A recombinant fusion protein, polynucleotide, recombinant expression plasmid, engineered host cell, and method of making a polypeptide of interest

Abstract

This invention relates to the field of biopharmaceutical preparation technology, specifically to a recombinant fusion protein, polynucleotide, recombinant expression plasmid, engineered host cell, and a method for preparing a target polypeptide. The recombinant fusion protein of this invention is structured from N-terminus to C-terminus as fusion peptide-target polypeptide-(linker peptide-target polypeptide)n, wherein the isoelectric point of the recombinant fusion protein is 4.0–5.0, the average hydrophilicity is -0.500–-0.905, the fusion peptide is SEQ ID NO.2 or SEQ ID NO.3, the linker peptide is adapter 1-spacer peptide-adaptor 2, adapter 1 is KR or RR, adapter 2 is SEQ ID NO.4, the spacer peptide is 4–10 amino acids connecting adapter 1 and adapter 2, the target polypeptide is a GLP-1 analog, and n is a positive integer not less than 1. The recombinant fusion protein of this invention is expressed in the form of inclusion bodies, simplifying subsequent processing, increasing the expression level of the recombinant fusion protein and the yield of the target polypeptide, and exhibiting high enzymatic digestion efficiency, thus reducing costs from multiple dimensions.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical preparation technology, specifically to a recombinant fusion protein, a polynucleotide sequence encoding the recombinant fusion protein, a recombinant expression plasmid containing the polynucleotide sequence, an engineered host cell containing the recombinant expression plasmid or a gene in which the polynucleotide is integrated, and a method for preparing a target polypeptide using the recombinant fusion protein. Background Technology

[0002] Lowering the unit price of therapeutic proteins and peptides can significantly improve public health and reduce the cost burden of healthcare. In the past few years, the production capacity of therapeutic proteins and peptides has been measured in grams or kilograms. Due to their high unit price, while production costs have been a focus, progress in cost control, such as improvements in production processes, has been slow. With the increasing demand for oral semaglutide, hundreds of companies worldwide attempting to enter the market have begun expanding production to dilute and reduce labor, utilities, management, and plant depreciation costs. Previously, process costs, including materials and fillers, accounted for 5% to 30% of the total cost in grams or kilograms; after expansion, this proportion has increased to 50% to 85%. How to improve the process flow to reduce overall production costs has become an urgent problem to be solved.

[0003] CN106434717A describes a fusion protein design comprising three parts: an N-terminal soluble tag such as TrxA, DsbA, DsbC, Sumo, GST, or Intein; a soluble tag linked to an enzyme cleavage site (e.g., enterokinase, thrombin, or Sumo protease); and the target peptide. Due to the large molecular weight of the soluble tag, the target peptide constitutes a small proportion of the entire fusion protein, approximately 20%, with an expression level of 15%–50%, yielding about 10g of fusion protein per liter of fermentation. Furthermore, during purification, because of the soluble expression, the fusion protein needs to be captured, and the target peptide is produced after 4–12 hours of enzymatic digestion. Precipitation of the target peptide with 20% ethanol yields a final purity of approximately 70%. The target peptide prepared using the method disclosed in this patent application has low purity as a raw material or starting material for synthesis and requires further purification.

[0004] CN110128521B's fusion protein design comprises three parts: an N-terminus soluble and thermostable tag such as mTrA; a soluble and thermostable tag linked to an enzyme cleavage site such as enterokinase, thrombin, tobacco erosion virus protease, 3C protease, trypsin, or lysine endopeptidase, and the target polypeptide. Due to the large molecular weight of the mTrA tag, the target polypeptide accounts for a relatively small proportion of the entire fusion protein, approximately 20%, resulting in a 20% expression level of the fusion protein.

[0005] CN110128552B's fusion protein design comprises three parts: an insoluble tag at the N-terminus linking an enterokinase recognition site and the target peptide. Due to the insoluble tag's approximately 7 kDa size, the target peptide constitutes about 30% of the entire fusion protein, and the expression level is unknown. High-density fermentation resulted in an OD600 of 180. After washing inclusion bodies, dissolving them in an alkaline solution easily led to the formation of epimers. Enzyme digestion at 37℃ for 12 hours was performed; the high temperature and long duration posed a significant risk of contamination. Ion exchange chromatography purification yielded a target peptide purity of 92% and a yield of 87%. Impurities were present before and after the target peak, and the low packing material loading made further improvement difficult.

[0006] Although existing technologies disclose the molecular design of various fusion proteins, problems still exist, such as low expression levels of fusion proteins, high reaction temperatures and long reaction times in the enzymatic digestion steps, high risk of reaction contamination, low yield and purity of the target peptides, and high production costs. Summary of the Invention

[0007] To address the shortcomings of the existing technology, this invention provides a recombinant fusion protein, a polynucleotide sequence encoding the recombinant fusion protein, a recombinant expression plasmid containing the polynucleotide sequence, an engineered host cell containing the recombinant expression plasmid or a gene incorporating the polynucleotide, and a method for preparing a target polypeptide from the recombinant fusion protein. The recombinant fusion protein of this invention exhibits high expression levels, is expressed in the form of inclusion bodies, and the formed inclusion bodies are easily soluble, simplifying subsequent processing. It also results in a high proportion of the target polypeptide in the recombinant fusion protein, high enzyme digestion yield, high enzyme digestion purity, high target polypeptide purification yield, and high purification purity, thereby reducing the cost of preparing target polypeptides from recombinant fusion proteins from multiple dimensions.

[0008] The technical solution of this invention is as follows: This invention provides a recombinant fusion protein, wherein the recombinant fusion protein is structured from N-terminus to C-terminus as fusion peptide-target polypeptide-(linker peptide-target polypeptide)n, wherein the isoelectric point of the recombinant fusion protein is 4.0-5.0, and the average hydrophilicity of the recombinant fusion protein is -0.500 to -0.905; the fusion peptide is SEQ ID NO.2 or SEQ ID NO.3; the linker peptide is adapter 1-spacer peptide-adaptor 2, wherein adapter 1 is KR or RR, adapter 2 is SEQ ID NO.4, and the spacer peptide is 4-10 amino acids connecting adapter 1 and adapter 2; The target polypeptide is a GLP-1 analog; n is a positive integer not less than 1.

[0009] Preferably, the isoelectric point of the recombinant fusion protein is 4.40 to 4.83, and the average hydrophilicity of the recombinant fusion protein is -0.858 to -0.905.

[0010] Preferably, the linker peptide is one of SEQ ID NO.5-6 and SEQ ID NO.8-12.

[0011] Preferably, the recombinant fusion protein is one of the following: the fusion peptide is SEQ ID NO.2, and the linker peptide is one of SEQ ID NO.9 to 12; the fusion peptide is SEQ ID NO.3, and the linker peptide is SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO.8.

[0012] Preferably, n is 5 to 9.

[0013] Preferably, the GLP-1 analog is a smegglutinin precursor peptide.

[0014] Preferably, the amino acid sequence of the recombinant fusion protein is one of SEQ ID NO.16 to 22.

[0015] The present invention provides a polynucleotide sequence that encodes the above-mentioned recombinant fusion protein.

[0016] The present invention provides a recombinant expression plasmid, which is formed by recombining an expression vector sequence and the above-mentioned polynucleotide sequence.

[0017] The present invention provides an engineered host cell containing the recombinant expression plasmid or having the polynucleotide sequence integrated into its genome.

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

[0019] The present invention also provides a method for preparing a target polypeptide, comprising the following steps: (a) Synthesize the polynucleotide encoding the recombinant fusion protein described herein according to the amino acid sequence; (b) Insert the polynucleotide into the vector to obtain a recombinant expression plasmid; (c) Transfect the recombinant expression plasmid into host cells to obtain engineered host cells; (d) Use resistance plates to screen engineered host cells containing the recombinant expression plasmid; (e) Fermenting the engineered host cell containing the recombinant expression plasmid 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.

[0020] 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.

[0021] Preferably, the host cell is Escherichia coli.

[0022] Preferably, the mass ratio of the Kex2 protease, carboxypeptidase B, and enterokinase to the recombinant fusion protein is 1:800 to 1:1500, the enzyme digestion temperature is 30 to 38°C, and the enzyme digestion time is 2 to 6 hours.

[0023] More preferably, the mass ratio of the enzyme to the recombinant fusion protein is Kex2 enzyme:recombinant fusion protein = 1:1000, carboxypeptidase B:recombinant fusion protein = 1:1200, WELQ enzyme:recombinant fusion protein = 1:1500, the enzyme digestion temperature is 30℃, and the enzyme digestion time is 6 hours.

[0024] Preferably, the purification includes ion exchange resin purification and / or hydrophobic resin purification and / or isoelectric point precipitation.

[0025] Reducing the cost of peptide production from recombinant fusion proteins is a multi-dimensional issue. For example, increasing the yield of the target peptide in the fermentation process requires increasing the expression level of the recombinant fusion protein and its proportion within the total recombinant fusion protein. This involves reducing costly steps in the production process, such as minimizing or eliminating the use of denaturing agents that dissolve inclusion bodies, avoiding extreme conditions like high-pH alkaline solutions, and reducing the generation of epimers, as epimers are very similar to the product, resulting in poor purification and removal, and increased subsequent purification costs. For enzymatic digestion, it is necessary to control the reaction time, reduce the amount of enzyme used, and use highly specific enzymes to minimize mis-digestion. After 99% 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 may allow the entire process to require only one purification step.

[0026] The recombinant fusion protein of this invention possesses a specific isoelectric point (pI) and average hydrophilicity (GRAVY). Sequence optimization of the recombinant fusion protein based on these isoelectric point and average hydrophilicity increases its expression level and the probability of inclusion body formation, reducing subsequent capture chromatography steps and significantly lowering production costs. Simultaneously, the inclusion bodies possess sufficient hydrophilicity, eliminating the need for special denaturing agents during refolding and reducing the use of denaturing agents or high-pH alkaline solutions. Furthermore, the recombinant fusion protein of this invention inserts a specific fusion peptide before the target peptide, which can immediately prevent or release ribosome arrest after translation occurs, thereby increasing the expression level of the recombinant fusion protein. Moreover, because the fusion peptide sequence is shorter, it constitutes a smaller proportion of the entire recombinant fusion protein; that is, at the same expression level, a smaller proportion of the fusion peptide results in a higher proportion of the target peptide. Simultaneously, the recombinant fusion protein of this invention contains multiple tandem target peptides, further increasing the proportion of the target peptide in the recombinant fusion protein. At the same expression level, a higher proportion of the target peptide in the recombinant fusion protein allows for the cleavage of more target peptides, increasing the target peptide yield. In the case of multiple target peptides tandemly, the linker peptides between the target peptides contain enzyme cleavage sites. The enzymes used in the process include Kex2 protease, carboxypeptidase B, and enterokinase, resulting in high enzyme yield and purity. The recombinant fusion protein of this invention, after enzyme digestion, can achieve a purity of over 96% using a one-step ion exchange resin purification process and over 95% purity using isoelectric point precipitation. The production process of this invention does not require ultrafiltration or reverse-phase purification; it requires only one step or no chromatographic purification, achieving a purity of over 95%, significantly reducing production costs compared to existing processes. Attached Figure Description

[0027] Figure 1 Molecular structure of recombinant fusion proteins; Figure 2 Plasmid structure expressing recombinant fusion protein; Figure 3 The SDS-PAGE pattern corresponding to the recombinant fusion protein GHB055 is shown below. M represents the protein standard (Thermofisher, 26616); 1 to 3 correspond to the GHB055 recombinant fusion protein. The concentrations of IPTG used to induce the expression of the recombinant fusion protein are 0 mM, 1 mM, and 1 mM in ascending order of the number, and the induction temperatures are 25℃, 25℃, and 37℃ in ascending order of the number. Figure 4 HPLC chromatogram of the target peptide after enzymatic digestion of recombinant fusion protein GHB055; Figure 5HPLC chromatogram of the target peptide after enzymatic digestion and purification of recombinant fusion protein GHB055; Figure 6 LC-MS map of the target peptide obtained from recombinant fusion protein number GHB055. Detailed Implementation

[0028] 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.

[0029] The recombinant fusion protein structure of this invention is as follows: Figure 1As shown, from the N-terminus to the C-terminus, the sequence is fusion peptide-target peptide-(linker peptide-target peptide)n, wherein the isoelectric point of the recombinant fusion protein is 4.0–5.0, and the average hydrophilicity of the recombinant fusion protein is -0.500 to -0.905. Preferably, the isoelectric point of the recombinant fusion protein is 4.40–4.83, and the average hydrophilicity is -0.858 to -0.905. This invention improves the expression level of the recombinant fusion protein and increases the expression probability of its inclusion bodies by giving the recombinant fusion protein the aforementioned specific range of isoelectric point and average hydrophilicity, and by adjusting the sequence composition of the fusion peptide and linker peptide using the isoelectric point and average hydrophilicity as a reference. Simultaneously, it ensures sufficient hydrophilicity in the inclusion bodies to reduce the use of special denaturing agents or high-pH alkaline solutions, thereby lowering subsequent processing costs. Furthermore, the fusion peptide is SEQ ID NO.2 or SEQ ID NO.3, and its amino acid sequence is shown in Table 1. The fusion peptide contains a Kex2 protease cleavage site, such as KR and RR, or an enterokinase cleavage site, such as SEQ ID NO.4 (DDDDK). The fusion peptide can immediately prevent or release ribosome arrest after translation occurs, thereby increasing the expression level of the recombinant fusion protein. In addition, the fusion peptide has a shorter sequence and accounts for a smaller proportion of the entire recombinant fusion protein, resulting in a higher proportion of the target peptide at the same expression level. The fusion peptide is linked to the target peptide, followed by the linking of one or more (linker peptide-target peptide), wherein the linker peptide is adapter 1-spacer peptide-linker 2, adapter 1 contains a Kex2 protease cleavage site and a carboxypeptidase B cleavage site, adapter 2 contains an enterokinase cleavage site, adapter 1 is KR or RR, adapter 2 is SEQ ID NO.4, and the spacer peptide is 4-10 amino acids connecting adapter 1 and adapter 2. Both the fusion peptide and the linker peptide contain protease cleavage sites, making them suitable for removal by cleavage with Kex2 protease, carboxypeptidase B, and enterokinase after expression of the recombinant fusion protein, thereby forming multiple free target peptides. Using the aforementioned proteases for cleavage results in high yield and purity; a single-step ion exchange resin purification step after cleavage achieves a purity of over 96%, and isoelectric point precipitation achieves a purity of over 95%. The target peptide is a GLP-1 analog, preferably a semaglutide precursor peptide, and more preferably, the amino acid sequence of the semaglutide precursor peptide is EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO.1). n is the number of repeating units in the recombinant fusion protein, and n is a positive integer not less than 1. That is, the recombinant fusion protein of the present invention contains multiple target peptides in tandem, increasing the proportion of target peptides in the recombinant fusion protein and enabling the cleavage of more target peptides after expression. In a preferred embodiment of the present invention, n is 5-9, and more preferably, n is 5.When n is within the above range, the target polypeptide accounts for a high proportion in the recombinant fusion protein, and the repetitive sequence is moderate, which is conducive to gene synthesis and suitable for engineered bacteria expression.

[0030] In a preferred embodiment of the present invention, the linker peptide is one of SEQ ID NO. 5-6 and SEQ ID NO. 8-12. More preferably, when the fusion peptide is SEQ ID NO. 2, the linker peptide is one of SEQ ID NO. 9-12; when the fusion peptide is SEQ ID NO. 3, the linker peptide is SEQ ID NO. 5, SEQ ID NO. 6, or SEQ ID NO. 8. The recombinant fusion protein formed by the above-mentioned fusion peptide and linker peptide has a higher expression level, is more easily expressed in the form of inclusion bodies, and the generated inclusion bodies are easier to process and have a higher enzyme digestion yield, resulting in a higher yield and purity of the final target polypeptide.

[0031] In a preferred embodiment of the present invention, the amino acid sequence of the recombinant fusion protein is one of SEQ ID NO. 16 to 22. More preferably, the amino acid sequence of the recombinant fusion protein is SEQ ID NO. 22.

[0032] In one embodiment of the present invention, a polynucleotide sequence encoding the recombinant fusion protein of the present invention is provided. The polynucleotide sequence may be a DNA and / or RNA sequence. Preferably, the polynucleotide sequence of the recombinant fusion protein is one of SEQ ID NO. 29 to 35. More preferably, the polynucleotide sequence of the recombinant fusion protein is SEQ ID NO. 35.

[0033] In another embodiment of the present invention, a recombinant expression plasmid is provided, which is formed by recombining a vector sequence and the polynucleotide sequence described in the present invention. The vector used in the present invention can be a prokaryotic expression vector or a eukaryotic expression vector, such as the pET series expression vectors commonly used in the art, such as the pET30a vector, pET30b vector, and other prokaryotic expression vectors.

[0034] 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, and in vivo recombination technology. The DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The recombinant expression plasmid also includes a ribosome binding site for translation initiation and a transcription terminator.

[0035] One embodiment of the present invention provides an engineered host cell comprising the above-described expression plasmid or a polynucleotide sequence of the recombinant fusion protein of the present invention integrated into its genome. The host cell is preferably *Escherichia coli*, such as *Escherichia coli* BL21(DE3).

[0036] In one embodiment of the present invention, the method for preparing the target polypeptide using the recombinant fusion protein of the present invention includes the following steps: (a) Synthesize the polynucleotide sequence encoding the recombinant fusion protein based on the amino acid sequence; (b) Insert the polynucleotide sequence into the vector sequence to obtain a recombinant expression plasmid; (c) Transfect the recombinant expression plasmid into host cells to obtain engineered host cells; (d) Use resistance plates to screen engineered host cells containing the recombinant expression plasmid; (e) Fermenting the engineered host cell containing the recombinant expression plasmid 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.

[0037] In step (a), according to the present invention, the amino acid sequence of the recombinant fusion protein and the host cell codon usage preference are optimized to synthesize the polynucleotide sequence encoding the recombinant fusion protein.

[0038] Those skilled in the art can prepare recombinant expression plasmids using methods well-known in the art, transfect the 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 ion exchange chromatography and / or hydrophobic chromatography 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.

[0039] 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 the inclusion bodies do not require denaturing agents or other extreme conditions, such as high pH values, which can reduce the generation of epimers, simplify subsequent processing steps, and reduce costs.

[0040] In a preferred embodiment of the present invention, the host cell used in the preparation method is *Escherichia coli* BL21(DE3). The preferred mass ratio of Kex2 protease, carboxypeptidase B, and enterokinase to the recombinant fusion protein is 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 30–38°C, and the digestion time is 2–6 hours, preferably 30°C, and the digestion time is 6 hours, improving the digestion efficiency.

[0041] Recombinant fusion protein

[0042] The recombinant fusion protein of the present invention is described below using the semaglutide precursor peptide of SEQ ID NO.1 as an example. For the recombinant fusion proteins in the embodiments and comparative examples of the present invention, the sequences and combinations of the fusion peptide and linker peptide are shown in Table 1, the amino acid sequence of the recombinant fusion protein is shown in Table 2, and the parameters of the recombinant fusion protein are shown in Table 3. Specifically, pI is obtained using an iterative pI calculation model based on the Bjellqvist method, GRAVY is calculated by summing the Kyte-Doolittle values ​​of each amino acid contained in the recombinant fusion protein, and the target peptide percentage = number of target peptides × molecular weight of a single target peptide / total molecular weight of the recombinant fusion protein × 100%.

[0043] Table 1

[0044] Table 2

[0045] Table 3

[0046] As can be seen from Table 3, the proportion of the target peptide in the recombinant fusion protein of the present invention is greater than 65%. Compared with the recombinant fusion protein in the prior art, the recombinant fusion protein of the present invention can cleave more target peptides and increase the yield of target peptides under the same expression level.

[0047] Codon optimization of recombinant fusion proteins, synthesis of multinucleotide sequences encoded by recombinant fusion proteins, and plasmid construction. Codon optimization and polynucleotide sequence synthesis were performed based on the recombinant fusion protein sequence and codon usage preferences of *E. coli* strain B. The polynucleotide sequence corresponding to the recombinant fusion protein in this invention was synthesized and the plasmid was constructed by Suzhou Hongxun Biotechnology Co., Ltd. The polynucleotide sequence was digested with NdeI and NotI endonucleases and then inserted into the pET30a(+) / pET30b(+) prokaryotic expression vector to construct the expression plasmid. The plasmid structure expressing the recombinant fusion protein is shown below. Figure 2 As shown in Table 4, the polynucleotide sequences corresponding to the recombinant fusion proteins are also shown.

[0048] Table 4

[0049] Plasmid transfection of BL21(DE3) competent cells and detection of expression levels

[0050] The plasmid containing the polynucleotide sequence corresponding to the recombinant fusion protein was transfected into competent *E. coli* BL21(DE3) cells (purchased from Shanghai Sangon Biotech Co., Ltd.) using calcium phosphate precipitation. 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. The cells were then heat-shocked at 42°C for 90 seconds, placed on ice for 5 minutes, and 700 μL of LB broth (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 cells were 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.

[0051] Pick a single clone of the strain and transfer it to 5 mL of LB liquid medium containing kanamycin sulfate (1.0 g soybean peptone / 100 mL, 0.5 g yeast extract / 100 mL, 1.0 g NaCl / mL, 50 µg kanamycin sulfate / mL, balance water). Incubate on a shaker at 37°C or 25°C at 200 rpm until 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 a concentration of 1 mM, and induce recombinant fusion protein expression for 8 hours. The relative expression level of the recombinant fusion protein was determined using 12% SDS-PAGE electrophoresis. The results are as follows: Figure 3 As shown in Table 5, the relative expression level of the recombinant fusion protein = gray level of the target band / total gray level of the lane × 100%.

[0052] Table 5

[0053] As shown in Table 5, the relative expression level of the recombinant fusion protein of the present invention is approximately between 60% and 90%. At different induction temperatures, the relative expression level of the recombinant fusion protein in the examples is significantly higher than that in the comparative examples. The present invention, by controlling the isoelectric point and average hydrophilicity, and by selecting a specific combination of fusion peptides and linker peptides, can increase the expression level of the recombinant fusion protein, thereby increasing the yield of the target peptide.

[0054] High-density fermentation 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.

[0055] 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.

[0056] High-density fermentation was carried out by autoclaving the fermentation medium (1% w / t soybean peptone, 1% w / t yeast extract, 0.4% w / t NaCl, 0.25% w / t K₂HPO₄·3H₂O, 0.1% w / t KH₂PO₄, pH adjusted to 7.0 with 20% NaOH solution) 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 100, cool the temperature to 30°C, add IPTG (final concentration 1mM) for induction, and continue culturing until OD reaches 100. 600 Fermentation should be stopped when the OD value reaches 150-200. Specific fermentation time and OD value at the end of fermentation are not detailed here. 600 The wet weight of the fermentation cells is shown in Table 6: Table 6

[0057] As shown in Table 6, the total bacterial count per unit volume of fermentation broth using the recombinant fusion protein of this invention is greater than 250 g / L, significantly higher than that of the recombinant fusion protein in the comparative example. The results in Tables 5 and 6 demonstrate that the recombinant fusion protein of this invention can maintain high expression in both conventional and high-density fermentation processes, thereby increasing the yield of the target peptide.

[0058] Preparation of recombinant fusion proteins and conversion and purification of target peptides The high-density fermentation-derived bacterial cells GHB055 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 then dissolved and renatured using buffer B (1% w / t 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 renatured sample to adjust the pH to the isoelectric point of the recombinant fusion protein. The mixture was centrifuged at 8000 rpm for 15 min, and the white precipitate was collected. This precipitate was then dissolved in buffer C (50mM Tris-HCl, pH 8.0), with the volume of buffer C being the same as that of buffer A. The dissolved recombinant fusion protein was then added to 100X restriction enzyme buffer (200mM CaCl2), along with enterokinase (Paijin Biotechnology) and Kex2. The enzymes used were protease (Paijin Biotechnology) and carboxypeptidase B (Paijin Biotechnology). The enzyme-to-recombinant fusion protein ratios were: Kex2 enzyme:recombinant fusion protein = 1:1000, carboxypeptidase B:recombinant fusion protein = 1:1200, and enterokinase:recombinant fusion protein = 1:1500. The digestion temperature was 30℃, the digestion time was 6 hours, the digestion efficiency was greater than 95%, and the purity of the target peptide was 95.8%. The HPLC chromatogram of the target peptide is shown below. Figure 4 As shown. After enzymatic digestion, the smegglutinin precursor peptide (18 g / L packing material) was purified by Q-FF ion exchange chromatography (Jiangsu Hanbang) and precipitated at the isoelectric point. The product purity was 97.42%, and the yield was 95.2%. The HPLC chromatogram of the target peptide is shown below. Figure 5 As shown. LC-MS (WatersXevo G3 QTof) confirmed that the molecular weight of the prepared smegglutinin precursor peptide was 1059.44 × 3 - 3 = 3175.32, which matches the molecular weight of the target peptide in SEQ ID NO. 1. The LC-MS spectrum is shown below. Figure 6 As shown in Table 7, the yield per unit volume of fermentation broth and the purity of the target peptide after enzymatic digestion / purification are presented.

[0059] Table 7

[0060] As can be seen from the above embodiments, the recombinant fusion protein of the present invention exists in the form of inclusion bodies, and no denaturing agents or other extreme conditions are required in post-processing operations such as dissolution and refolding, which reduces the generation of epimeric impurities. The purity of the target peptide after enzymatic digestion and before purification has reached 95.8%, and only simple purification is required to achieve a purity of more than 97%, which reduces the difficulty and cost of subsequent purification.

[0061] The target peptide of this invention accounts for more than 65% of the recombinant fusion protein, and the relative expression level of the recombinant fusion protein is approximately 60%~90%, which is higher than that of existing technologies. The recombinant fusion protein of this invention has a high expression level; each liter of fermentation broth, after treatment to the point of enzymatic digestion, yields no less than 10g of recombinant fusion protein, which is higher than that of existing technologies. Furthermore, the technical solution of this invention increases the expression of the recombinant fusion protein in inclusion bodies. After enzymatic digestion and before purification, the purity of the target peptide is no less than 95%, reducing downstream chromatography steps. In this invention, the dissolution and refolding of inclusion bodies do not require denaturing agents or other extreme conditions, such as pH, reducing the generation of epimers and lowering subsequent purification costs. This invention not only improves the yield and productivity of the target peptide but also reduces the cost of preparing the target peptide from recombinant fusion proteins from multiple dimensions.

[0062] 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 recombinant fusion protein, characterized in that, The recombinant fusion protein, from its N-terminus to its C-terminus, is structured as fusion peptide-target polypeptide-(linker peptide-target polypeptide)n, where, The recombinant fusion protein has an isoelectric point of 4.0 to 5.0 and an average hydrophilicity of -0.500 to -0.

905. The fusion peptide is SEQ ID NO.3; The linker peptide is a linker 1-spacer peptide-linker 2, wherein linker 1 is KR or RR, linker 2 is SEQ ID NO.4, and the spacer peptide is 4 to 10 amino acids that connect linker 1 and linker 2; The target polypeptide is a GLP-1 analog; The n is a positive integer not less than 1; The amino acid sequence of the recombinant fusion protein is SEQ ID NO.

22.

2. The recombinant fusion protein of claim 1, wherein, The isoelectric point of the recombinant fusion protein is 4.40 to 4.83, and the average hydrophilicity of the recombinant fusion protein is -0.858 to -0.

905.

3. An isolated polynucleotide, comprising, The polynucleotide encodes the recombinant fusion protein of claim 1 or 2.

4. A recombinant expression plasmid, characterized in that, The recombinant expression plasmid is formed by recombination of the expression vector and the polynucleotide described in claim 3.

5. An engineered host cell, characterized in that, The engineered host cell contains the recombinant expression plasmid of claim 4, or the genome is integrated with the polynucleotide of claim 3.

6. The engineered host cell according to claim 5, characterized in that, The host cell is Escherichia coli.

7. A method for preparing a target polypeptide, characterized in that, Includes the following steps: (a) Synthesizing a polynucleotide encoding the recombinant fusion protein of claim 1 or 2 according to the amino acid sequence; (b) Insert the polynucleotide into the vector to obtain a recombinant expression plasmid; (c) Transfect the recombinant expression plasmid into host cells to obtain engineered host cells; (d) Use resistance plates to screen engineered host cells containing the recombinant expression plasmid; (e) Fermenting the engineered host cell containing the recombinant expression plasmid 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.

8. The method for preparing the target polypeptide according to claim 7, 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.

9. The method for preparing the target polypeptide according to claim 7, characterized in that, The host cell is Escherichia coli.

10. The method for preparing the target polypeptide according to claim 7, characterized in that, The mass ratio of the Kex2 protease, carboxypeptidase B, and enterokinase to the recombinant fusion protein is 1:800 to 1:1500, the digestion temperature is 30 to 38°C, and the digestion time is 2 to 6 hours.

11. The method for preparing the target polypeptide according to claim 10, characterized in that, The mass ratio of the enzyme to the recombinant fusion protein is as follows: Kex2 enzyme: recombinant fusion protein = 1:1000, carboxypeptidase B: recombinant fusion protein = 1:1200, and enterokinase: recombinant fusion protein = 1:1500. The enzyme digestion temperature is 30°C, and the enzyme digestion time is 6 hours.

12. The method for preparing the target polypeptide according to claim 7, characterized in that, The purification includes ion exchange resin purification and / or hydrophobic resin purification and / or isoelectric point precipitation.

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