Method for efficiently expressing and producing recombinant parathyroid hormone in escherichia coli

Abstract

Aiming at the long-term challenges that recombinant parathyroid hormone (PTH1-34) is difficult to express, easy to degrade and high in cost in escherichia coli, a brand-new fusion protein framework is designed: efficient secretion is ensured through a signal peptide SP6, high solubility and stable expression are realized by utilizing an optimized special soluble protein tag, and then the parathyroid hormone (PTH1-34) is obtained through an exquisite linker sequence. And purifying and releasing the target active polypeptide PTH1-34. According to the technical scheme, the expression quantity of the fusion protein is greatly improved, protease degradation of host bacteria is effectively resisted, the yield of PTH1-34 is remarkably improved, and the production cost is greatly reduced. The optimized strain is subjected to induced expression for 16 hours, the expression quantity of the fusion protein reaches 39.2%, the thallus yield is 167g / L, and the fermentation yield of the fusion protein is 22g / L, which is increased by at least 400 times compared with 50mg / L in the prior art; the stable, efficient and large-scale production of the therapeutic polypeptide is realized.

Description

Technical Field

[0001] This invention relates to a method for the efficient expression and production of recombinant parathyroid hormone in Escherichia coli, belonging to the field of biotechnology. Background Technology

[0002] Osteoporosis is a metabolic bone disease characterized by decreased bone density and destruction of bone microstructure, leading to an increased risk of fractures. Parathyroid hormone (PTH) is a core hormone secreted by the parathyroid glands in the neck. Its core function is to precisely regulate the balance of calcium and phosphorus metabolism in the body, maintaining bone health and normal neuromuscular function. It is known as the "core regulator of calcium and phosphorus metabolism."

[0003] Parathyroid hormone (PTH) is a single-chain polypeptide composed of 84 amino acids. However, studies have found that its biological activity mainly depends on amino acid residues 1-34 at its N-terminus (PTH1-34). PTH1-34 is the active fragment of PTH and has the same biological activity as the full-length PTH (1-84). In the treatment of osteoporosis, teriparatide is a recombinant expression of the PTH1-34 fragment. It can promote bone formation and reduce fracture risk, and has been used in many countries around the world to treat postmenopausal women with high fracture risk of osteoporosis. It is one of the main drugs currently used clinically to treat osteoporosis.

[0004] Existing methods for producing PTH1-34 include chemical synthesis or recombinant PTH1-34 production via E. coli genetic engineering expression. Chemical synthesis of PTH1-34 is costly and environmentally problematic, and is only used for producing control standards or constructing small-scale mutant libraries. Currently, commercial PTH production is achieved through E. coli expression, which includes two methods: inclusion body renaturation and soluble cytoplasmic expression. Patents CN1417231A and CN1262649C describe inclusion body expression renaturation and enzyme digestion processes for PTH1-34 production. However, these processes are complex, have low yields, long cycles, and high costs due to variable renaturation design. Patents such as ZL200610070995.4, CN100484958C, CN117567640B, or CN1163510C design soluble expression of PTH1-34 fusion protein. The PTH1-34 peptide produced by the methods provided in these patents has a low yield, with only 50 mg / L of fusion protein expressed. Summary of the Invention

[0005] The inventors of this application observed in their research that when PTH1-34 or its fusion protein is directly expressed using a conventional E. coli cytoplasmic expression system, the target protein is easily degraded within the host cell and during subsequent disruption and purification processes. This leads to a significant reduction in the effective accumulation of the target fusion protein and a significantly low final product yield, severely restricting the large-scale industrial production of PTH1-34. The inventors of this application unexpectedly discovered a novel vesicle nucleation signal peptide, SP6. This signal peptide can efficiently direct the target therapeutic peptide to the peripheral space of E. coli and induce the formation of outer membrane vesicles. This not only significantly increases the expression level of the fusion protein but also prevents its degradation, thereby increasing PTH yield and reducing production costs.

[0006] This invention first provides a vesicle nucleation signal peptide SP6, wherein the signal peptide SP6 comprises or is composed of (a) or (b): (a) A polypeptide with the amino acid sequence shown in SEQ ID NO.1; (b) A derivative polypeptide derived from (a) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (a) while retaining the biological function of the sequence from which it originated.

[0007] The present invention further provides a fusion protein comprising the above-mentioned signal peptide SP6 and target polypeptide.

[0008] In one embodiment of the present invention, the fusion protein comprises, from the N-terminus to the C-terminus, the following: (1) Signal peptide SP6; (2) Solubilizing proteins; (3) Connector; (4) Target polypeptide.

[0009] The target polypeptide of the present invention can be a polypeptide or protein of any length. The target polypeptide that can be generated and purified by the method of the present invention can be 20-200, 25-150, 30-120, or 30-100 amino acid residues, for example, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 amino acid residues.

[0010] In one embodiment of the present invention, the target polypeptide is selected from parathyroid hormone (PTH), glucagon-like peptide-1 (GLP-1), brain natriuretic peptide (BNP), insulin secretion peptide (Ex-4), chemokine (CCL5), stromal cell-derived factor (SDF-1α), growth factor (IGF-1α), obesity hormone (Lep), calcitonin, sermorelin, thymosin, hirudin, cecropin, human histone, defensin, and human plasminogen 1-5, or their biologically active fragments.

[0011] In one embodiment of the present invention, the target polypeptide is a PTH active fragment, comprising (c) or (d) or composed of thereof: (c) PTH1-34 shown in SEQ ID NO: 5; (d) A derivative polypeptide derived from (c) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (c) while retaining the biological function of the sequence from which it originated.

[0012] In one embodiment of the present invention, the lysing protein is selected from P53TD and its mutants, GB1, StefinA, NusA, GST, Trx, SUMO, DsbC, Z, MBP, T7PK, or combinations thereof. Preferably, the P53TD mutant is selected from P53TD / K, more preferably P53TD / E2K.

[0013] In one embodiment of the present invention, the solubilizing protein comprises or is composed of (e) or (f): (e) A polypeptide with an amino acid sequence as shown in any one of SEQ ID NO: 2 to SEQ ID NO: 4; (f) A derivative polypeptide derived from (e) in which one or more amino acids are substituted, deleted or added in the amino acid sequence defined in (e) while retaining the biological function of the sequence from which it originated.

[0014] In one embodiment of the present invention, the adapter includes a purification tag and a cleavage site; preferably, the purification tag is a His tag (HHHHHH) or a glutathione transferase tag (GST-tag); preferably, the cleavage site is a chemical cleavage site, an autocleavage site, or an enzyme cleavage site; more preferably, the enzyme cleavage site includes an enterokinase cleavage site, a TEV enzyme cleavage site, a thrombin cleavage site, and / or a 3C protease cleavage site, as well as functionally equivalent variants of the above sites that retain specific cleavage function.

[0015] The present invention also provides a nucleic acid molecule that encodes the above-mentioned signal peptide or fusion protein.

[0016] In one embodiment of the present invention, the nucleic acid molecule comprises: (1) The first polynucleotide encoding the signal peptide; (2) The second polynucleotide encoding a lysin; (3) The third polynucleotide encoding the linker; (4) The fourth polynucleotide encoding the target polypeptide; The first to fourth polynucleotides are sequentially linked to express a fusion protein comprising a signal peptide, a lysin, a linker, and a target polypeptide.

[0017] The present invention also provides an expression plasmid vector that expresses the above-mentioned signal peptide or fusion protein, or contains the above-mentioned nucleic acid molecule.

[0018] In one embodiment of the present invention, the expression plasmid vector includes pET28a plasmid, pET9a plasmid, pET21a plasmid, pET24a plasmid, pET30a plasmid and / or pET32a plasmid.

[0019] The present invention further provides a host cell that expresses the above-mentioned signal peptide or fusion protein, or contains the above-mentioned nucleic acid molecule or one or more of the above-mentioned expression plasmid vectors.

[0020] In one embodiment of the invention, the host cell is a transformant, which is a host cell that has been altered by introducing one or more expression plasmid vectors into the host cell. In some embodiments, the transformant is obtained by introducing the plasmid vector into a host cell that exhibits competence with the expression plasmid vector.

[0021] In one embodiment of the present invention, the host cell is a recombinant host cell containing the aforementioned nucleic acid molecule integrated into the host cell chromosome. Preferably, the nucleic acid molecule contains a first to a fourth polynucleotide, wherein the first to the fourth polynucleotide are sequentially linked to express a fusion protein comprising a signal peptide-lysin-adaptor-target polypeptide.

[0022] In one embodiment of the present invention, the host cell includes fungi, bacteria, plant cells and / or animal cells.

[0023] In one embodiment of the present invention, the bacteria include Escherichia coli, Bacillus subtilis, Lactococcus lactis and / or Corynebacterium glutamicum.

[0024] In a specific embodiment of the present invention, the host strain of the transformed or mutant host cell is selected from bacteria or fungi; optionally, the host strain is selected from wild-type or genetically engineered *Escherichia coli*, *Bacillus subtilis*, *Bacillus megaterium*, *Bacillus amyloliquefaciens*, *Lactococcus lactis*, *Corynebacterium glutamicum*, *Saccharomyces cerevisiae*, *Candida utilis*, or *Pichia pastoris*; optionally, the host strain is selected from wild-type or genetically engineered *Escherichia coli*. *Escherichia coli* can be any strain derived from the *Escherichia coli* K-12 or B lineage, or a derivative thereof, such as *Escherichia coli* BL21(DE3) or BL21star(DE3).

[0025] The present invention also provides a method for preparing a target polypeptide, the method comprising: a) The above-mentioned host cells are inoculated into a culture medium for fermentation to obtain a fermentation broth; b) The fusion protein was isolated and purified from the fermentation broth; c) Separate the target polypeptide and the remainder of the fusion protein by cutting the connector; d) Remove the remaining parts of the fusion protein to obtain the purified target peptide.

[0026] This invention also provides the application of the above-mentioned signal peptide, fusion protein, nucleic acid molecule, expression plasmid vector, and host cell in the preparation of target polypeptides.

[0027] The technical solution of this invention has the following advantages: This invention constructs a highly efficient secretory expression system by designing a novel SP6 vesicle nucleation signal peptide system. Under high-density fermentation conditions, the volume yield of the fusion protein reaches up to 22 g / L, accounting for 39.2% of the total cellular protein, which is at least 400 times higher than the 50 mg / L of the prior art. This enables low-cost, large-scale biomanufacturing of therapeutic peptides. Attached Figure Description

[0028] Figure 1 Schematic diagram of the PTH1-34 molecular expression vector Figure 2 Expression of lysinogens GB1 and Stefin A in *E. coli*, where: M. standard molecular weight of protein; 1. expression after 5 h of induction with Stefin A expression vector; 2. expression after 5 h of induction with GB1 expression vector; 3. expression after 20 h of induction with Stefin A expression vector; and 4. expression after 20 h of induction with GB1 expression vector. Figure 3Expression of the p53TD / E2K molecular expression vector for the lysin in *E. coli*, where: M. standard molecular weight of protein; 1. expression after 5 h of induction by strain 1; 2. expression after 5 h of induction by strain 2; 3. expression after 20 h of induction by strain 1; and expression after 20 h of induction by strain 2. Figure 4 Fermentation expression of a molecular expression vector containing the lysosomal protein p53TD / E2K in Escherichia coli Detailed Implementation

[0029] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0030] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used in this invention are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0031] In this invention, the term "amplification" refers to the enhancement of the intracellular activity of one or more enzymes encoded by appropriate DNA in a microorganism, for example, by increasing the gene copy number, using a strong promoter or using a gene encoding an appropriate enzyme with high activity, and selectively combining these methods.

[0032] The nucleotide sequences or polynucleotides used in this invention may include double-stranded DNA or single-stranded DNA (i.e., the sense and antisense strands that make up double-stranded DNA) or RNA. Polynucleotides containing a specific polynucleotide sequence may include fragments and / or mutants of that specific polynucleotide sequence. A fragment of a polynucleotide refers to a portion of a polynucleotide that encodes a polypeptide that provides substantially the same function as the polypeptide encoded by the complete polynucleotide sequence. Examples of mutants of a specific polynucleotide sequence include naturally occurring allelic mutants, artificial mutants, and polynucleotide sequences obtained by deleting, substituting, adding, and / or inserting one or more nucleotides into said specific polynucleotide sequence. It should be understood that such fragments and / or mutants of a specific polynucleotide sequence encode a polypeptide that has substantially the same function as the polypeptide encoded by the original specific polynucleotide sequence.

[0033] In this invention, the term "gene synthesis" refers to the generation of DNA using recombinant DNA technology or the acquisition using synthetic DNA or amino acid sequence techniques available and known in the art. "Encoding" refers to the inherent property of using a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, as a template to synthesize other polymers and macromolecules in biological processes, said polymers and macromolecules having either a defined sequence of nucleotides or a defined sequence of amino acids and the biological properties produced therefrom. Therefore, if the transcription and translation of the mRNA of a particular gene produces a particular polypeptide or protein in a cell or other biological system, then that particular gene encodes that particular polypeptide or protein.

[0034] In this invention, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0035] In this invention, the term "vector" or "expression vector" refers to a genetic material composition comprising isolated nucleic acids, which can be used to deliver the isolated nucleic acids into the cell. The vector may contain sequences that guide autonomous replication within the cell or sequences sufficient to allow integration into the host cell's DNA; in specific embodiments of this invention, the vector is heterologous relative to the host cell. Many vectors are known in the art, including, but not limited to, plasmids, phage particles, artificial chromosomes, bacterial phages, and animal viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses.

[0036] In this invention, the term "purification" refers to the separation of fusion proteins or target polypeptides from production culture media using known methods. Examples of such known methods include crystallization, chromatography, ion exchange resin methods, activated carbon adsorption-elution methods, and solvent extraction methods.

[0037] In this invention, the term "about" refers to a measurable value such as a quantity, a period of time, etc., and indicates a variation of ±10% from a given value, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1%, provided that such variation is suitable for implementing the disclosed method.

[0038] The term "vesicle-forming signal peptide" is known in the art, such as the VNp peptide derived from full-length endogenous Hsα-synuclein disclosed in CN118488963A.

[0039] In this invention, a "fusion protein" refers to a fusion tag that, upon fusion with a target polypeptide, helps the target polypeptide fold correctly and improves the solubility of the fusion protein. Many such "fusion proteins" are known to those skilled in the art, such as P53TD and its mutants, GB1, StefinA, NusA, GST, Trx, SUMO, DsbC, Z, MBP, T7PK, or combinations thereof.

[0040] According to the present invention, the target polypeptide is linked to the signal peptide and the fusion protein moiety via a linker, wherein the linker includes a cleavage site. The "cleavage site" includes sequences required to achieve cleavage, such as protease recognition sequences for enzymatic cleavage, integrin sequences for self-cleavage, etc.

[0041] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0042] In this invention, the expression protein of recombinant PTH1-34 whole bacteria was detected as follows: 5 mL of fermentation broth was taken, 30 mL of PBS buffer was added and mixed well, and after sonication, it was centrifuged at 4℃ and 9000 rpm for 30 min, and the supernatant was collected; the purity of the supernatant was detected by SDS-PAGE, and the concentration was detected by Lowry's method.

[0043] Protein expression detection of recombinant PTH1-34 cells in this invention: Take 5 mL of fermentation broth and centrifuge at 4℃ and 9000 rpm for 30 min. Transfer the supernatant after centrifugation to a clean 50 mL centrifuge tube, seal and store for later use. Add 30 mL of PBS buffer to the precipitated cells, vortex thoroughly to mix, and sonicate for 50 min. Then centrifuge again at 4℃ and 9000 rpm for 30 min and collect the supernatant for subsequent detection.

[0044] Example 1: Strain Construction This invention first constructs a molecular expression vector for the PTH1-34 fusion protein, the specific structure of which is shown in Figure 1. This vector contains four functional domains from the N-terminus to the C-terminus: the first is a vesicle-forming signal peptide SP6; the second is a lysosome-promoting protein used to enhance the expression efficiency of the target polypeptide; the third is an adapter sequence containing restriction enzyme sites, such as commonly used restriction enzyme sites like TEV, bovine enterokinase, and thrombin; and the fourth is the PTH1-34 polypeptide. Through screening and optimization of the lysosome-promoting protein, a molecular expression vector capable of efficiently expressing PTH1-34 is finally obtained.

[0045] This invention selects GB1, StefinA, and p53TD / E2K as target lysosomal proteins, and constructs corresponding PTH1-34 fusion protein expression vectors. Using the molecular biology software Jcat, the DNA sequences of the above genes were optimized based on the codon bias of *E. coli*. After sequence synthesis was completed by Shanghai Sangon Biotech Co., Ltd., the synthesized fragments were inserted into plasmid pET28a using the restriction enzyme sites NcoI and XhoI. The optimized sequences are as follows: The amino acid sequence of the signal peptide SP6 is shown in SEQ ID NO: 1: MEVFKAGFSIADESVIGAVEKTDQGVTDAAEKTKESVM The sequence of the lysosomal protein GB1 is shown in SEQ ID NO: 2: YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE The sequence of the solubilizing protein StefinA is shown in SEQ ID NO: 3: IPGGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVVAGTNYYIKVRAGDNKYMHLKVFKSLPGQNEDLVLTGYQVDKNKDDELTGF The sequence of the lysosomal protein p53TD / E2K is shown in SEQ ID NO: 4: GEYFTLQIRGRERRFEMFRKLNKALELKDAQA The recombinant PTH1-34 polypeptide sequence is shown in SEQ ID NO: 5: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF The adapter sequence contains a His tag and a TEV enzyme cleavage site, wherein: His tag sequence is shown in SEQ ID NO: 6: HHHHHH The TEV enzyme cleavage site is shown in SEQ ID NO: 7: ENLYFQ.

[0046] The gene expression plasmid, verified by sequencing, was transformed into *E. coli* BL21 (DE3) competent cells. Two single-clone strains, numbered 1# and 2#, were picked and inoculated into Erlenmeyer flasks containing 10 mL of LB medium and cultured overnight at 37°C with shaking. The next day, the plasmids were transferred at a 1:50 volume ratio to Erlenmeyer flasks containing 50 mL of fresh LB medium and cultured at 37°C and 200 rpm with shaking for approximately 2 hours, until the bacterial culture reached OD500. 600 When the protein expression reached a concentration of 0.8–1.0, IPTG at a final concentration of 0.1 mM was added to induce protein expression. Samples were taken at 5 h and 20 h after induction, and the bacterial cell pellet and culture supernatant were separated by centrifugation. Both were analyzed by SDS-PAGE. The grayscale analysis of the SDS-PAGE gel images of the fusion protein was performed using analytical software to obtain the proportion of the expressed fusion protein to the total protein.

[0047] The proportion of fusion proteins in the total protein expression in the bacterial cells is shown in Figures 2 and 3 and Table 1. The results show that the expression level of the lysing protein GB1 was 32% at 5 h, decreasing to 19% at 20 h; StefinA expression was 32.2% at 5 h and 21.6% at 20 h; p53TD / E2K had the highest expression level, reaching 54% at 5 h and further increasing to 59.4% at 20 h. Based on these results, subsequent studies will focus on fermentation expression of strains transformed with the p53TD / E2K expression vector.

[0048] Table 1

[0049] Example 2: Fermentation expression of recombinant parathyroid hormone PTH1-34 fusion protein in a 5L fermenter.

[0050] 1. Primary Seed Preparation 1.1 Preparation of the plate: Bacterial suspensions were extracted from glycerol-preserved bacterial strains and serially diluted to 10⁻⁶ using sterilized LB medium. -5 Concentration. Take 100 μL of the diluted bacterial suspension and spread it evenly on the prepared LB solid medium plate. Incubate overnight at 37°C. After a single, uniform colony has grown on the plate, transfer it to a refrigerator at 4°C for storage.

[0051] 1.2 Primary activation: Pick a plump single colony from a solid culture medium plate and inoculate it into LB liquid medium to prepare a primary seed culture. The volume of the culture is 50 mL / 250 mL Erlenmeyer flask, and 50 μL of kanamycin stock solution with a concentration of 50 mg / mL is added.

[0052] 1.3 Cultivation: After inoculation, the Erlenmeyer flasks were placed in a constant-temperature shaker, with the shaking speed set to 250 rpm and the temperature at 30°C, and cultured for 9 hours to obtain the primary seed culture. The OD of the bacterial culture was... 600 The value is approximately 1.5.

[0053] 2. Secondary seed preparation 2.1. Vaccination: Remove the primary seed culture flask from the constant temperature shaking incubator, and inoculate 270 μL of bacterial culture in a clean bench. Transfer the culture to the secondary seed culture medium at a 1‰ volume ratio. The secondary seed culture volume is 270 mL / L, with 300 μL of kanamycin stock solution added simultaneously.

[0054] 2.2. Cultivation: After inoculation, the Erlenmeyer flasks were placed in a constant-temperature shaker, set to 250 rpm and 30°C, and incubated for 10 hours to obtain secondary seed cultures. The OD of the bacterial culture was... 600 The value is approximately 4.0.

[0055] 2.3. Microscopic examination: Aseptic sampling was performed in a clean bench. After aspirating 1 mL of bacterial suspension, 10 μL was spread onto a glass slide for microscopic examination. During microscopic examination, the size and morphology of the bacteria in the field of view were observed to confirm the absence of contamination. Gram staining and microscopic examination showed that the bacteria were red and had the typical short rod-shaped morphology of Escherichia coli.

[0056] 3. Fermentation 3.1. Preparations before fermentation 3.1.1 Check the equipment status and confirm that the equipment is usable; 3.1.2 Check steam pipes, valves, motors, power supplies, air filters, water pipes, etc. for leaks or disconnections; 3.1.3 Clean the inner and outer walls of the fermentation tank with distilled water; 3.1.4 Add fermentation medium and calibrate the pH and DO electrodes; pH electrode calibration: Connect the pH electrode to the fermentation control system. First, place the pH electrode probe in 7.00. Wait until the pH in the system displays 7.00 and the calibration is stable. Then, place the pH electrode probe in 4.01. Wait until the pH in the system displays 4.01 and the calibration is stable.

[0057] Dissolved oxygen electrode calibration: Zero-point calibration: Connect the dissolved oxygen electrode to the fermentation control system, place the electrode tip in a 5% saturated sodium sulfite solution, and the calibration is complete when the system zero point displays 0% or stabilizes near zero. Dissolved oxygen 100% calibration: At a temperature of 37℃, set the aeration rate to 10.0 SLPM and the stirring speed to 1000 rpm. Begin calibrating to 100% dissolved oxygen and wait for it to stabilize before completing the calibration.

[0058] 3.1.5 Tighten the top cover bolts and clamp all pipes, leaving only the vent for ventilation; 3.1.6 Insert the probe into the fermenter; 3.1.7 Place the fermenter into an autoclave for off-site sterilization; 3.1.8 After sterilization, tighten the vent, turn on the motor and all probes, and turn on the cold water to cool it down; 3.2. Vaccination Under flame protection, aseptically transfer the base glucose, magnesium sulfate, and secondary seed culture into the fermenter, and connect the acid, alkali, defoamer, and feed culture medium. Set the fermentation parameters, enter the fermentation batch number, and begin fermentation.

[0059] 3.3. Temperature control during fermentation during the induction phase (explanation) 3.4. Dissolved oxygen control The rotation speed is always controlled in conjunction with the aeration rate, with an initial speed of 300 rpm and a maximum speed of 800 rpm. Dissolved oxygen is controlled at 35% before induction and at 25% after induction. Adjustment is made when lowering the fermenter temperature, also adjusting the dissolved oxygen to 25%.

[0060] 3.5. Feeding Add 1‰ of trace elements to the fed culture medium before fermentation.

[0061] Feeding method: When the microbial cells in the fermenter grow to the OD level... 600 Feeding was initiated at a rate of 27.6 ml / h from 12 to 14 days post-fermentation. After IPTG induction, the feeding rate was increased to 30 ml / h. The feeding rate was finely adjusted according to dissolved oxygen levels during fermentation and induction until fermentation was complete.

[0062] 4. Induction The culture temperature before induction was 37℃, and OD was induced. 600 The value is 70-80, the induction temperature is 30℃, and the OD is measured before reaching the induction value. 600 At 65°C, the temperature was adjusted to 30°C, and the dissolved oxygen was adjusted to 32%. The inducer was added initially at 0.1 mM, and then 0.1 mM was added again after 4 hours, for a total of four additions, inducing for a total of about 16 hours.

[0063] 5. Sampling of fermentation broth, post-induction monitoring, and harvesting of cells / supernatant. (1) Timed sampling and expression detection during induction process After adding IPTG to initiate induction, sample 1 mL of fermentation broth every hour until induction is complete. Simultaneously, retain the sample from the 0-hour induction period as a control. Divide each 1 mL sample into two tubes, 0.5 mL each, for subsequent cell processing and supernatant processing, respectively.

[0064] (2) Bacterial cell treatment Bacterial cell treatment was used to detect total expression levels: based on bacterial culture OD... 600 Add an appropriate amount of purified water to adjust 0.5 ml of bacterial suspension to OD value. 600 =10; then add an appropriate amount of 5×SDS loading buffer, heat and incubate at 100℃ for 5 min, centrifuge at 9000 rpm for 3 min, and take 13 μL of supernatant for SDS-PAGE electrophoresis.

[0065] (3) Supernatant treatment The supernatant was used to detect the secretion expression level: Take another tube of 0.5 mL of the post-induction fermentation broth, centrifuge at 4℃ and 12,000 rpm for 10 min, and collect the supernatant. Subsequent processing and electrophoresis methods were the same as for the bacterial cell samples.

[0066] (4) Method for retaining fermentation broth After fermentation, 1 L of fermentation broth was taken from each fermentation tank and centrifuged at 9000 rpm for 20 min at 4℃. The supernatant and bacterial precipitate were collected separately and stored at -20℃.

[0067] 6. After fermentation is complete, remove the fermentation liquid and clean and disinfect the fermentation tank and experimental environment; 7. Fill out the instrument usage record.

[0068] 8. Fermentation results.

[0069] The proportion of fusion protein expression in bacterial cells to total protein expression is shown in the figure. Figure 4 According to Table 2, after 16 h of induction, the proportion of fusion protein in the target strain reached 39.2%; the cell yield during fermentation was 167 g / L, and the fermentation yield of fusion protein was 22 g / L.

[0070] Table 2

[0071] This invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A vesicle nucleation signal peptide SP6, comprising or consisting of (a) or (b): (a) a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 1; (b) a derivative polypeptide derived from (a) with one or several amino acids substituted, deleted or added in the amino acid sequence defined in (a) and retaining the biological function of the original sequence.

2. A fusion protein comprising the signal peptide SP6 of claim 1 and a target polypeptide.

3. The fusion protein of claim 2, comprising in order from N-terminus to C-terminus: (1) the signal peptide SP6; (2) a solubility enhancing protein; (3) a linker; (4) a target polypeptide. Preferably, the target polypeptide can be a polypeptide and a protein of any length; further preferably, the target polypeptide can have a length of 20-200, 25-150, 30-120, 30-100 amino acid residues, for example, about 30, about 40, about 50, about 60, about 70, about 80, about 90 amino acid residues. Preferably, the target polypeptide is selected from parathyroid hormone PTH, glucagon-like peptide GLP-1, brain natriuretic peptide BNP, exendin-4, chemokine CCL5, stromal cell-derived factor SDF-1a, growth-promoting factor IGF-1a, obesity hormone Lep, calcitonin, sermorelin, thymosin, lepirudin, cecropin, human histatin, defensin, and human plasminogen Kringle 1-5, or a biologically active fragment thereof. Preferably, the target polypeptide is a PTH active fragment, comprising or consisting of (c) or (d): (c) PTH1-34 as set forth in SEQ ID NO: 5; (d) a derivative polypeptide derived from (c) with one or several amino acids substituted, deleted or added in the amino acid sequence defined in (c) and retaining the biological function of the original sequence.

4. The fusion protein of claim 3, wherein the solubility enhancing protein is selected from P53TD and mutants thereof, GB1, StefinA, NusA, GST, Trx, SUMO, DsbC, Z, MBP, T7PK, or a combination thereof; preferably, the P53TD mutant is selected from P53TD / K, more preferably P53TD / E2K. Preferably, the solubility enhancing protein comprises or consists of (e) or (f): (e) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NO. 2 to SEQ ID NO. 4; (f) a derivative polypeptide derived from (e) with one or several amino acids substituted, deleted or added in the amino acid sequence defined in (e) and retaining the biological function of the original sequence. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 5. The fusion protein of claim 3, wherein the linker comprises a purification tag and a cleavage site; preferably, the purification tag is a His tag (HHHHHH) or a glutathione transferase tag (GST-tag); preferably, the cleavage site is a chemical cleavage site, a self-cleavage site or an enzymatic cleavage site; further preferably, the enzymatic cleavage site comprises an enterokinase cleavage site, a TEV protease cleavage site, a thrombin cleavage site and / or a 3C protease cleavage site and functional equivalent variants of the above sites and retain the specific cleavage function.

6. A nucleic acid molecule encoding the signal peptide of claim 1 or the fusion protein of any one of claims 2-5; preferably, the nucleic acid molecule comprises: (1) a first polynucleotide encoding the signal peptide; (2) a second polynucleotide encoding the solubility enhancing protein; (3) a third polynucleotide encoding the linker; (4) a fourth polynucleotide encoding the polypeptide of interest; the first to fourth polynucleotides are linked in sequence to express a fusion protein comprising the signal peptide-solubility enhancing protein-linker-polypeptide of interest.

7. An expression plasmid vector expressing the signal peptide of claim 1 or the fusion protein of any one of claims 2-5, or comprising the nucleic acid molecule of claim 6; preferably, the expression plasmid vector comprises a pET28a plasmid, a pET9a plasmid, a pET21a plasmid, a pET24a plasmid, a pET30a plasmid and / or a pET32a.

8. A host cell expressing the signal peptide of claim 1 or the fusion protein of any one of claims 2-5, or comprising the nucleic acid molecule of claim 6 or one or more expression plasmid vectors of claim 7; preferably, the host cell is a transformant, which is a host cell that has been altered by the introduction of one or more expression plasmid vectors into the host cell; preferably, the host cell is a recombinant host cell comprising the above nucleic acid molecule integrated into the chromosome of the host cell; preferably, the nucleic acid molecule comprises first to fourth polynucleotides linked in sequence to express a fusion protein comprising the signal peptide-solubility enhancing protein-linker-polypeptide of interest; preferably, the host cell comprises a fungus, a bacterium, a plant cell and / or an animal cell; preferably, the host strain of the transformant or mutant host cell is selected from a bacterium or a fungus; alternatively, the host strain is selected from a wild type or genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Lactococcus lactis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; alternatively, the host strain is selected from a wild type or genetically engineered Escherichia coli; the Escherichia coli can be any strain derived from the Escherichia coli K-12 line or B line, or a derivative strain thereof (e.g. Escherichia coli BL21(DE3) or BL21star(DE3).

9. A method for preparing a polypeptide of interest, the method comprising: a) inoculating the host cell of claim 8 into a culture medium for fermentation to obtain a fermentation broth; b) isolating and purifying the fusion protein from the fermentation broth; c) separating the target polypeptide from the rest of the fusion protein by cleaving the linker; d) removing the rest of the fusion protein to obtain the purified target polypeptide.

10. Use of the signal peptide of claim 1, the fusion protein of any one of claims 2-5, the nucleic acid molecule of claim 6, the expression plasmid vector of claim 7, or the host cell of claim 8 in the preparation of a target polypeptide.

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