to 3, characterized in that The recombinant plasmid contains the nucleotide sequence shown in SEQ ID NO:

3.

5. A method for preparing linaclotide, characterized in that: The preparation method comprises: The linaclotide is obtained by removing the Sumo tag protein from the soluble intermediate of the linaclotide prepared by the preparation method of any one of claims 1 to 4.

6. The preparation method according to claim 5, characterized in that removing the Sumo tag protein from the soluble intermediate of linaclotide to obtain crude linaclotide; The crude linaclotide is purified to obtain the linaclotide.

7. The preparation method according to claim 5 or 6, characterized in that: The excision comprises: using an enzyme to cut the soluble intermediate of linaclotide to obtain the separated Sumo-tagged protein and the crude linaclotide.

8. The preparation method according to claim 6, characterized in that The purification comprises: Adjusting the pH of the system containing the crude linaclotide to the isoelectric point of the Sumo-tagged protein to obtain a tagged protein precipitation system; The tagged protein precipitation system is mixed with an organic solution and centrifuged, and the supernatant is separated to obtain the linaclotide.

9. The preparation method according to claim 8, characterized in that The isoelectric point is pH 5.4-5.

8.

10. The preparation method according to claim 9, characterized in that The isoelectric point is pH 5.

6.

11. The preparation method according to claim 8, characterized in that The organic solution includes one or more of an acetonitrile solution, a hexafluoroisopropanol solution or an acetone solution.

12. The preparation method according to claim 11, characterized in that The acetonitrile solution is an acetonitrile aqueous solution with an acetonitrile volume fraction of 50%-70%.

13. The preparation method according to claim 12, characterized in that The acetonitrile aqueous solution is a 60% acetonitrile aqueous solution.

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