Canagliflozin precursor peptide tandem recombinant fusion protein, polynucleotide, recombinant expression plasmid, engineered recombinant host cell and method for preparing target polypeptide

Abstract

The invention relates to the field of biological medicine preparation, and particularly provides canagliflozin precursor peptide tandem recombinant fusion protein, polynucleotide, recombinant expression plasmid, engineered host cell and a method for preparing target polypeptide. The recombinant fusion protein is a fusion peptide-target polypeptide-(linker peptide-target polypeptide) n from an N terminal to a C terminal, n is a positive integer of 4-6, the fusion peptide is SEQ ID NO.1 or SEQ ID NO.2, the target polypeptide is a canagliptin precursor peptide, the linker peptide comprises a spacer peptide and a protease restriction enzyme cutting site, the spacer peptide is SEQ ID NO.3, the isoelectric point of the recombinant fusion protein is 5.5-6.5, and the average hydrophilic value is-0.85--0.96. The invention creatively provides a method for preparing the canagliflozin precursor peptide by using a gene recombination technology, the proportion of the target polypeptide in the recombinant fusion protein is high, the use of a solvent under extreme conditions is avoided in the production process, the enzyme digestion efficiency is high, and the product purity and yield are high.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a tandem recombinant expression of caglitazone precursor peptide, more specifically to a recombinant fusion protein containing caglitazone precursor peptide, a polynucleotide encoding the recombinant fusion protein, a recombinant expression plasmid containing the polynucleotide, an engineered recombinant host cell containing the recombinant expression plasmid or whose genome integrates the polynucleotide, and a method for preparing a target polypeptide using the recombinant fusion protein. Background Technology

[0002] Canagliptin is a novel, long-acting acylated amylin analogue, a non-selective amylin receptor (AMYR) and calcitonin G protein-coupled receptor (CTR) agonist. Canagliptin can reduce energy intake, regulate food choices and preferences, and exert glucose regulation by co-secreting with insulin. It also inhibits postprandial glucagon release and delays gastric emptying. It shows significant advantages in the treatment of diabetes, obesity, metabolic syndrome, and cardiovascular diseases, and has broad application prospects.

[0003] Canagliflozin is a cyclic polypeptide composed of 38 amino acids and contains a single disulfide bond. Currently, the methods for preparing canagliflozin are all total chemical synthesis methods, including solid-phase synthesis (SPPS) or a combination of solid and liquid phase synthesis (SPPS-LPPS). The basic idea of ​​chemical synthesis is to use resin to sequentially couple individual amino acids and then attach side chains to prepare canagliflozin, or to first use resin to prepare several large fragments and then couple these large fragments to prepare canagliflozin. However, these methods have drawbacks such as the need for deprotection, long reaction steps, difficulty in impurity control, low overall yield, high preparation cost, and difficulty in industrial-scale production.

[0004] Lowering the unit price of therapeutic protein and peptide products can significantly improve public health and reduce the cost burden of healthcare. In recent years, due to the high unit price of protein and peptide drugs, while production costs have been a focus, progress in cost control, such as improvements to production processes, has been slow. With increasing demand for protein and peptide drugs, more manufacturers are expanding production, thereby diluting and reducing labor, utilities, management, and plant depreciation costs. The proportion of process costs, including raw materials, preparation methods, and purification methods, is increasing. How to improve the process flow to reduce overall production costs has become an urgent problem to be solved.

[0005] Genetic engineering is a biosynthetic method for synthesizing peptides. It offers advantages such as highly targeted expression, safety and hygiene, and a wide range of raw material sources. However, there are currently no reports of using genetic engineering to prepare canagliflozin precursor peptides. Furthermore, while existing technologies have disclosed molecular designs for various fusion proteins for other peptides, they still suffer from problems such as low expression levels, high and time-consuming enzymatic digestion steps, high risk of reaction contamination, low yield and purity of the target peptide, and high production costs. Summary of the Invention

[0006] To address the shortcomings of the existing technology, this invention provides a multi-tandem recombinant fusion protein targeting canagliflozin precursor peptide, a polynucleotide encoding the recombinant fusion protein, a recombinant expression plasmid containing the polynucleotide, an engineered recombinant host cell containing the recombinant expression plasmid or whose genome integrates the polynucleotide, and a method for preparing the target polypeptide from the recombinant fusion protein.

[0007] The technical solution of this invention is as follows: In one aspect, the present invention provides a casagliptin precursor peptide tandem recombinant fusion protein, wherein the recombinant fusion protein is defined from the N-terminus to the C-terminus as fusion peptide-target polypeptide-(linker peptide-target polypeptide)n, wherein n is a positive integer from 4 to 6, the fusion peptide is SEQ ID NO.1 or SEQ ID NO.2, the target polypeptide is a casagliptin precursor peptide, the linker peptide comprises a spacer peptide and a protease cleavage site, the spacer peptide is SEQ ID NO.3, and the recombinant fusion protein has an isoelectric point of 5.5 to 6.5 and an average hydrophilicity of -0.85 to -0.96.

[0008] Preferably, the linker peptide is a linker 1-spacer peptide-linker 2, wherein the linker 1 is KR or RR, and the linker 2 is SEQ ID NO.4.

[0009] More preferably, the linker peptide is SEQ ID NO.5.

[0010] Preferably, the amino acid sequence of the canagliflozin precursor peptide is shown in SEQ ID NO.6.

[0011] Preferably, the fusion peptide is SEQ ID NO.1.

[0012] Preferably, the recombinant fusion protein is SEQ ID NO.7 or SEQ ID NO.8.

[0013] In another aspect, the present invention provides a polynucleotide that encodes any of the above-described recombinant fusion proteins.

[0014] In another aspect, the present invention provides a recombinant expression plasmid, which is formed by recombining an expression vector with the aforementioned polynucleotide.

[0015] In another aspect, the present invention provides an engineered host cell containing the above-mentioned recombinant expression plasmid or having the above-mentioned polynucleotides integrated into its genome.

[0016] Preferably, the host cell is Escherichia coli.

[0017] In another aspect, the present invention provides a method for preparing a target polypeptide, comprising the following steps: (a) Synthesize a polynucleotide encoding any of the above-mentioned recombinant fusion proteins based on the amino acid sequence; (b) Insert the polynucleotide into the expression vector to obtain a recombinant expression plasmid; (c) The recombinant expression plasmid is transformed into a host cell to obtain an engineered host cell; (d) Using resistance plates to screen engineered host cells containing the recombinant expression plasmid of the polynucleotide; (e) Fermenting the engineered host cell containing the recombinant expression plasmid containing the polynucleotide to induce the expression of the recombinant fusion protein in the form of intracellular insoluble inclusion bodies; (f) The fermented engineered host cells are homogenized by high pressure to break down the bacteria, the inclusion bodies are collected, and then the inclusion bodies are refolded. (g) The target polypeptide is obtained by enzymatic digestion, transformation and purification, wherein the proteases used for enzymatic digestion are Kex2 protease, carboxypeptidase B and enterokinase.

[0018] Preferably, the host cell is Escherichia coli.

[0019] Preferably, the inclusion bodies are refolded using a buffer solution containing 1% w / t Triton X-100 in 50 mM Tris-HCl at pH 8.0.

[0020] Preferably, the mass ratio of the Kex2 protease, carboxypeptidase B, and enterokinase to the recombinant fusion protein is independently 1:800 to 1:1500, the enzyme digestion temperature is 30℃ to 38℃, and the enzyme digestion time is 2 to 6 hours.

[0021] Preferably, the purification includes one or more of ion exchange resin purification, hydrophobic resin purification, and isoelectric point precipitation.

[0022] The technical effects achieved by this invention are as follows: First, this invention innovatively provides a method for preparing canagliflozin precursor peptide using gene recombination technology, offering a new approach to canagliflozin production. Compared to chemical synthesis methods, the method of this invention has advantages such as strong expression directionality, safety and hygiene, and a wide range of raw material sources.

[0023] Secondly, reducing the cost of producing peptides from recombinant fusion proteins is a multi-dimensional issue in gene recombination technology. For example, in fermentation processes, increasing the yield of the target peptide per unit volume of host cell broth can reduce costs. Increasing the yield of the target peptide requires increasing the expression level of the recombinant fusion protein and the proportion of the target peptide in the recombinant fusion protein. However, since the expression of recombinant fusion proteins is a burden on host cells, the cell broth concentration is often lower when the recombinant fusion protein is highly expressed compared to when it is low. Therefore, it is necessary to increase the concentration of high-density fermentation broth. During the production process, it is also important to reduce costly steps, such as reducing or eliminating the use of denaturing agents that dissolve inclusion bodies, and avoiding extreme conditions such as high-pH alkaline solutions to reduce the generation of epimers. Epimers are very similar to the product, resulting in poor purification and removal, which increases subsequent purification costs. In the enzymatic digestion reaction, it is necessary to control the reaction time and reduce the amount of enzyme used. The enzyme used in the process should have high specificity to reduce the proportion of mis-digestion. After 99% enzymatic digestion of the recombinant fusion protein, the mis-digestion rate should be controlled below 2%. Achieving a certain level of purity for the target peptide after digestion allows the entire process to require only one purification step or use a low-cost purification process to obtain high-purity target peptides. The recombinant fusion protein of this invention has a high proportion of target peptides, and at the same expression level, it can cleave more target peptides. Furthermore, the recombinant fusion protein of this invention avoids the use of extreme solvent conditions for dissolution and refolding during production, has high enzymatic digestion efficiency, and can obtain high-purity and high-yield canagliflozin precursor peptides. Attached Figure Description

[0024] Figure 1 This relates to the molecular design of the recombinant fusion protein in this invention; Figure 2 This is an example of a recombinant expression plasmid of the present invention; Figure 3 The results of SDS-PAGE analysis of the recombinant fusion protein in Example 2 are shown below, where M represents the protein standard (ThemoFisher #26616); 1-4 correspond to the recombinant fusion protein of SEQ ID No. 7, wherein the concentrations of IPTG used to induce the expression of the recombinant fusion protein are 0 mM, 1 mM, 1.5 mM and 2 mM in ascending order of the numbers; 8-11 correspond to the recombinant fusion protein of SEQ ID No. 8, wherein the concentrations of IPTG used to induce the expression of the recombinant fusion protein are 0 mM, 1 mM, 1.5 mM and 2 mM in ascending order of the numbers. Figure 4 The images show the HPLC chromatograms of the canagliflozin precursor peptide obtained by the recombinant fusion protease in Example 1, where A is the HPLC chromatogram before and after enzyme digestion and purification, and B is the HPLC chromatogram after and after enzyme digestion and purification. Figure 5 The images show the HPLC chromatograms of the canagliflozin precursor peptide obtained by the recombinant fusion protease in Example 2, where A is the HPLC chromatogram before and after enzyme digestion and purification, and B is the HPLC chromatogram after and after enzyme digestion and purification. Figure 6 The LC-MS map of the canagliflozin precursor peptide prepared by the recombinant fusion protein of Example 1 (6A) and Example 2 (6B). Detailed Implementation

[0025] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Unless otherwise specified, the methods used in this invention are conventional production methods; the raw materials used, unless otherwise specified, are conventional commercially available products. Unless otherwise specified, all percentages appearing in this invention are by mass percentages.

[0026] In this invention, a fusion peptide refers to an amino acid sequence located at the N-terminus of a recombinant fusion protein, starting from the first amino acid at the N-terminus and ending at the first protease cleavage site (including the cleavage site). Protease cleavage can separate the fusion peptide from other parts of the recombinant fusion protein. In this invention, a linker peptide refers to an amino acid sequence located between two target peptides and containing a protease cleavage site. Protease cleavage can separate the two target peptides linked by the linker peptide. Using the protease cleavage sites contained in the fusion peptide and linker peptide, after expression of the recombinant fusion protein, the fusion peptide and linker peptide can be removed by protease cleavage to form multiple free target peptides.

[0027] like Figure 1As shown, the caglitazone precursor peptide tandem recombinant fusion protein of the present invention is structured from the N-terminus to the C-terminus as fusion peptide-target polypeptide-(linker peptide-target polypeptide)n, where n is a positive integer from 4 to 6. The fusion peptide is SEQ ID NO.1 or SEQ ID NO.2, and contains a protease cleavage site, thereby enabling separation from the target polypeptide under the action of an enzyme. The target polypeptide is a caglitazone precursor peptide. The linker peptide contains a spacer peptide and a protease cleavage site. The spacer peptide is SEQ ID NO.3. The isoelectric point of the recombinant fusion protein is 5.5-6.5, and the average hydrophilicity is -0.85 to -0.96. The recombinant fusion protein of the present invention inserts a fusion peptide before the target polypeptide, including MQHHHHAEAEAEAEKR (SEQ ID NO.1) or MSKIKR (SEQ ID NO.2), which can immediately prevent or release ribosome arrest after the recombinant fusion protein is generated during translation, thereby increasing the expression level of the recombinant fusion protein. Preferably, the fusion peptide is SEQ ID NO.1, and the recombinant fusion protein containing this fusion peptide has a higher expression level. Furthermore, by using suitable fusion peptide, linker peptide, and spacer peptide sequences to regulate the isoelectric point (pI) and average hydrophilicity (GRAVY) of the recombinant fusion protein to suitable values, this invention can increase the probability of inclusion body formation, ensuring the inclusion bodies have sufficient hydrophilicity so that no additional denaturing agent is needed to dissolve them during refolding, and ensuring the resulting inclusion bodies have a suitable acid deposition pH, reducing subsequent capture chromatography steps and significantly lowering production costs. On the other hand, because the fusion peptide sequence is relatively short, it accounts for a smaller proportion of the entire recombinant fusion protein; that is, with the same expression level, the tag proportion is smaller, thus increasing the proportion of the target peptide. This invention designs multiple target peptides tandemly in the recombinant fusion protein, increasing the proportion of the target peptide in the fusion protein. With the same expression level, the higher the proportion of the target peptide in the fusion protein, the more target peptides can be cleaved. When n is 4 to 6, the target polypeptide accounts for a high proportion of the fusion protein, and the repetitive sequence is moderate, which is conducive to gene synthesis and suitable for engineered host cell expression.

[0028] In a preferred embodiment of the present invention, the linker peptide is a linker 1-spacer peptide-linker 2, wherein the linker 1 can be KR or RR, and the linker 2 is SEQ ID NO.4 (DDDDK).

[0029] In the recombinant fusion protein of this invention, linkers 1 and 2 are the cleavage sites for Kex2 protease, carboxypeptidase B, and enterokinase, respectively. By using Kex2 protease, carboxypeptidase B, and enterokinase, the recombinant fusion protein of this invention can achieve a purity of over 93% after enzymatic digestion using one-step ion exchange resin purification or isoelectric point precipitation. The production process of this invention does not require ultrafiltration and reverse-phase purification; it only requires one step or no chromatography purification, achieving a purity of over 93%. This avoids the use of expensive purification methods and instruments, significantly reducing production costs compared to existing processes. The spacer peptide is used for spatial separation of linker 1 and linker 2, and its length is preferably 6-9 amino acids. In this invention, the spacer peptide is SEQ ID NO.3 (EAGSEA). More preferably, the linker peptide is SEQ ID NO.5 (KREAGSEADDDDK). By using the above-mentioned spacer peptide, mutual interference between linker 1 and linker 2 can be avoided during enzymatic digestion, resulting in higher digestion efficiency.

[0030] The recombinant fusion protein of the present invention is applicable to various canagliflozin precursor peptides. Preferably, the amino acid sequence of the canagliflozin precursor peptide is shown in SEQ ID NO. 6. In a preferred embodiment of the present invention, the fusion peptide is SEQ ID NO. 1, the target peptide is SEQ ID NO. 6, and the linker peptide is SEQ ID NO. 5. In another preferred embodiment of the present invention, the fusion peptide is SEQ ID NO. 2, the target peptide is SEQ ID NO. 6, and the linker peptide is SEQ ID NO. 5. In another preferred embodiment of the present invention, the recombinant fusion protein is SEQ ID NO. 7, with a pI of 5.73 and a GRAVY of -0.945. In another preferred embodiment of the present invention, the recombinant fusion protein is SEQ ID NO. 8, with a pI of 6.47 and a GRAVY of -0.899.

[0031] SEQ ID NO.6: KCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNT SEQ ID NO.7: MQHHHHAEAEAEAEKRKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNT SEQ ID NO.8: MSKIKRKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTPKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNT。

[0032] This invention also provides a polynucleotide encoding the recombinant fusion protein of this invention. In a preferred embodiment, the polynucleotide encoding the recombinant fusion protein of this invention includes SEQ ID NO. 9 and SEQ ID NO. 10. This invention also provides a recombinant expression plasmid composed of an expression vector and the above-mentioned polynucleotide, and an engineered host cell containing the above-mentioned recombinant expression plasmid or whose genome integrates the above-mentioned polynucleotide. The vector used in this invention can be a pET series expression vector commonly used in the art, such as pET30a vector, pET30b vector, and other prokaryotic expression vectors. Those skilled in the art can construct recombinant expression plasmids containing the DNA sequence encoding the recombinant fusion protein of this invention and suitable transcription / translation control signals using well-known methods, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The host cell suitable for this invention is preferably Escherichia coli, such as Escherichia coli BL21(DE3).

[0033] The present invention also provides a method for preparing a target polypeptide using the recombinant fusion protein of the present invention, comprising the following steps: (a) synthesizing a polynucleotide encoding the recombinant fusion protein of the present invention according to the amino acid sequence; (b) inserting the polynucleotide into an expression vector to obtain a recombinant expression plasmid; (c) transforming the recombinant expression plasmid into a host cell to obtain an engineered host cell; (d) screening engineered host cells containing the recombinant expression plasmid of the polynucleotide using an antibiotic plate; (e) fermenting the engineered host cells containing the recombinant expression plasmid of the polynucleotide to induce the expression of the recombinant fusion protein in the form of intracellular insoluble inclusion bodies; (f) subjecting the fermented engineered host cells to high-pressure homogenization to lysis, collecting the inclusion bodies, and then refolding the inclusion bodies; (g) obtaining the target polypeptide by enzymatic digestion and purification, wherein the proteases used for enzymatic digestion are Kex2 protease, carboxypeptidase B, and enterokinase.

[0034] Those skilled in the art can prepare recombinant expression plasmids using methods well-known in the art, transfect the recombinant expression plasmids into host cells, and ferment the recombinant engineered cells selected through resistance plate screening in a fermentation medium. After fermentation, the cells are homogenized under high pressure to obtain inclusion bodies. The inclusion bodies are refolded and purified using a buffer solution containing a nonionic surfactant to obtain the recombinant fusion protein. The recombinant fusion protein is then cleaved into multiple independent target peptides by enzymatic digestion. The recombinant fusion protein is digested using three enzymes: Kex2 protease, carboxypeptidase B, and enterokinase. The obtained target peptides are then separated and purified using various methods, preferably using ion exchange resin purification and / or hydrophobic resin purification and / or isoelectric point precipitation. The preparation method of the present invention enables the preparation of target peptides with high yield, high purity, and low cost.

[0035] In a preferred embodiment of the present invention, the inclusion bodies are refolded using a buffer solution containing 1% w / t Triton X-100, 50 mM Tris-HCl, and pH 8.0. In the preparation method of the present invention, the dissolution and refolding of inclusion bodies do not require denaturing agents or other extreme conditions, such as high pH values, which can reduce the generation of epimers, thereby simplifying the subsequent purification process and reducing the process cost.

[0036] In a preferred embodiment of the present invention, the host cell used in the preparation method is *Escherichia coli* BL21(DE3). The mass ratio of Kex2 protease, carboxypeptidase B, and enterokinase to the recombinant fusion protein is preferably 1:800 to 1:4000, more preferably 1:800 to 1:1500, reducing the amount of enzymes used in the process. The enzyme digestion temperature is preferably 30 to 38°C, and the digestion time is preferably 2 to 6 hours, improving the digestion efficiency. Recombinant fusion protein

[0037] The amino acid sequences of the recombinant fusion protein, fusion peptide, and linker peptide in the embodiments and comparative examples of this invention, as well as the relevant parameters of the recombinant fusion protein, are shown in Table 1. The pI value was obtained using an iterative pI calculation model based on the Bjellqvist method. The GRAVY value was calculated by summing the Kyte-Doolittle values ​​of each amino acid contained in the recombinant fusion protein. The target peptide percentage was calculated as: (Molecular weight of the target peptide × Number of target peptides / Total molecular weight of the recombinant fusion protein) × 100%.

[0038] Table 1

[0039] In Comparative Example 1, the number of target peptides was greater than the scope to be protected by this invention, and in Comparative Example 2, a spacer peptide different from that of this invention was used. The amino acid sequences of the recombinant fusion proteins of Comparative Examples 1 and 2 are as follows: SEQ ID NO.11: MQHHHHAEAEAEKRKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGS NTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKREAGSEADDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNT SEQ ID NO.12: MSKIKRKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKRADAEASDDDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKRADAEASDDDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKRADAEASDDDDKKCNTATCATQRLAEF LRHSSNNFGPILPPTNVGSNTKRADAEASDDDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKRADAEASDDDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNTKRADAEASDDDDDKKCNTATCATQRLAEFLRHSSNNFGPILPPTNVGSNT. The polynucleotide sequence of the recombinant fusion protein

[0040] Codon optimization of polynucleotide sequences was performed based on the recombinant fusion protein sequence and codon usage preferences of *E. coli* strain B. The polynucleotide sequences corresponding to the recombinant fusion proteins in the examples and comparative examples were synthesized by Suzhou Hongxun Biotechnology Co., Ltd. The polynucleotide sequences were digested with NdeI and NotI endonucleases and then inserted into the pET30b(+) prokaryotic expression vector to construct recombinant expression plasmids. An example of one of the recombinant expression plasmids is shown below. Figure 2 As shown in Table 2, the polynucleotide sequences corresponding to the recombinant fusion proteins are also shown.

[0041] Table 2 Plasmid transfection of BL21(DE3) competent cells and detection of expression levels

[0042] The plasmids containing the polynucleotide sequences corresponding to the recombinant fusion protein were transfected into competent *E. coli* BL21(DE3) cells (purchased from Shanghai Sangon Biotech Co., Ltd.) using the calcium phosphate precipitation method. 1 ng of plasmid aqueous solution was added to 100 µL of competent *E. coli* BL21(DE3) cells, gently mixed, and placed on ice for 30 minutes. A heat shock at 42°C for 90 seconds was performed, followed by 5 minutes on ice. Then, 700 µL of LB liquid medium (1.0 g / 100 mL soybean peptone, 0.5 g / 100 mL yeast extract, 1.0 g / mL NaCl, antibiotic-free, balance water) was added. The culture was incubated at 37°C and 200 rpm for 1 hour using a shaker. After centrifugation at 5000 rpm for 2 minutes, 700 µL of supernatant was aspirated. The remaining liquid was used to resuspend the centrifuged cells. 100 µL of the resuspended cells were plated onto LB agar plates containing kanamycin sulfate (1.0 g / 100 mL soybean peptone, 0.5 g / 100 mL yeast extract, NaCl...). 1.0 g / mL, 1.5 g / 100 mL agar powder, 50 µg / mL kanamycin sulfate, and the remainder water) were incubated in a 37°C incubator for 13 h.

[0043] Pick a single clone of the strain and transfer it to a shaker tube containing 5 mL of LB liquid medium (1.0 g / 100 mL soybean peptone, 0.5 g / 100 mL yeast extract, 1.0 g / mL NaCl, 50 µg / mL kanamycin sulfate, and the remainder water). Incubate at 37°C and 200 rpm on a shaker until the OD reaches 100°C. 600≈0.5, transfer 0.5 mL of bacterial culture to a new sterile 1.5 mL centrifuge tube and store at 4°C. Add isopropyl-β-D-thiogalactoside (IPTG) to the remaining bacterial culture to achieve concentrations of 1 mM, 1.5 mM, and 2 mM, respectively. Induce recombinant fusion protein expression at 30°C for 8 hours. The relative expression level of the recombinant fusion protein was determined using 12% SDS-PAGE electrophoresis. The results are shown below. Figure 3 As shown, the relative expression level of the recombinant fusion protein = gray level of the target band / total gray level of the lane × 100%, and the relative expression levels are shown in Table 3. "——" indicates no expression.

[0044] Table 3

[0045] As can be seen from Table 3, the number of repeats of the target peptide in Comparative Example 1 is outside the scope of protection of this invention, and it cannot be expressed. Comparative Example 2 used other fusion peptides, and their relative expression levels at different IPTG concentrations were all lower than those in Examples 1 and 2. This invention can achieve high expression using lower IPTG concentrations, thus reducing production costs. Preparation of recombinant fusion proteins and conversion and purification of target peptides

[0046] 0.5 mL of bacterial culture stored at 4 °C was inoculated into 50 mL of LB medium (1.0 g / 100 mL soybean peptone, 0.5 g / 100 mL yeast extract, 1.0 g / mL NaCl, 50 µg / mL kanamycin sulfate, and the remainder water), and incubated at 37 °C for 200 rpm until OD reached 100%. 600 The bacterial culture value is approximately between 0.4 and 0.8. Transfer the bacterial culture to a 50 mL sterile centrifuge tube, centrifuge at 3000 g for 5 min, collect the bacterial cells, and resuspend the bacterial cells in an appropriate amount of the above LB medium until the OD value is reached. 600 ≈2, add an equal volume of sterile 50% glycerol, mix well, and dispense 1 mL / tube into cryovials and store at -80°C.

[0047] Take one bacterial culture from the -80℃ freezer and inoculate it into LB medium (1.0g / 100mL soybean peptone, 0.5g / 100mL yeast extract, 1.0g / mL NaCl, 50µg / mL kanamycin sulfate, and the remainder water) at a volume ratio of 1 / 100. Incubate at 37℃ and 200rpm for 5 hours to obtain a well-recovered bacterial culture.

[0048] High-density fermentation was carried out by autoclaving the fermentation medium (1% soybean peptone, 1% yeast extract, 0.4% NaCl, 0.25% K₂HPO₄·3H₂O, 0.1% KH₂PO₄, pH adjusted to 7.0 with 20% NaOH solution, the remainder being water) and glucose feed (50% glucose aqueous solution), followed by cooling to 37°C. The revived inoculum was inoculated at a 1 / 100 volume ratio. The fermentation broth OD... 600 When the concentration reaches approximately 100, cool the temperature to 30°C, add IPTG (final concentration 1 mM) for induction, and continue culturing until the OD reaches 100. 600 Fermentation should be stopped when the OD value reaches 150-200. Specific fermentation time and OD values ​​at the end of fermentation are not detailed here. 600 The wet weight of the fermentation cells is shown in Table 4.

[0049] Table 4

[0050] The bacterial cells obtained from high-density fermentation were resuspended in buffer A (50mM Tris-HCl, pH 7.0) at 11 times their weight. The cells were homogenized three times using an ATS (AH-PILOT 2018) at 750 bar. Inclusion bodies were collected by centrifugation at 8000 rpm for 60 min. The inclusion bodies were dissolved and refolded using buffer B (1% Triton X-100, 50mM Tris-HCl, pH 8.0), with the volume of buffer B being the same as that of buffer A. 2N HCl was added to the refolded sample to adjust the pH to the isoelectric point of the fusion protein. The sample was centrifuged at 8000 rpm for 15 min, and the white precipitate was collected. The white precipitate was dissolved in buffer C (50 mM Tris-HCl, pH 8.0), the volume of which was the same as that of buffer A. The dissolved recombinant fusion protein was then added to 100X digestion buffer (200 mM CaCl2), along with enterokinase (Pajin Bio), Kex2 protease (Pajin Bio), and carboxypeptidase B (Pajin Bio). The enzyme-to-fusion protein ratio was: Kex2 enzyme:fusion protein = 1:1000, carboxypeptidase B:fusion protein = 1:1200, and enterokinase:fusion protein = 1:1500. The digestion temperature was 30°C, the digestion time was 6 hours, the digestion efficiency was greater than 95%, and the purity of the target peptide was >35% (Waters 2695 RP-HPLC). Figure 4 As shown in A and 5A. After enzymatic digestion, the canagliflozin precursor peptide was purified by SP-FF ion exchange chromatography (BorgLoughlin) and then precipitated at the isoelectric point. The purity was >93% and the yield was >86.9% (Waters 2695RP-HPLC). Figure 4As shown in B and 5B, the molecular weight of the prepared canagliflozin precursor peptide was confirmed by LC-MS (Waters Xevo G3 QTof). The molecular weights of the canagliflozin precursor peptides prepared from the recombinant fusion proteins of SEQ ID NO.7 and SEQ ID NO.8 were (965.83-1.01)×4=3859.28 Da and (965.81-1.01)×4=3859.2 Da, respectively, both consistent with the molecular weight (3859.316 Da) of the target peptide SEQ ID NO.6. The LC-MS spectra are shown below. Figure 6 As shown in (A, B). The yield per unit volume of fermentation broth and the purity of the target peptide after enzymatic digestion / purification are shown in Table 5.

[0051] Table 5

[0052] This invention innovatively provides a method for the biosynthesis of canagliflozin precursor peptide. Compared with chemical synthesis methods, the method of this invention has advantages such as strong expression directionality, safety and hygiene, and wide availability of raw materials. Furthermore, this invention can reduce the cost of preparing target peptides from recombinant fusion proteins in multiple dimensions. The target peptide in this invention accounts for 70%~80% of the recombinant fusion protein, and the relative expression level of the recombinant fusion protein is 40%~50%. The recombinant fusion protein of this invention has a high expression level; after high-density fermentation, 220~250g of wet cells can be obtained per liter of fermentation broth, and more than 10g of recombinant fusion protein can be obtained per liter of fermentation broth before enzymatic digestion. In addition, the technical solution of this invention increases the expression of recombinant fusion protein in inclusion bodies, resulting in high purity of the target peptide before enzymatic digestion, reducing downstream chromatography steps. The dissolution and refolding of inclusion bodies in this invention do not require denaturing agents or other extreme conditions, such as pH, reducing the generation of epimers and lowering subsequent purification costs.

[0053] This invention is not limited to the optional embodiments described above, and anyone can derive other various forms of products based on the inspiration of this invention. The specific embodiments described above should not be construed as limiting the scope of protection of this invention; the scope of protection of this invention should be determined by the claims, and the specification can be used to interpret the claims.

Claims

1. A canagliflozin precursor peptide tandem recombinant fusion protein, characterized in that, The recombinant fusion protein is defined from the N-terminus to the C-terminus as fusion peptide-target polypeptide-(linker peptide-target polypeptide)n, where n is a positive integer from 4 to 6. The fusion peptide is SEQ ID NO.1 or SEQ ID NO.2, the target polypeptide is a canagliflozin precursor peptide, the linker peptide contains a spacer peptide and a protease cleavage site, the spacer peptide is SEQ ID NO.3, and the recombinant fusion protein has an isoelectric point of 5.5 to 6.5 and an average hydrophilicity of -0.85 to -0.

96.

2. The recombinant fusion protein according to claim 1, characterized in that, The linker peptide is a linker 1-spacer peptide-linker 2, wherein linker 1 is KR or RR, and linker 2 is SEQ ID NO.

4.

3. The recombinant fusion protein according to claim 2, characterized in that, The linker peptide is SEQ ID NO.

5.

4. The recombinant fusion protein according to claim 1, characterized in that, The amino acid sequence of the canagliflozin precursor peptide is shown in SEQ ID NO.

6.

5. The recombinant fusion protein according to claim 1, characterized in that, The fusion peptide is SEQ ID NO.

1.

6. The recombinant fusion protein according to claim 1, characterized in that, The recombinant fusion protein is SEQ ID NO.7 or SEQ ID NO.

8.

7. A polynucleotide, characterized in that, The polynucleotide encodes the recombinant fusion protein according to any one of claims 1 to 6.

8. A recombinant expression plasmid, characterized in that, The recombinant expression plasmid is formed by recombination of the expression vector and the polynucleotide as described in claim 7.

9. An engineered host cell, characterized in that, The engineered host cell contains the recombinant expression plasmid of claim 8, or the genome is integrated with the polynucleotide of claim 7.

10. The engineered host cell according to claim 9, characterized in that, The host cell is Escherichia coli.

11. A method for preparing a target polypeptide, characterized in that, Includes the following steps: (a) Synthesizing, according to the amino acid sequence, the polynucleotide encoding the recombinant fusion protein of any one of claims 1 to 6; (b) Insert the polynucleotide into the expression vector to obtain a recombinant expression plasmid; (c) The recombinant expression plasmid is transformed into a host cell to obtain an engineered host cell; (d) Using resistance plates to screen engineered host cells containing the recombinant expression plasmid of the polynucleotide; (e) Fermenting the engineered host cell containing the recombinant expression plasmid containing the polynucleotide to induce the expression of the recombinant fusion protein in the form of intracellular insoluble inclusion bodies; (f) The fermented engineered host cells are homogenized by high pressure to break down the bacteria, the inclusion bodies are collected, and then the inclusion bodies are refolded. (g) The target polypeptide is obtained by enzymatic digestion, transformation and purification, wherein the proteases used for enzymatic digestion are Kex2 protease, carboxypeptidase B and enterokinase.

12. The preparation method according to claim 11, characterized in that, The host cell is Escherichia coli.

13. The method according to claim 11, characterized in that, The inclusion bodies were refolded using a buffer solution containing 1% w / t Triton X-100 in 50 mM Tris-HCl at pH 8.

0.

14. The method according to claim 11, characterized in that, The mass ratio of the Kex2 protease, carboxypeptidase B, and enterokinase to the recombinant fusion protein is independently 1:800 to 1:1500, the digestion temperature is 30℃ to 38℃, and the digestion time is 2 to 6 hours.

15. The method according to any one of claims 11 to 14, characterized in that, The purification includes one or more of ion exchange resin purification, hydrophobic resin purification, and isoelectric point precipitation.

Images