A method for preparing a recombinant human granulocyte colony stimulating factor

Abstract

The application provides a preparation method of recombinant human granulocyte colony stimulating factor (rhG-CSF). Specifically, the rhG-CSF is expressed by using SUMO fusion. The soluble expression of the fusion protein in Escherichia coli is realized by using an Escherichia coli expression system. And high-purity rhG-CSF is obtained through fermentation, pretreatment and purification and the like. Different from a traditional inclusion body process, the preparation method can directly obtain the rhG-CSF with biological activity, and has the characteristics of high yield, simple operation and low cost, and is suitable for industrialized and efficient production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. Specifically, this invention relates to a method for preparing recombinant human granulocyte colony-stimulating factor. Background Technology

[0002] Granulocyte colony-stimulating factor (G-CSF) promotes the proliferation and differentiation of granulocyte-lineage hematopoietic stem cells and enhances the function of mature cells. It can promote neutrophil proliferation in bone marrow transplantation and has significant therapeutic effects on severe neutropenia caused by cancer chemotherapy and neutropenia associated with aplastic anemia.

[0003] In 1985, Welte K successfully purified and refined human granulocyte colony-stimulating factor (hG-CSF) from the culture supernatant of human bladder cancer cell line 5637. Welte K and Souza LM of Amgen in the United States further determined the N-terminal amino acid sequence of this hG-CSF and cloned the hG-CSF gene from the 5637 cell line. Using genetic engineering technology, the gene was inserted into E. coli to obtain hG-CSF, and recombinant human granulocyte colony-stimulating factor (rhG-CSF) was developed, commercially known as Filgratin (Souza LM, et al, Sicense, 1986, 232:61-65).

[0004] Because the sources of hG-CSF are very limited, large-scale industrial production relies on genetic engineering methods. Currently, most pharmaceutical companies, both domestically and internationally, use the widely adopted *E. coli* system to express rhG-CSF. The rhG-CSF molecule contains a free cysteine ​​residue at position 17 and two disulfide bonds, namely CyS36-Cys42 and Cys64-Cys74, which are essential for its activity. Due to the reducing environment within *E. coli* cells, which is unfavorable for disulfide bond formation, rhG-CSF misfolds and easily aggregates, existing primarily as biologically inactive inclusion bodies within the cell. Denaturation and renaturation are necessary to obtain active rhG-CSF. However, due to aggregate formation and disulfide bond mismatches during renaturation, the renaturation rate is low, and multiple subsequent purification steps are required to purify the renatured protein (CN103228672), resulting in low production efficiency.

[0005] Fusion expression technology is a commonly used technique for preparing recombinant proteins. Introducing a tag protein for fusion expression can play an important role in preventing the target protein from being degraded, shielding the host from the toxic effects of exogenous proteins, and increasing expression yield. In the production process, the tag protein can be removed by a matching protease to obtain the target protein.

[0006] Therefore, there is an urgent need in this field for a simple, high-yield, low-cost, and high-volume method for preparing recombinant human granulocyte colony-stimulating factor. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing recombinant human granulocyte colony-stimulating factor. This method not only needs to be simple to operate, have a high yield, low cost, and high production volume, but also requires that the prepared recombinant human granulocyte colony-stimulating factor can be correctly folded, thereby having activity.

[0008] Another objective of this invention is to provide a fusion protein for the preparation of recombinant human granulocyte colony-stimulating factor.

[0009] In a first aspect, the present invention provides a method for preparing rhG-CSF, characterized in that the method comprises the following steps:

[0010] (a) Culture host cells containing the gene encoding the fusion protein shown in Formula I under suitable expression conditions;

[0011] (b) Obtain the fusion protein from the host cells cultured in step (a); and

[0012] (c) Optionally process the fusion protein to obtain rhG-CSF;

[0013] ABC (I)

[0014] In this context, region A may be an optional signal peptide or may not exist.

[0015] Section B is the SUMO fusion tag;

[0016] The C segment is the amino acid sequence of rhG-CSF;

[0017] "-" indicates an optional connector.

[0018] In a specific embodiment, the amino acid sequence of the SUMO fusion tag is shown in SEQ ID NO:1.

[0019] In a specific embodiment, the amino acid sequence of the rhG-CSF is shown in SEQ ID NO:2.

[0020] In a specific embodiment, the amino acid sequence of the fusion protein shown in Formula I is as shown in SEQ ID NO:3.

[0021] In a preferred embodiment, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:4.

[0022] In a preferred embodiment, the host cell includes a eukaryotic cell or a prokaryotic cell.

[0023] In a preferred embodiment, the eukaryotic cells include mammalian cells, yeast cells, or insect cells.

[0024] In a preferred embodiment, the mammalian cells include: Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, COS cells, mouse NSO thymoma cells, and mouse myeloma SP2 / 0 cells.

[0025] In a preferred embodiment, the yeast cells include: Pichia pastoris and Saccharomyces cerevisiae.

[0026] In a preferred embodiment, the insect cells include Sf21 and Sf9 ovarian cells of the fall armyworm.

[0027] In a preferred embodiment, the prokaryotic cell is a host cell derived from bacteria, such as Escherichia coli or Bacillus subtilis; preferably, Escherichia coli cells, such as Escherichia coli BL21(DE3).

[0028] In a preferred embodiment, step (b) includes releasing the fusion protein by rupturing engineered E. coli bacteria.

[0029] In a preferred embodiment, the method for breaking down the engineered bacteria of the fusion protein can be chemical, osmotic pressure impact, enzymatic, ultrasonic, freeze-thaw, bead milling, high-pressure homogenization, or spray impact, preferably ultrasonic or high-pressure homogenization, and more preferably high-pressure homogenization.

[0030] In a preferred embodiment, the solution prior to high-pressure homogenization can be obtained by resuspending engineered Escherichia coli in a homogenization buffer containing additives, wherein the volume ratio of the buffer to the wet weight of the engineered bacteria is 10:1 (ml:g).

[0031] In a preferred embodiment, the homogenate buffer contains tris(hydroxymethyl)aminomethane, disodium hydrogen phosphate, and sodium dihydrogen phosphate as buffering components, preferably tris(hydroxymethyl)aminomethane.

[0032] In a preferred embodiment, the buffer solution has a pH of 7.5-8.5, preferably a pH of 8.0, and contains 5 mM disodium ethylenediaminetetraacetate.

[0033] In a preferred embodiment, the homogenization pressure is 800 bar, and the homogenization is performed three times to obtain a homogenate containing the fusion protein.

[0034] In a preferred embodiment, step (c) includes:

[0035] ①Ulp1 enzyme was used to digest the fusion protein to obtain a pair of disulfide-paired rhG-CSF;

[0036] ② Pairing, causing another pair of disulfide bonds to pair;

[0037] ③ Acid precipitation is used for coarse separation, the precipitate is the impurity, and the target product is in the supernatant;

[0038] ④ Use anion exchange chromatography, flow through rhG-CSF, and collect the flow-through solution;

[0039] ⑤ Using cation chromatography, rhG-CSF is adsorbed on the chromatography column and eluted with a high-salt mobile phase;

[0040] ⑥ Use ammonium sulfate precipitation to precipitate the target protein and further separate impurities;

[0041] ⑦ Redissolve in buffer solution, and then replace with ultrafiltration in the storage solution to obtain rhG-CSF stock solution.

[0042] In a preferred embodiment, the pH of the Ulp1 enzyme digestion in step ① can be 5.5-10.5, preferably 6-8, and most preferably 8.0; the digestion temperature can be 4-37℃, preferably 30℃; and the enzyme dosage of Ulp1 enzyme digestion can be 3000:1, 2000:1, 1000:1, 500:1, or 300:1 (V). 匀浆液 :V Ulp1 The preferred enzyme ratios are 500:1 and 300:1, with the optimal ratio being 500:1. The Ulp1 enzyme digestion time can be 10-60 min, preferably 30 or 40 min, with the most preferred being 30 min.

[0043] In a preferred embodiment, the disulfide bond pairing described in step ② can be achieved by stirring in a buffer solution with a pH of 8.0, 8.5, 9.0, 9.5, 10.0, preferably 8.5, 9.0, 9.5, and most preferably 8.5.

[0044] In a preferred embodiment, the disulfide bond pairing described in step ② can be carried out in a buffer solution with a pH of 8.0, 8.5, 9.0, 9.5, or 10.0, preferably 8.5, 9.0, or 9.5, and most preferably 8.5.

[0045] In a preferred embodiment, step ② involves adding an additive during disulfide bond pairing. The additive may be hydrogen peroxide, purified water, cystine, or glutathione, preferably purified water and cystine, and most preferably an equal volume of purified water.

[0046] In a preferred embodiment, the pairing time in step ② can be 1-4 hours, preferably 2 hours.

[0047] In a preferred embodiment, the acid used in step ③ for acid precipitation to remove impurities can be hydrochloric acid or glacial acetic acid, preferably hydrochloric acid.

[0048] In a preferred embodiment, the acid precipitation can be carried out at a pH of 7.0, 6.0, 5.0, 4.0, 3.0, or 2.0, preferably 4.0 or 3.0, and most preferably 4.0.

[0049] In a preferred embodiment, the packing material used in the anion exchange chromatography in step ④ can be a strong anion or weak anion packing material such as QFF, Capto Q, Poly PEI, or DEAE, with QFF packing material being the most preferred; the sample loading and buffer solution pH of the anion exchange chromatography can be 7.0-9.0, preferably 7.0, 7.5, or 8.0, with 8.0 being the most preferred.

[0050] In a preferred embodiment, the packing material used in step ⑤ of the cation exchange chromatography can be a strong cation or weak cation packing material such as SPFF, Capto S, or CM FF, with SPFF being the most preferred. The sample loading and buffer solution pH of the cation exchange chromatography can be 3.0-5.0, preferably 4.0 or 4.5, with 4.5 being the most preferred. The buffer solution system can be acetate-sodium acetate or citric acid-sodium citrate, with acetate-sodium acetate being the most preferred.

[0051] In a preferred embodiment, the ammonium sulfate precipitate in step ⑥ has an ammonium sulfate concentration of 10-60%, preferably 30-40%, and most preferably 33%.

[0052] In a preferred embodiment, the reconstitution buffer solution in step ⑦ can be Tris-HCl or PBS, preferably a PBS buffer solution.

[0053] In a preferred embodiment, the preservation solution described in step ⑦ is a PBS buffer solution.

[0054] In a preferred embodiment, the preparation method further includes step (d) performing mass spectrometry analysis, peptide mapping analysis, and cell activity assay on the obtained rhG-CSG.

[0055] In a preferred embodiment, the rhG-CSF is a soluble expression.

[0056] In a preferred embodiment, the disulfide bond pairing rate of rhG-CSF obtained by the preparation method is above 80%, preferably above 90%, and most preferably above 98%.

[0057] In a preferred embodiment, the expression level of rhG-CSF obtained by the preparation method is 2.1 g / L, preferably 2.2 g / L, and most preferably 2.3 g / L.

[0058] In a preferred embodiment, the purity of rhG-CSF obtained by the preparation method is greater than 99%.

[0059] In a preferred embodiment, the preparation method does not involve denaturation and refolding steps.

[0060] In a second aspect, the present invention provides the fusion protein shown in Figure I:

[0061] ABC(I)

[0062] In this context, region A may be an optional signal peptide or may not exist.

[0063] Section B is the SUMO fusion tag;

[0064] The C segment is the amino acid sequence of rhG-CSF;

[0065] "-" indicates an optional connector.

[0066] In a specific embodiment, the amino acid sequence of the SUMO fusion tag is shown in SEQ ID NO:1.

[0067] In a specific embodiment, the amino acid sequence of the rhG-CSF is shown in SEQ ID NO:2.

[0068] In a specific embodiment, the amino acid sequence of the fusion protein shown in Formula I is as shown in SEQ ID NO:3.

[0069] In a third aspect, the present invention provides an isolated polynucleotide molecule that encodes the fusion protein described in the second aspect.

[0070] In a preferred embodiment, the nucleotide sequence of the polynucleotide molecule is shown in SEQ ID NO:4.

[0071] In a fourth aspect, the present invention provides an expression vector comprising the polynucleotide molecule described in the third aspect.

[0072] In a preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof; preferably pET28a.

[0073] In a fifth aspect, the present invention provides a host cell containing the expression vector described in the fourth aspect, or an exogenous polynucleotide molecule described in the third aspect integrated into the chromosome, or expressing the fusion protein described in the first aspect.

[0074] In a preferred embodiment, the host cell includes a eukaryotic cell or a prokaryotic cell.

[0075] In a preferred embodiment, the eukaryotic cells include mammalian cells, yeast cells, or insect cells.

[0076] In a preferred embodiment, the mammalian cells include: Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, COS cells, mouse NSO thymoma cells, and mouse myeloma SP2 / 0 cells.

[0077] In a preferred embodiment, the yeast cells include: Pichia pastoris and Saccharomyces cerevisiae.

[0078] In a preferred embodiment, the insect cells include Sf21 and Sf9 ovarian cells of the fall armyworm.

[0079] In a preferred embodiment, the prokaryotic cell is a host cell derived from bacteria, such as Escherichia coli or Bacillus subtilis; preferably, Escherichia coli cells, such as Escherichia coli BL21(DE3).

[0080] In a sixth aspect, the present invention provides the use of the fusion protein described in the second aspect in the preparation of recombinant human granulocyte colony-stimulating factor.

[0081] In a seventh aspect, the present invention provides a kit for preparing recombinant human granulocyte colony-stimulating factor, the kit comprising:

[0082] 1) The fusion protein described in the second aspect;

[0083] 2) Instructions for use in preparing recombinant human granulocyte colony-stimulating factor using this fusion protein; and

[0084] 3) Optional other necessary reagents for the preparation of recombinant human granulocyte colony-stimulating factor using this fusion protein.

[0085] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0086] Figure 1 The plasmid map of the fusion protein is displayed;

[0087] Figure 2 The electrophoresis results show the amount of enzyme added to the fusion protein digested;

[0088] Figure 3 The data map of enzyme digestion time of fusion protein is displayed;

[0089] Figure 4 The liquid phase analysis chromatograms show the validation of the enzyme digestion process;

[0090] Figure 5 The electrophoresis results at different pH values ​​during the acid precipitation process are shown.

[0091] Figure 6 The liquid phase analysis chromatograms for reviewing process verification are displayed;

[0092] Figure 7 The mass spectrum of rhG-CSF prepared by the present invention is shown;

[0093] Figure 8 The RP-HPLC chromatograms of rhG-CSF prepared by the present invention and commercial rhG-CSF are shown.

[0094] Figure 9 The peptide mapping analysis of rhG-CSF prepared by the present invention is shown;

[0095] Figure 10 Cell growth curves obtained using rhG-CSF control and rhG-CSF prepared according to the present invention are shown. Detailed Implementation

[0096] Through extensive and in-depth research, and through numerous screenings and tests, the inventors have provided a method for preparing rhG-CSF. This method involves constructing a genetically engineered bacterium expressing the rhG-CSF fusion protein, performing high-density fermentation, extracting the fusion protein, removing the tag, and purifying it to obtain rhG-CSF. Unlike traditional methods using inclusion bodies, this invention uses engineered bacteria to solublely express the fusion protein intracellularly, followed by treatment of the fusion protein to obtain rhG-CSF. This method offers advantages such as simple operation, high yield, low cost, and high production volume. Based on this, the present invention was completed.

[0097] the term

[0098] To facilitate understanding of this invention, certain technical terms are specifically defined below. Unless otherwise specified herein, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0099] A numerical range includes the numbers within that range. Therefore, each numerical range disclosed herein is intended to include every narrower numerical range falling within such a wider numerical range, as if such narrower numerical ranges were explicitly written herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein include every lower (or higher) numerical limit, as if such lower (or higher) numerical limit were explicitly written herein.

[0100] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”. And vice versa.

[0101] Unless otherwise stated, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in the direction from amino to carboxyl.

[0102] The terms “protein,” “polypeptide,” and “peptide” as used herein are used interchangeably to refer to polymers in which at least two amino acids are covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0103] The term "amino acid" as used in this article is represented by its commonly known three-letter symbol or the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides can also be represented by their commonly accepted single-letter codes.

[0104] As used herein, the terms “engineered” and “recombinant”, when applied to cells, polynucleotides, or polypeptides, refer to a material or a material corresponding to the natural or natural form of a material that has been modified in a manner not present in or similar to that found in nature, but which is produced or derived from synthetic materials and / or through the use of recombinant techniques.

[0105] The term "acid precipitation" as used in this article refers to the precipitation of impurities under low pH conditions by taking advantage of the differences in isoelectric points and stability of different proteins, while the target protein remains in the supernatant.

[0106] As used herein, the term "vector" is a DNA construct used to introduce a DNA sequence into a cell. In some embodiments, the vector is an expression vector operatively linked to a suitable control sequence capable of expressing a polypeptide encoded in the DNA sequence in a suitable host. In some embodiments, the "expression vector" has a promoter sequence operatively linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0107] As used herein, the term "expression" includes any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes the secretion of peptides from cells.

[0108] As used herein, the term "production" refers to the generation of proteins and / or other compounds by cells. This term includes any steps involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes the secretion of peptides from cells.

[0109] As used herein, the terms "host cell" and "host strain" refer to a suitable host for the expression vector (e.g., a polynucleotide encoding a mucin-binding fusion protein variant) used to bind to the DNA provided herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA technologies known in the art.

[0110] As used in this article, the term "culture" refers to the growth of a microbial cell population under any suitable conditions (e.g., using liquid, gel, or solid culture media).

[0111] As used herein, the terms “isolated” and “purified” refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components removed from at least one other component naturally associated with them. The term “purified” does not require absolute purity, but is used as a relative definition.

[0112] As used in this article, "homogeneous mixture" refers to the process of lysing or mechanically lysing host cells to release intracellular substances from the host cells. The system containing lysed host cells and intracellular substances obtained after homogenizing and disrupting cells is called a "homogeneous mixture".

[0113] The term "SUMO fusion tag" used in this article refers to a polypeptide that is added to the N-terminus of a target protein using DNA recombination technology. This allows the fusion protein to be expressed in a soluble form, thereby improving the expression level of the target protein, preventing protease hydrolysis, and increasing product purity.

[0114] The fusion protein and its preparation method of the present invention

[0115] The inventors have discovered that after rhG-CSF is made into a fusion protein with a specific fusion tag, a rhG-CSF fusion protein with a correctly paired disulfide bond can be obtained by culturing host cells containing the encoding gene of the fusion protein. After enzymatic digestion and simple disulfide bond pairing, biologically active rhG-CSF can be obtained.

[0116] In a specific embodiment, the fusion protein of the present invention is a fusion protein as shown in Formula I:

[0117] ABC (I)

[0118] In this sequence, region A is an optional signal peptide or may not be present; region B is a SUMO fusion tag; region C is the amino acid sequence of rhG-CSF; and "-" is an optional linker.

[0119] In a specific embodiment, the amino acid sequence of the SUMO fusion tag is shown in SEQ ID NO:1; the amino acid sequence of the rhG-CSF is shown in SEQ ID NO:2. In a preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO:3.

[0120] As is well known to those skilled in the art, mammalian-derived proteins, especially human proteins, often require expression in eukaryotic host cells. However, the culture of eukaryotic host cells, such as mammalian cells, often suffers from drawbacks such as low yield, high cost, and complex operation. Therefore, it would be ideal to express mammalian-derived proteins, especially human proteins, in prokaryotic host cells. rhG-CSF is often expressed in prokaryotic cells as intracellular inclusion bodies, requiring denaturation and renaturation processes to form rhG-CSF with correctly paired disulfide bonds. The inventors have discovered that after rhG-CSF is formulated into the fusion protein of this invention, the fusion protein can be solublely expressed in prokaryotic cells. The expressed rhG-CSF obtained from this fusion protein possesses a correctly paired disulfide bond, and since the rhG-CSF is in a soluble state, denaturation is not required; the correct pairing of the two disulfide bonds can be achieved through simple renaturation conditions. Therefore, the fusion protein of this invention can be expressed in various host cells to obtain rhG-CSF. For example, the host cells include eukaryotic cells or prokaryotic cells. In a preferred embodiment, the eukaryotic cells include mammalian cells, such as Chinese hamster ovary (CHO) cells, small hamster kidney (BHK) cells, COS cells, mouse NSO thymoma cells, and mouse myeloma SP2 / 0 cells; yeast cells, such as Pichia pastoris and Saccharomyces cerevisiae; insect cells, such as fall armyworm ovary cells Sf21 cells and fall armyworm ovary cells Sf9 cells; the prokaryotic cells are host cells derived from bacteria, such as Escherichia coli and Bacillus subtilis; preferably Escherichia coli cells, such as Escherichia coli BL21(DE3).

[0121] To remove the SUMO fusion tag from the fusion protein of this invention, the tool enzyme U1p1 can be used. The tool enzyme U1p1 can recognize the three-dimensional structure of SUMO proteins, thus removing the SUMO tag from the fusion protein with extremely high specificity. The cleavage site of the U1p1 enzyme is located on the carboxyl side of the C-terminus Gly of the SUMO-tagged protein. After cleavage by U1p1, the N-terminus of the target protein does not contain any extra amino acid residues (Tauseef R. Butt, et al. Protein Expression and Purification 43.1(2005):1-9).

[0122] Based on the teachings of this invention and conventional techniques in the art, those skilled in the art can express the fusion protein of this invention in various ways known in the art. For example, a plasmid containing the coding gene of the fusion protein of this invention can be introduced into a host cell, or the coding gene can be integrated into the genome of a host cell, and the host cell can be cultured accordingly.

[0123] Based on the fusion protein of the present invention, the present invention provides a method for preparing rhG-CSF, the method comprising the steps of culturing host cells containing the encoding gene of the fusion protein shown in Formula I; obtaining the fusion protein from the cultured host cells; and processing the fusion protein to obtain rhG-CSF.

[0124] Those skilled in the art can process the obtained fusion protein using various known methods, but a preferred processing method includes the following steps: ① digesting the fusion protein with Ulp1 enzyme to obtain rhG-CSF with one disulfide bond pair; ② pairing, and pairing another disulfide bond pair; ③ performing coarse separation by acid precipitation, precipitating impurities, with the target product in the supernatant; ④ performing anion exchange chromatography, allowing rhG-CSF to flow through, and collecting the flow-through; ⑤ performing cation exchange chromatography, adsorbing rhG-CSF onto the chromatography column, and eluting with a high-salt mobile phase; ⑥ precipitating the target protein by ammonium sulfate precipitation, and further separating impurities; ⑦ reconstituted with a buffer solution, and then replaced with a storage solution by ultrafiltration to obtain the rhG-CSF stock solution.

[0125] Based on the description of this invention, those skilled in the art will understand that the fusion protein of this invention can be used to prepare rhG-CSF. Therefore, the scope of protection of this invention includes the fusion protein, as well as the method for directly preparing rhG-CSF using the fusion protein of this invention; that is, the method for preparing rhG-CSF of this invention may or may not include the step of preparing the fusion protein of this invention itself.

[0126] Furthermore, the fusion protein of the present invention can be formulated into a kit for preparing rhG-CSF. The kit includes the fusion protein of the present invention and instructions for use in preparing rhG-CSF using the fusion protein. The kit may also include other necessary reagents for preparing rhG-CSF using the fusion protein, such as reagents required for the above-mentioned acid precipitation, enzymatic digestion, and purification of the fusion protein. These reagents are well known to those skilled in the art and can be selected independently according to the specific fusion protein.

[0127] The main advantages of this invention are:

[0128] 1. Soluble expression of rhG-CSF was achieved through fusion expression with SUMO;

[0129] 2. Soluble-expressed rhG-CSF does not require denaturation and already has one pair of correctly paired disulfide bonds. The other pair of disulfide bonds can be correctly paired by simple water dilution and stirring, thus obtaining active rhG-CSF with a yield much higher than that of traditional denaturation and renaturation processes.

[0130] 3. For the first time, after chromatographic purification of rhG-CSF, ammonium sulfate precipitation was used for further purification to obtain rhG-CSF with a purity greater than 99%.

[0131] 4. The preparation process of the present invention is simple to operate, low in cost and high in yield.

[0132] The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0133] The abbreviations used in the following embodiments are as follows:

[0134] SOB (Super Optimal Broth)

[0135] Kanamycin

[0136] LB (Luria-Bertani, broth culture medium)

[0137] PBS (phosphate buffered saline)

[0138] Tris (Tris(hydroxymethyl)aminomethane)

[0139] IPTG (Isopropyl-β-D-1-thiogalactopyranoside)

[0140] SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)

[0141] U1p1 (Ubiquitin-like-specific protease 1)

[0142] SUMO (small ubiquitin-related modifier)

[0143] Detection methods

[0144] Protein electrophoresis analysis

[0145] The speed at which a protein moves in an electric field depends on its charge, the strength of the electric field, and the resistance it encounters. Therefore, different proteins move at different speeds under the same conditions. For example, different proteins in human serum have different isoelectric points and move at different speeds in solutions with the same pH. Thus, during electrophoresis, individual proteins can be distinguished on an electrophoresis support (such as filter paper or agar). After staining, a clear band pattern is obtained. This invention uses sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for detection. A 12.5% ​​gel was prepared according to the method in the *Protein Purification Guide*, and reduction electrophoresis was performed for detection.

[0146] High-performance liquid chromatography (HPLC)

[0147] Chromatographic column: ACE C4, 150×4.6mm, 300μm;

[0148] Column temperature: 50℃;

[0149] Detection wavelength: 215nm;

[0150] Flow rate: 0.6 ml / min;

[0151] Mobile phase: A) 0.05% trifluoroacetic acid / aqueous solution (v / v);

[0152] B) 0.05% trifluoroacetic acid / acetonitrile solution (v / v);

[0153] The gradient elution method is as follows:

[0154] t / min A％ B％ 5 80 20 10 50 50 18 30 70 30 20 80 32 80 20

[0155] Liquid chromatography-mass spectrometry (LC-MS)

[0156] Ultra-high performance liquid chromatograph: Waters Acquity UPLC; Mass spectrometer detector: Waters ACQUITY QDa;

[0157] Chromatographic column: Waters ACQUITY UPLC Protein BEH C4 ( (1.7μm, 2.7×100mm);

[0158] Flow rate: 0.3 ml / min;

[0159] Mobile phase: A) 0.1% formic acid / water solution (v / v);

[0160] B) 0.1% formic acid / acetonitrile solution (v / v);

[0161] The elution gradient is as follows:

[0162] t / min A％ B％ 0 80 20 2 80 20 14 20 80 15 80 20

[0163] Example 1. Construction of recombinant Escherichia coli expressing rhG-CSF

[0164] (1) DNA sequence design

[0165] Following the preferred codons of *E. coli* BL21(DE3), the amino acid sequence of rhG-CSF was translated into a DNA sequence, named G-CSF, and fused with the SUMO protein for expression, thus constructing the target gene sequence SUMO-G-CSF. Restriction enzyme sites NcoI (C / CATGG) and BamHI (G / GATCC) were added to the 5' and 3' ends of the sequence, respectively, resulting in a total sequence length of 6092 bp. After synthesis by Shanghai Jierui Biotechnology, the target gene was transformed into the vector pET28a and named pET28a-SUMO-G-CSF.

[0166] The amino acid sequence of the rhG-CSF is SEQ ID NO:2;

[0167] The amino acid sequence of the SUMO fusion tag is SEQ ID NO:1;

[0168] The amino acid sequence of the SUMO-GCSF fusion protein is SEQ ID NO:3;

[0169] The nucleic acid sequence of the fusion protein is SEQ ID NO:4.

[0170] (2) Plasmid extraction

[0171] ① Inoculate Escherichia coli containing plasmid pET28a-SUMO-G-CSF into 10 ml of liquid LB medium containing antibiotic Kan, and culture at 37°C with shaking at 220 r / min for 16 h;

[0172] ② Centrifuge four 2ml centrifuge tubes at 12000r / min for 1min to collect all the bacterial cells;

[0173] ③ Add 100 μl of solution I to suspend the bacterial cells; add 300 μl of solution II and mix well; then add 300 μl of solution III and mix well. Incubate at -20℃ for 20 min, centrifuge at 12000 r / min for 10 min, and take the supernatant into 1.5 ml centrifuge tubes.

[0174] ④ Add 2 / 3 volume of isopropanol, mix well, place at -20℃ for 20 min, centrifuge at 12000 r / min for 10 min, and collect the precipitate;

[0175] ⑤ Add 1 ml of 75% ethanol, wash the precipitate, and discard the supernatant; after drying the precipitate at 37℃, add 50 μL of deionized water to each tube to dissolve the precipitate. Then collect the solutions from each tube into one tube, which is the plasmid pET28a-SUMO-G-CSF, and store it at -20℃ for later use.

[0176] (3) Transformation of competent cells

[0177] After mixing 2 μL of plasmid pET28a-SUMO-GCSF with competent Escherichia coli BL21(DE3) cells, the mixture was transferred to a pre-chilled 1 mm electroporation cuvette and electroporated. Then, 100 μL of SOB medium was quickly added and mixed thoroughly. The bacterial culture was then transferred to a sterile 1.5 mL centrifuge tube and incubated at 37°C with shaking for 60 min. Finally, the bacterial culture was aspirated and spread evenly on a Petri dish containing 25 μg / mL Kane solid LB medium and incubated at 37°C for 16–20 h.

[0178] (4) Screening and culture of engineered bacteria

[0179] Single colonies of the recombinant strain *Escherichia coli* BL21(DE3) / pET28a-SUMO-G-CSF were picked and inoculated into 10 mL of liquid LB medium containing 25 μg / mL Kan, and cultured at 37°C with shaking at 220 rpm for 5 h. Then, the culture was transferred to 30 mL of liquid SM504 medium containing 25 μg / mL Kan at an inoculation rate of 3% (3 mL of the above culture was added to every 100 mL of SM504 fermentation medium), and cultured at 37°C with shaking at 220 rpm for 3 h. IPTG was added to the culture to a final concentration of 0.2 mM for induction, and the culture was cultured at 30°C with shaking at 220 rpm for 6 h.

[0180] Collect 4 mL of bacterial culture in a 5 mL centrifuge tube, centrifuge at 12,000 rpm for 1 min, collect the bacterial cells, resuspend and wash once with 1 mL PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), centrifuge at 12,000 rpm for 1 min, discard the supernatant, add 1 mL PBS buffer to resuspend, pre-cool on ice, and then disrupt the cells using an ultrasonic cell disruptor (400 W, 5 s operation time, 10 s interval, 7 cycles), followed by centrifugation at 12,000 rpm for 10 min. The supernatant after centrifugation was found to contain SUMO-G-CSF fusion protein. Separately, mix 0.5 mL of the bacterial culture with 0.5 mL of 50% (v / v) glycerol in a glycerol tube and store at -80°C.

[0181] Example 2. Fermentation

[0182] (1) Preparation of primary seed liquid:

[0183] Recombinant Escherichia coli expressing the fusion protein, stored at -80℃, was inoculated into LB liquid medium (containing 25 mg / L kanamycin) and cultured in a shaker at 37℃ for 6 h.

[0184] (2) Preparation of secondary seed liquid:

[0185] After confirming that the primary seed culture was free of contamination under a microscope, the primary seed culture was inoculated into LB liquid medium (containing 25 mg / L kanamycin) and cultured at 37°C in a shaker for 12 h.

[0186] (3) Preparation of fermentation medium and fed medium

[0187] Fermentation medium composition: 30 g / L glycerol, 8.6 g / L Na2HPO4·12H2O, 4.4 g / L NaH2PO4·2H2O, 4.0 g / L KCl, 5.2 g / L (NH4)2SO4, 5.9 g / L Na3C6H5O7·2H2O, 1.0 g / L MgSO4, 0.2 g / L CaCl2 (sterilized separately), 1 mL / L trace elements, pH 7.0; sterilized at 121℃ for 30 min.

[0188] Trace element stock solution: 67.2 g / L FeSO4·7H2O, 20.0 g / L ZnSO4·7H2O, 24.4 g / L MnCl2·4H2O, 7.5 g / L CuSO4·5H2O, 1.8 g / L CoCl2·6H2O, 2.5 mL / L concentrated hydrochloric acid; store at 4℃.

[0189] Feeding medium composition: 75% glycerol (w / v), sterilized at 121°C for 30 min.

[0190] (4) Fermentation process regulation and cell collection

[0191] After confirming that the secondary seed culture was free of contamination under a microscope, it was inoculated into 3L of fermentation medium at an inoculation rate of 1%-3% (1-3ml of secondary seed culture per 100ml of fermentation medium). The initial fermentation parameters were set as follows:

[0192] Temperature 37℃, pH 6.95, rotation speed 300 rpm, aeration rate 3 L / min, tank pressure 0.1-0.12 MPa. Dissolved oxygen was controlled to be greater than 30% by adjusting the rotation speed, aeration rate, and tank pressure. Feeding was initiated after approximately 14 hours of fermentation when dissolved oxygen and pH showed a significant increase. After approximately 16 hours of fermentation, when OD... 600 When the pH value reaches 60-120, add IPTG to a final concentration of 0.1-0.3 mmol / L to begin induction. Stop fermentation after 6 hours to obtain a large volume of *E. coli* expressing the fusion protein. Centrifuge the culture at 7000 rpm for 15 minutes, collect the cells, and store at -20°C.

[0193] Example 3. High-pressure homogenization

[0194] The bacterial cells collected after fermentation were resuspended in homogenization buffer (25 mM Tris-HCl, 5 mM EDTA, pH 8.0) at a ratio of 1 g of bacterial cells to 10 ml of buffer. After resuspending, the cells were filtered through gauze and homogenized three times at 800-900 bar to dystrophy. The homogenate was collected. 1 mL of the homogenate was centrifuged at 15000 r / min for 5 min, and the supernatant was used for RP-HPLC analysis.

[0195] Example 4. Ulp1 enzyme cleavage fusion protein dosage

[0196] Since the fusion protein was in the homogenate and no standard was available, its content could not be quantified. Therefore, the appropriate enzyme dosage was determined by the volume ratio of the homogenate to the crude U1p1 enzyme. The homogenate from Example 3 was taken and the pH was adjusted to 8.0 with 1M NaOH. Enzyme dosages were then added at ratios of 3000:1, 2000:1, 1000:1, 500:1, and 300:1 (V... 匀浆液 / V Ulp1 Add Ulp1 solution (enzyme activity: 2115 U / mL), stir, and digest at 30℃ for 1 hour. Sample processing is the same as in Example 3. Separate samples are taken without centrifugation and analyzed directly by electrophoresis.

[0197] like Figure 2 As shown, the enzyme digestion rate is 97.4% when the enzyme ratio is 500:1. When the ratio is increased to 300:1, the enzyme digestion rate does not increase. Therefore, the enzyme ratio of 500:1 is preferred.

[0198] Example 5. Time for Ulp1 cleavage of fusion protein

[0199] Take the homogenate from Example 3 and adjust the pH to 8.0 with 1M NaOH. Mix according to a 500:1 (V) ratio. 匀浆液 / V Ulp1 Add Ulp1 solution (enzyme activity: 2115 U / mL), and perform enzymatic digestion under stirring and controlled temperature of 30℃. Samples are taken at 10, 20, 30, 40, 50, and 60 minutes. The sample processing method is the same as in Example 3.

[0200] like Figure 3 As shown, the digestion rate was 97.4% when the digestion time was 30 min, and 98.7% when the digestion time was 40 min. The digestion rate did not increase significantly with the extension of time, so the preferred digestion time was 30 min.

[0201] Example 6. Verification of U1p1 enzyme digestion conditions

[0202] Take the homogenate from Example 3 and adjust the pH to 8.0 with 1M NaOH. Mix according to a 500:1 (V) ratio. 匀浆液 / V Ulp1 Add Ulp1 solution (enzyme activity: 2115 U / mL), and perform enzyme digestion under stirring and controlled temperature of 30℃. Samples were taken 30 minutes after digestion, and the sample processing method was the same as in Example 3. Results are as follows: Figure 4 As shown, the fusion protein was completely cleaved, producing rhG-CSF and a tag.

[0203] Example 7. Disulfide bond pairing pH

[0204] The rhG-CSF in the enzyme digestion solution has only one pair of disulfide bonds, which needs to be paired correctly through oxidation. The enzyme digestion solution from Example 6 was taken, and the pH was adjusted to 8.5, 9.0, 9.5, and 10.0 with 1M NaOH, respectively, and then stirred. Samples were taken for testing at 1h, 2h, and 3h, respectively. The sample processing method was the same as in Example 3.

[0205] Based on the RP-HPLC results, the pairing rate was calculated as the ratio of the paired peak area to the 0h peak area. The pairing rates for each condition are shown in the table below.

[0206]

[0207] Example 8. Oxidizing Additive

[0208] At pH 8.5, an additive was added to shorten the pairing time. The enzyme digest from Example 6 was taken, and equal volumes of purified water, 0.5 mM cysteine, 1 mM cysteine, and 2 mM cysteine ​​were added respectively. The pH was adjusted to 8.5 with 1 M NaOH, and the mixture was stirred. Samples were taken and analyzed at 1 h and 2 h. The sample processing method was the same as in Example 3.

[0209] According to RP-HPLC results, the pairing rate of a sample with an equal volume of purified water added and stirred for 1 hour was 87.6%, and the pairing rate was 98.6% after stirring for 2 hours. However, the addition of the oxidant cystine caused the solution to become turbid and the target protein to precipitate.

[0210] Example 9. Verification of disulfide bond pairing conditions

[0211] Take 1000 ml of the enzyme digestion solution from Example 6, add 1000 ml of purified water, adjust the pH to 8.5 with 1 M NaOH, and stir for 2 hours. The sample processing method is the same as in Example 3. The results are attached. Figure 5 As shown, the pairing rate was 97.5%.

[0212] Example 10. Acid precipitation

[0213] Take six 50 mL samples from Example 9, and slowly add 6 mol / L HCl under stirring to adjust the pH to 2.0, 3.0, 4.0, 5.0, and 6.0 respectively. After the pH stabilizes, continue stirring for 30 min, centrifuge at 10000 r / min for 15 min, and take the supernatant for SDS-PAGE analysis.

[0214] like Figure 6 As shown, at pH 4.0, the supernatant contains fewer impurities and has the best clarity.

[0215] Example 11. QFF column purification of rhG-CSF

[0216] Adjust the pH of the acid precipitation supernatant to 8.0 with 1 mol / L NaOH, and add 0.1% poloxamer 188 (V). 上清 :g P188 Stir for 20 minutes. Filter using a 0.22 μm pore size membrane.

[0217] Equilibration 1: Equilibrate the QFF column with buffer B (25 mmol / L Tris-HCl + 1 mol / L Nacl, pH 8.0) at a flow rate of 150 cm / h until the conductivity stabilizes at the conductivity of buffer B.

[0218] Equilibration 2: Equilibrate the column again with buffer A (25 mmol / L Tris-HCl, pH 8.0) at a flow rate of 150 cm / h until the conductivity stabilizes to that of buffer A and the pH stabilizes at 8.0.

[0219] Sample loading: Load the filtered refolded liquid sample at a flow rate of 150 cm / h and a loading of 15 mg / ml, and collect the flow-through liquid.

[0220] Washing: Equilibrate the column again with buffer A at a flow rate of 150 cm / h for 4 column volumes.

[0221] Elution: Elute the proteins adsorbed on the column with buffer B at a flow rate of 150 cm / h;

[0222] The purification results showed that the target protein had a yield of 92% in the flow-through buffer and a purity of 62% by RP-HPLC.

[0223] Example 12. SP FF column purification of rhG-CSF

[0224] The flow-through solution from Example 11 was adjusted to pH 4.5 with 1 mol / L HCl, diluted with purified water to a conductivity of 3.0-4.0 ms / cm, and TCEP was added to a final concentration of 0.1 mmol, and poloxamer 188 was added to a final concentration of 0.1%.

[0225] Equilibration 1: Equilibrate the SPFF chromatography column with buffer B (25 mmol / L NaAC-HAC + 1 mol / L NaCl, pH 4.5) at a flow rate of 150 cm / h until the conductivity stabilizes at the conductivity of buffer B.

[0226] Equilibration 2: Equilibrate the chromatography column again with buffer A (25 mmol / L NaAC-HAC, pH 4.5) at a flow rate of 150 cm / h until the conductivity stabilizes to that of buffer A and the pH stabilizes at 4.5.

[0227] Sample loading: Load the sample at a flow rate of 150 cm / h and a loading rate of 15 mg / ml;

[0228] Washing: Equilibrate the column again with buffer A at a flow rate of 150 cm / h for 4 column volumes.

[0229] Elution: Elute the proteins adsorbed on the column with buffer B at a flow rate of 150 cm / h. The elution gradient is shown in the table below.

[0230] Column volume A％ B％ type 0 100 0 6 90 10 isometry 4 87 13 linear 12 82 18 linear 12 75 25 linear 4 0 100 linear

[0231] The purification results showed that the target protein was in the eluent, and the eluents were combined according to the purity obtained from RP-HPLC. The final yield was 85%, and the RP-HPLC purity was 82%.

[0232] Example 13. Ammonium sulfate precipitation

[0233] The combined solution from Example 12 was slowly added to ammonium sulfate (AS) powder at 25°C with stirring until saturation levels of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60% (w / v), respectively. After stirring for 30 min, the solution was centrifuged at 10,000 rpm for 15 min. The precipitate and supernatant were collected, and the precipitate was reconstituted with an equal volume of buffer. Analysis revealed that the target protein was present in the precipitate at AS concentrations of 30% and 35%, with impurities present in the supernatant.

[0234] Example 14. Optimization of Ammonium Sulfate Concentration

[0235] The combined solution from Example 12 was slowly added to AS powder at 25°C with stirring until saturation levels reached 30%, 33%, and 35%, respectively. After stirring for 30 min, the solution was centrifuged at 10000 rpm for 15 min. The precipitate and supernatant were collected and reconstituted with different buffer solutions. Buffer 1 was a 20 mmol PB solution at pH 7.4, and buffer 2 was a 25 mmol Tris-HCl solution at pH 8.0. The results are shown in the table below. A preferred AS concentration is 33%, and a 20 mmol PB solution is preferred for reconstitution.

[0236]

[0237]

[0238] Example 15. RP-HPLC and LC-MS

[0239] The rhG-CSF stock solution obtained in this invention and commercially available pharmaceutical products were simultaneously analyzed by RP-HPLC. The results are as follows: Figure 8 As shown, the self-made rhG-CSF has the same retention time as commercially available drugs, good symmetry, and high purity.

[0240] The theoretical molecular weight of rhG-CSF is 18667.65. LC-MS analysis of the rhG-CSF obtained through this invention yielded the following mass spectrometry results: Figure 7 As shown, the deconvolution molecular weight is 18666.09, which is basically consistent with the theoretical value.

[0241] Example 16. Peptide Mapping Analysis

[0242] Take rhG-CSF stock solution, add guanidine hydrochloride to a final concentration of 6M and react for half an hour for denaturation, then add iodoacetamide to a final concentration of 30mM for alkylation of the protein, replace the protein buffer solvent with 100mM Tris-HCl (pH=8) for desalting, and then add 1:40 (m 酶 :m 蛋白 The enzyme was digested with V8 enzyme, and after 14 hours of reaction, the results were analyzed by LC-MS. The results are shown in the table below. Figure 9 .

[0243]

[0244] Example 17. In vitro activity

[0245] Using the commercially available rhG-CSF as an activity control, its activity was 6.0 × 10⁻⁶. 6 IU (100 μg). Take the standard and the rhG-CSF stock solution obtained in this invention, and dilute to 100.0 ng / ml with basal culture medium in a 96-well cell culture plate, then perform a 5-fold serial dilution. Take the cultured NFS-60 cell line, wash three times with RPM1640 medium, and collect the cell suspension for cell counting before the third centrifugation. After counting, resuspend the cells in basal culture medium to prepare a 2.0 × 10⁻⁶ cell suspension. 5 Cells / ml. Add 50 μl of cell suspension to each well of 50 μl standard / homemade rhG-CSF and incubate at 37°C and 5% CO2 for 48 hours. Then, add 10 μl of WST-1 solution (5 mg / ml) to each well and incubate at 37°C and 5% CO2 for 4 hours. Measure the cells using a microplate reader at wavelengths of 450 nm and 630 nm (reference wavelength). See the cell growth curve below. Figure 10 The specific activity of the self-made rhG-CSF was calculated to be 6.12 × 10⁻⁶. 6 IU (100μg).

[0246] The sequence involved in this invention

[0247]

[0248] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing rhG-CSF, characterized in that, The method includes the following steps: (a) Culture host cells containing the gene encoding the fusion protein shown in Formula I under suitable expression conditions; (b) Obtain the fusion protein from the host cells cultured in step (a); and (c) Optionally process the fusion protein to obtain rhG-CSF; ABC(I) In this context, region A may be an optional signal peptide or may not exist. Section B is the SUMO fusion tag; The C segment is the amino acid sequence of rhG-CSF; "-" indicates an optional connector.

2. The preparation method according to claim 1, characterized in that, The amino acid sequence of the SUMO fusion tag is shown in SEQ ID NO:

1.

3. The preparation method according to claim 1, characterized in that, The amino acid sequence of the rhG-CSF is shown in SEQ ID NO:

2.

4. The preparation method according to claim 1, characterized in that, The amino acid sequence of the fusion protein shown in Formula I is shown in SEQ ID NO:

3. 5.I. Fusion protein: ABC(I) in, Segment A may be an optional signal peptide or may not exist; Section B is the SUMO fusion tag; The C segment is the amino acid sequence of rhG-CSF; "-" indicates an optional connector.

6. The fusion protein as described in claim 5, characterized in that, The amino acid sequence of the SUMO fusion tag is shown in SEQ ID NO:

1.

7. The fusion protein as described in claim 5, characterized in that, The amino acid sequence of the rhG-CSF is shown in SEQ ID NO:

2.

8. The fusion protein as described in claim 5, characterized in that, The amino acid sequence of the fusion protein shown in Formula I is shown in SEQ ID NO:

3.

9. An isolated polynucleotide molecule, said polynucleotide molecule encoding the fusion protein of any one of claims 5-8.

10. An expression vector comprising the polynucleotide molecule of claim 9.

11. A host cell containing the expression vector of claim 10, or an exogenous polynucleotide molecule of claim 9 integrated into a chromosome, or expressing a fusion protein of any one of claims 1-4.

12. Use of the fusion protein according to any one of claims 5-8 in the preparation of recombinant human granulocyte colony-stimulating factor.

13. A kit for preparing recombinant human granulocyte colony-stimulating factor, the kit comprising: 1) The fusion protein according to any one of claims 5-8; 2) Instructions for use in preparing recombinant human granulocyte colony-stimulating factor using this fusion protein; and 3) Optional other necessary reagents for the preparation of recombinant human granulocyte colony-stimulating factor using this fusion protein.

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