Preparation method of soluble RANKL protein

Abstract

The present invention provides a method for preparing a soluble RANKL protein. Specifically, the present invention provides a codon-optimized RANKL nucleotide sequence, which can be transferred into cells for expression to achieve a significant increase in RANKL production.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to a method for preparing a soluble RANKL protein. Background Art

[0002] RANKL, also known as TNFSF11, is a member of the tumor necrosis factor superfamily (TNFSF).

[0003] RANKL is a type II transmembrane protein and a ligand for the receptor activator of NF-κB (RANK). When RANKL binds to RANK, it induces the differentiation of monocyte / macrophage lineage cells into osteoclasts and further leads to the maturation of osteoclast precursors.

[0004] Therefore, RANKL is essential for osteoclast maturation, bone modeling and bone remodeling, and the development of lymph nodes (LN). In bone tissue, RANKL is expressed by osteoblasts, osteocytes, and immune cells, especially in osteoblasts and osteocytes. RANKL is also expressed by T cells and can promote the proliferation and survival of dendritic cells.

[0005] As a membrane-bound protein, RANKL is structurally composed of a cytoplasmic domain, a helical domain, and an extracellular domain. The extracellular domain of RANKL can fall off and form a soluble protein, namely soluble RANKL (sRANKL), which belongs to the TNF family cytokine 1. sRANKL has many different functions, such as playing a key role in osteoclast formation and immune cells. Therefore, sRANKL is of great significance for the study of biological processes such as bone metabolism and immune cell differentiation, and for the development of drugs to treat diseases such as osteoporosis and rheumatoid arthritis.

[0006] At present, the expression of soluble RANKL protein is mostly carried out in Escherichia coli. However, the recombinant sRANKL protein expressed in prokaryotic Escherichia coli requires denaturation and renaturation of inclusion bodies, and the prepared sRANKL is a non-glycosylated polypeptide chain, lacking modifications such as glycosylation in eukaryotic organisms.

[0007] In summary, there is still a lack of methods for large-scale production of human sRANKL using non-yeast mammalian eukaryotic cells in the art. Therefore, there is an urgent need to develop an efficient method suitable for large-scale production of human sRANKL in the art. Summary of the invention

[0008] The present invention provides a highly efficient method suitable for large-scale production of human sRANKL.

[0009] In a first aspect of the present invention, an optimized polynucleotide encoding a soluble RANKL (sRANKL) recombinant protein is provided, wherein the nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 2 or 7.

[0010] In another preferred example, the polynucleotide encodes a soluble RANKL protein with an amino acid sequence as shown in SEQ ID NO: 5.

[0011] In another preferred embodiment, the polynucleotide encodes a soluble sRANKL protein having an amino acid sequence as shown in SEQ ID NO: 5; or a sRANKL precursor protein having an amino acid sequence as shown in SEQ ID No: 6.

[0012] In another preferred embodiment, the nucleotide sequence of the polynucleotide is as shown in SEQ ID No: 7.

[0013] In another preferred example, the soluble RANKL protein is human soluble RANKL protein.

[0014] In the second aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide described in the first aspect.

[0015] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0016] In another preferred embodiment, a protein tag is further inserted into the vector, such as a Flag tag, a Halo tag, a SNAP tag, a His-tag tag, etc., preferably a histidine (His-tag) tag.

[0017] In another preferred embodiment, the protein tag is a 6his tag.

[0018] In another preferred embodiment, the vector is a eukaryotic cell expression vector.

[0019] In another preferred embodiment, the vector is a pCDNA3.4 vector.

[0020] In the third aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the second aspect of the present invention, or the polynucleotide described in the first aspect of the present invention is integrated into its genome.

[0021] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0022] In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

[0023] In another preferred embodiment, the host cell is a mammalian cell.

[0024] In another preferred embodiment, the host cell is a somatic cell derived from human.

[0025] In another preferred embodiment, the host cell is selected from the following group: HEK cells, HEK293 cells, 293T cells, Vero cells, or a combination thereof.

[0026] In another preferred embodiment, the host cell is HEK293 cell.

[0027] In a fourth aspect of the present invention, a method for preparing a soluble sRANKL protein is provided, the method comprising the steps of:

[0028] (a) culturing the host cell according to the third aspect of the present invention under conditions suitable for culture, thereby obtaining a culture; and

[0029] (b) isolating and purifying soluble sRANKL protein from the culture.

[0030] In another preferred embodiment, step (b) includes:

[0031] (b1) isolating sRANKL precursor protein from the culture supernatant, wherein the sequence of the sRANKL precursor protein is shown in SEQ ID No: 6;

[0032] (b2) performing an enzyme digestion treatment on the sRANKL precursor protein to obtain an enzyme digestion mixture containing the sRANKL protein, wherein the amino acid sequence of the sRANKL protein is shown in SEQ ID No: 5;

[0033] (b3) separating the sRANKL protein from the enzyme cleavage mixture containing the sRANKL protein.

[0034] In another preferred embodiment, in step (b1), the sRANKL precursor protein is separated from the culture supernatant by a nickel column affinity purification method.

[0035] In another preferred embodiment, in step (b2), the sRANKL precursor protein is cleaved by using EK enzyme.

[0036] In another preferred embodiment, step (b3) comprises:

[0037] (b3a) Enzyme digestion and reverse purification: using nickel column affinity purification to adsorb and remove the 6His tag or impurity fragments containing the 6His tag in the enzyme digestion mixture, thereby obtaining the sRANKL protein; and

[0038] (b34) Optional size exclusion chromatography: Purify the sRANKL protein using a Superdex 200 pg chromatography column to obtain the sRANKL protein with high purity.

[0039] In a preferred aspect, the present invention provides a method for preparing RANKL protein, comprising the steps of:

[0040] (i) synthesizing a nucleic acid construct corresponding to the RANKL recombinant protein, and constructing it into a eukaryotic expression vector to obtain a RANKL recombinant expression vector;

[0041] (ii) transfecting HEK293 cells with the RANKL recombinant expression vector via a transient expression system;

[0042] (iii) culturing the transfected HEK293 cells under conditions suitable for culture to obtain a culture;

[0043] (iv) centrifuging the culture to collect the supernatant, and filtering and purifying to obtain the RANKL recombinant protein;

[0044] Among them, the nucleic acid construct sequence corresponding to the RANKL protein is shown in SEQ ID NO: 2.

[0045] In another preferred example, the amino acid sequence of the RANKL recombinant protein is shown in SEQ ID NO: 5.

[0046] In the fifth aspect of the present invention, a RANKL recombinant protein is provided. The recombinant protein is produced by the cell described in the third aspect of the present invention, or is prepared by the method described in the fourth aspect of the present invention.

[0047] In the sixth aspect of the present invention, there is provided the use of the polynucleotide as described in the first aspect of the present invention, the expression vector as described in the second aspect of the present invention, the host cell as described in the third aspect of the present invention, or the recombinant protein as described in the fifth aspect of the present invention, characterized in that it is used for preparing a preparation or composition.

[0048] In another preferred embodiment, the preparation is a laboratory preparation.

[0049] In another preferred embodiment, the composition is a pharmaceutical composition.

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 An alignment diagram of the original RANKL codons and the optimized codons selected according to the present invention is shown.

[0052] Figure 2 The SDS-PAGE gel electrophoresis pattern of Ni column purification is shown. The target protein is mainly eluted at a concentration of 250mM imidazole. Among them, M: Marker; S: cell secretion fluid; Ft: Ni column flow-through fraction; F0: re-equilibrium fraction; 114: Triton X-114 elution fraction; W1: Buffer A washing fraction; W2: 20mM imidazole elution minute; E1-E7: 250mM imidazole elution fraction and separate collection; R: residual fraction on the column.

[0053] Figure 3 The SDS-PAGE gel electrophoresis pattern after the target protein was cleaved by EK enzyme is shown, and the target protein was completely cleaved. The lanes are as follows: M: Marker; 443B: before EK cleavage; 443A: after EK cleavage.

[0054] Figure 4 The SDS-PAGE gel electrophoresis pattern during the nickel column anti-capture process is shown. The target protein after enzyme digestion is mainly in the flow-through fraction and the 20mM imidazole elution fraction. The lanes are as follows: M: Marker; S: Ni column pre-load fraction; Ft: Ni column flow-through fraction; F0: Buffer A wash fraction and separate collections 1-4; W1: Buffer B elution fraction and separate collections 1-6; W2: Buffer C elution fraction; R: Residual fraction on the column.

[0055] Figure 5 The Superdex200pg chromatogram is shown. There are two main peaks. The first peak has a retention volume of 54 ml, which is mainly composed of polymers and high molecular weight impurities. The second peak has a retention volume of 80 ml, which is the main peak of the target protein.

[0056] Figure 6 The SDS-PAGE gel electrophoresis pattern after molecular exclusion chromatography purification is shown. After purification by Superdex200pg molecular sieve, the purity of the target protein after enzyme digestion reaches more than 95%. Among them, M: Marker; S: component before molecular sieve loading; A1, A8-A12, B12, B11: Buffer elution tube collection components.

[0057] Figure 7 The SDS-PAGE gel electrophoresis diagram for purity detection is shown. The final protein purity is more than 95%. Due to glycosylation, the target protein appears fuzzy and diffuse on the electrophoresis; M: Marker; R: reduced electrophoresis of the final purified component.

[0058] Figure 8 The components and contents of the blank control, standard substance and sample in Example 5 are shown.

[0059] Fig. 9 A graph of protein concentration versus OD405 is shown.

[0060] Fig.10 The SDS-PAGE gel electrophoresis pattern of Ni column purification is shown. Among them, S: cell fluid; M: Marker; FT: Ni column flow-through; F0: Buffer A wash; 114: TX-114 wash; W1: Buffer A wash TX-114 fraction; W2: 20mM imidazole fraction; E250: 250mM imidazole elution fraction; E500: 500mM imidazole elution fraction; R: column residue.

[0061] Fig.11 The SDS-PAGE gel electrophoresis pattern of Ni column purification is shown. Among them, S: cell sap; FT: Ni column flow-through; E20: 20mM imidazole elution; E250: 250mM Imi imidazole elution; NR: 250mM Imi imidazole elution non-reducing; E500: 500mM Imi imidazole elution; R: column residue; M: Marker.

[0062] Fig.12 The SDS-PAGE gel electrophoresis pattern of Ni column purification is shown, where M: Marker; S: cell fluid; FT: Ni column flow-through; E20: 20mM imidazole elution; E250: 250mM Imi imidazole elution; E500: 500mM Imi imidazole elution; R: column residue; NR: 250mM Imi imidazole elution without reduction. DETAILED DESCRIPTION

[0063] After extensive and in-depth research, the present invention unexpectedly obtained a coding sequence of human sRANKL with high production performance in eukaryotic cells derived from humans through a large number of screenings (referred to as "the human sRANKL coding sequence of the present invention"). The human sRANKL coding sequence of the present invention is a sRANKL nucleotide sequence obtained after optimizing various factors such as codon preference and mRNA secondary structure. The optimal human sRANKL coding sequence of the present invention (SEQ ID No: 2) can achieve a significant increase in the production of RANKL (or sRANKL) (relative expression level is about 3 compared to the wild-type coding sequence). The recombinant sRANKL protein of the present invention has almost the same glycosylation and other modified forms as the natural human RANKL protein, and has excellent solubility and biological activity. On this basis, the present invention was completed.

[0064] the term

[0065] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0066] As used herein, "isolated" or "isolated and purified" means that a substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist with them in their natural state.

[0067] Soluble RANKL protein

[0068] As used herein, the terms "sRANKL protein of the present invention", "soluble sRANKL protein of the present invention", "sRANKL protein with glycosylation modification of the present invention", "polypeptide of the present invention", or "sRANKL polypeptide of the present invention" are used interchangeably to refer to a soluble sRANKL protein in the correct glycosylation form expressed by a eukaryotic host cell (somatic cell) derived from a human and using the optimized sequence of the present invention. sRANKL is a truncated protein of the RANKL protein.

[0069] RANKL is a type II transmembrane protein and a ligand for the receptor activator of NF-κB (RANK). RANKL is preferentially expressed in osteoblasts and stromal cell lineages. RANKL's receptor, RANK, is preferentially expressed in osteoclast lineages.

[0070] RANKL is produced in these cells as a type II transmembrane protein that can be cleaved by specific metalloproteinases into an extracellular water-soluble form, also known as soluble RANKL (sRANKL) or soluble TNFSF11.

[0071] RANKL is structurally composed of a cytoplasmic domain, a helical domain, and an extracellular domain. Taking the mouse RANKL protein as an example, it is composed of a cytoplasmic domain (1-47), a helical domain (48-68), and an extracellular domain (69-317). The human RANKL protein is highly homologous to the mouse RANKL protein.

[0072] The length of human RANKL protein is 317 amino acids, and its sequence information can be found in GenBank: AAB86811.1 or SEQ ID No: 8.

[0073] 1 MRRASRDYTK YLRGSEEMGG GPGAPHEGPL HAPPPPAPHQ PPAASRSMFV ALLGLGLGQV

[0074] 61 VCSVALFFYF RAQMDPNRIS EDGTHCIYRI LRLHENADFQ DTTLESQDTK LIPDSCRRIK

[0075] 121 QAFQGAVQKE LQHIV GSQHI RAEKAMVDGS WLDLAKRSKL EAQPFAHLTI NATDIPSGSH

[0076] 181 KVSLSSWYHD RGWAKISNMT FSNGKLIVNQ DGFYYLYANI CFRHHETSGD LATEYLQLMV

[0077] 241 YVTKTSIKIP SSHTLMKGGS TKYWSGNSEF HFYSINVGGF FKLRSGEEIS IEVSNPSLLD

[0078] 301 PDQDATYFGA FKVRDID (SEQ ID No: 8)

[0079] A preferred amino acid sequence of human sRANKL protein is positions 136-317 in SEQ ID No: 8 or as shown in SEQ ID No: 5.

[0080] As an important cytokine, sRANKL plays a key role in biological processes such as bone metabolism and immune cell differentiation. It binds to the receptor RANK, activates the protein kinase signal transduction pathway, and regulates the proliferation of mesenchymal cells, the maturation and function of osteoblasts in bone metabolism, etc.

[0081] Optimization of sRANKL coding sequence

[0082] In the present invention, the inventors optimized the coding sequence of sRANKL by various different optimization methods, including but not limited to: redesigning and synthesizing the sRANKL gene, such as eliminating rare codons, optimizing codon sequences, GC content, destabilizing sequences, minimizing DNA secondary structures, adjusting the GC content of DNA, optimizing the gene translation start frame, optimizing the translation termination sequence frame, etc.

[0083] There are 64 genetic codes, but most organisms tend to use a subset of these codons. Those that are used most frequently are called optimal codons, and those that are not used frequently are called rare or low-usage codons. Different biological species show some degree of difference or preference in codon usage, so the expression of recombinant proteins may be affected by codon usage. The redesign of genes by synthesizing genes using preferred codons and avoiding low-usage or rare codons is called codon optimization.

[0084] In the present invention, human host cells (such as HEK cells, HEK293 cells or 293T cells) are used to express human proteins, and the translation system of human host cells is already very compatible with the human wild-type coding sequence (or optimized by natural evolution). Therefore, when certain codons are replaced, the improvement effect will not be obvious, or the protein production will decrease instead of increase (such as the sequence of SEQ ID No: 4 causes the expression level to decrease). However, there is little room for codon optimization based on biological species (i.e., human cells).

[0085] To this end, the applicant has analyzed and optimized some other factors that may affect protein expression besides codon preference, including (but not limited to): eliminating sequences that destabilize mRNA, minimizing DNA secondary structure, adjusting DNA GC content, optimizing gene translation start frame, optimizing translation termination sequence frame, etc. The redesigned multi-optimized sRANKL coding sequence of the present invention can effectively remove or change these unfavorable factors, thereby significantly increasing the expression of sRANKL in human host cells, making the production of exogenous proteins more efficient and economical.

[0086] Preparation and purification of sRANKL protein

[0087] The sRANKL protein of the present invention (polypeptide of the present invention) is a recombinant polypeptide, which is produced from a eukaryotic host cell (e.g., a host cell derived from a human somatic cell) using recombinant technology. The polypeptide of the present invention is glycosylated, preferably the glycosylation is a human glycosylation pattern. In addition, the polypeptide of the present invention may or may not include an initial methionine residue.

[0088] Once the optimized coding sequence of the present invention is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0089] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0090] The method of using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The DNA / RNA fragments amplified can be separated and purified by conventional methods such as by gel electrophoresis.

[0091] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the sRANKL coding sequence, and a method for producing the polypeptide of the present invention by recombinant technology.

[0092] The polynucleotide sequence of the present invention can be used to express or produce recombinant sRANKL polypeptides by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally speaking, the following steps are involved:

[0093] (1) Transforming or transducing a suitable host cell with the polynucleotide encoding human sRANKL of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0094] (2) Host cells cultured in a suitable culture medium;

[0095] (3) Isolate and purify proteins from culture medium or cells.

[0096] In the present invention, the human sRANKL polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as they can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0097] Methods well known to those skilled in the art can be used to construct expression vectors containing human sRANKL encoding DNA sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0098] In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance.

[0099] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0100] In the present invention, the host cell is a higher eukaryotic cell, such as a mammalian cell, especially a host cell derived from a human. Representative examples include: HEK cells, CHO, COS, 293T cells and other animal cells.

[0101] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0102] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0103] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0104] The obtained transformant can be cultured by conventional methods to express the sRANKL polypeptide of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0105] In the present invention, the sRANKL recombinant protein of the present invention is secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0106] In a preferred example, by using the optimized coding sequence shown in SEQ ID No: 2 of the present invention, compared with the wild-type coding sequence, under the condition of transient transfection of HEK293 cells, the relative expression level of sRANKL protein can reach about 289%, and after separation and purification, a sRANKL protein with a concentration of about 5.8 mg / ml can be obtained.

[0107] The main advantages of the present invention include:

[0108] (a) The codon-optimized nucleotide sequence of the present invention can significantly increase the production of sRANKL protein in human host cells (increased by about 2 times under transient transfection conditions).

[0109] (b) In the present invention, human cells such as HEK293 are used for eukaryotic expression, and the glycosylation pattern of the prepared sRANKL protein is more consistent with the glycosylation pattern of natural human RANKL.

[0110] (c) In the present invention, when human cells such as HEK293 are used for production, the recombinant sRANKL protein will be secreted into the culture supernatant, which makes the subsequent purification very simple and helps to obtain high-purity (up to 99% or higher) and highly active sRANKL protein.

[0111] (d) When codon preference alone could not effectively increase the expression level, the inventors analyzed and optimized some other factors that may affect protein expression besides codon preference, thereby significantly increasing the expression level of sRANKL in human host cells.

[0112] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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 according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0113] Example 1 Codon optimization of recombinant protein nucleotide sequence and construction of recombinant plasmid

[0114] In this example, the coding sequence was optimized based on the original nucleic acid sequence (NC_000013.11 or SEQ ID No: 1) of the soluble form of human RANKL (sRANKL).

[0115] The original nucleotide sequence (or wild-type sequence or natural sequence) of RANKL is shown below:

[0116] GGATCACAGCACATCAGAGCAGAGAAAGCGATGGTGGATGGCTCATGGTTAGATCTGGCCAAGAGGAGCAAGCTTGAAGCTCAGCCTTTTGCTCATCTCACTATTAATGCCACCGACATCCCATCTGGTTCCCATA AAGTGAGTCTGTCCTCTTGGTACCATGATCGGGGGTGGGGTAAGATCTCCAACATGACTTTTAGCAATGGAAAACTAATAGTTAATCAGGATGGCTTTTATTACCTGTATGCCAACATTTGCTTTCGACATCATGAA ACTTCAGGAGACCTAGCTACAGAGTATCTTCAACTAATGGTGTACGTCACTAAAACCAGCATCAAAATCCCAAGTTCTCATACCCTGATGAAAGGAGGAAGCACCAAGTATTGGTCAGGGAATTCTGAATTCCATT TTTATTCCATAAACGTTGGTGGATTTTTTAAGTTACGGTCTGGAGAGGAAATCAGCATCGAGGTCTCCAACCCCTCCTTACTGGATCCGGATCAGGATGCAACATACTTTGGGGCTTTTAAAGTTCGAGATATAGAT (SEQ ID NO: 1)

[0117] In sequence optimization, multiple optimizations (or multiple rounds of optimization) were performed, including (a) optimization based on codon preference, and (b) analysis and optimization of factors other than codon preference, including (but not limited to): eliminating sequences that destabilize mRNA, minimizing DNA secondary structure, adjusting the GC content of DNA, optimizing the gene translation start frame, optimizing the translation termination sequence frame, etc., to eliminate factors that are unfavorable to the efficient expression of sRANKL.

[0118] After optimization, dozens of optimized coding sequences were obtained. For these coding sequences, vectors were constructed after artificial synthesis and their expression performance was tested (see Example 2), and coding sequences with excellent performance were selected.

[0119] The optimized coding sequences of the present invention include: 3 coding sequences shown in SEQ ID No: 2-4:

[0120] Optimized coding sequence 1:

[0121] GGCAGCCAACACATCAGAGCCGAGAAGGCCATGGTGGACGGCAGCTGGCTGGACCTGGCCAAGAGAAGCAAGCTGGAGGCTCAGCCCTTCGCCCACCTGACCATCAACGCCACCGACATCCCTAGCGGCAGCCACA AGGTGAGCCTGAGCAGCTGGTACCACGACAGAGGCTGGGGCAAGATCAGCAACATGACCTTCAGCAACGGCAAGCTGATCGTGAACCAAGACGGCTTCTACTACCTGTACGCCAACATCTGCTTCAGACACCACGAG ACAAGCGGCGACCTGGCCACCGAGTACCTGCAGCTGATGGTGTACGTGACCAAGACAAGCATCAAGATCCCTAGCAGCCACACCCTGATGAAGGGCGGCAGCACCAAGTACTGGAGCGGCAACAGCGAGTTCCACT TCTACAGCATCAACGTGGGCGGCTTCTTCAAGCTGAGAAGCGGCGAGGATCAGCATCGAGGTGAGCAACCCTAGCCTGCTGGACCCCGACCAAGACGCCACCTACTTCGGCGCCTTCAAGGTGAGAGACATCGAC (SEQ ID NO: 2)

[0122] Optimized coding sequence 2:

[0123] GGTAGCCAGCATATTCGTGCAGAAAAAGCAATGGTTGATGGTAGCTGGCTGGATCTGGCAAAACGTAGCAAACTGGAAGCACAGCCGTTTGCACATCTGACCATTAATGCAACCGATATTCCGAGCGGTAGCCATAAAGTTAGCCTGAGCAGCTGGTATCATGATCGTGGTTGGGGTAAAATTAGCAACATGACCTTTAGCAACGGCAAACTGATTGTTAATCAGGATGGCTTCTATTACCTGTATGCCAATATTTGCTTTCGCCATCATGAAACCAGTGGTGATCTGGCCACCGAATATCTGCAGCTGATGGTTTATGTTACCAAAACCAGCATTAAAATCCCGAGCAGTCATACCCTGATGAAAGGTGGTAGCACAAAATATTGGAGCGGCAATAGCGAATTTCACTTCTATAGCATTAATGTCGGTGGGTTTTTCAAACTGCGTAGCGGTGAAGAAATTAGCATTGAAGTTAGCAATCCGAGCCTGCTGGATCCGGACCAGGATGCAACCTATTTTGGTGCATTTAAAGTGCGCGATATCGAT (SEQ ID NO: 3)

[0124] Optimized coding sequence 3:

[0125] GGATCACAACACATAAGGGCTGAAAAAGCAATGGTTGATGGCTCCTGGCTGGACTTGGCGAAACGTTCCAAATTGGAGGCGCAGCCGTTTGCGCACCTGACCATTAACGCCACGGATATCCCGTCAGGTTCTCACA AAGTGTCCCTGAGCAGCTGGTATCACGATCGCGGTTGGGGTAAGATCTCCAACATGACCTTCAGCAACGGCAAATTAATTGTGAACCAGGACGGCTTCTACTACCTGTATGCGAATATTTGCTTTCGTCATCATGAA ACCTCTGGCGACTTGGCTACTGAGTATCTGCAACTGATGGTCTATGTGACCAAGACCAGCATTAAAATCCCATCTAGCCACACCCTCATGAAAGGTGGTAGCACCAAGTACTGGAGCGGTAATAGCGAATTTCATT TTTACAGCATTAATGTTGGTGGCTTCTTCAAGCTGCGTAGTGGTGAGGAGATCTCGATCGAAGTTAGCAACCCGAGCCTGCTGGACCCGGATCAAGACGCTACGTACTTCGGCGCATTTAAGGTACGCGACATCGAT (SEQ ID NO: 4)

[0126] Among them, the optimized coding sequence 1 has a consistency of 72.53% with the original nucleotide sequence, and its sequence alignment is shown in the figure below: Figure 1 shown.

[0127] The sRANKL proteins encoded by the above optimized coding sequences are the same as those encoded by the wt coding sequence (SEQ ID No: 1), and their amino acid sequences are shown in SEQ ID NO: 5:

[0128] GSQHIRAEKAMVDGSWLDLAKRSKLEAQPFAHLTINATDIPSGSHKVSLSSWYHDRGWGKISNMTFSNGKLIVNQDGFYYLYANICFRHHETSGDLATEYLQLMVYVTKTSIKIPSSHTLMKGGSTKYWSGNSEFHFYSINVGGFFKLRSGEEISIEVSNPSLLDPDQDATYFGAFKVRDID (SEQ ID NO: 5).

[0129] Example 2 Expression of recombinant protein

[0130] The wild-type coding sequence (SEQ ID No: 1) and multiple optimized coding sequences (including SEQ ID NO: 2-4) in Example 1 were respectively introduced into plasmid pCDNA3.4 (purchased from Suzhou Jinweizhi Biotechnology Co., Ltd.), and recombinant plasmids were constructed accordingly.

[0131] Among them, the optimized coding sequence will be introduced into the recombinant plasmid in the form of a nucleotide sequence corresponding to a signal peptide, a 6his tag and an EK restriction site. Taking the optimized coding sequence 1 as an example, when the optimized coding sequence 1 is introduced into the recombinant plasmid in the form of a nucleotide sequence corresponding to a signal peptide, a 6his tag and an EK restriction site, the corresponding nucleotide sequence is:

[0132] (SEQ ID NO: 7)

[0133] Finally, the amino acid sequence corresponding to the nucleotide sequence introduced into the plasmid is:

[0134] METDTLLLWVLLLWVPGSTG HHHHHH DDDDKGSQHIRAEKAMVDGSWLDLAKRSKLEAQPFAHLTINATDIPSGSHKVSLSSWYHDRGWGKISNMTFSNGKLIVNQDGFYYLYANICFRHHETSGDLATEYLQLMVYVTKTSIKIPSSHTLMKGGSTKYWSGNSEFHFYSINVGGFFKLRSGEEISIEVSNPSLLDPDQDATYFGAFKVRDID (SEQ IDNO: 6)

[0135] The italic part is the signal peptide, the underlined part is the 6His tag, and the bold part is the cleavage site of enterokinase (EK enzyme), which forms a truncated RANKL (or sRANKL) after cleavage.

[0136] The fusion protein with the amino acid sequence shown in SEQ ID NO: 6 is called sRANKL precursor protein.

[0137] 2.1 Conversion

[0138] (1) Thaw DH5α competent cells from -80°C on ice, and add 40 μL of sterile water to the plasmid powder to dissolve the plasmid;

[0139] (2) Gently blow the DH5α competent cells evenly;

[0140] (3) Take 2 μL of plasmid and add it into DH5α competent cells;

[0141] (4) Place on ice for 30 min, activate at 42°C for 90 s, place back on ice for 2-5 min, and add 100-500 μL of antibody-free LB medium;

[0142] (5) Incubate the cells at 37°C and 220 rpm for about 1 h until the OD value reaches 0.6-0.8.

[0143] (6) Take 100 μL of the transformed bacterial solution and spread it evenly on an LB plate containing Ampicilin antibiotics. Incubate the plate upside down in a 37°C constant temperature incubator overnight.

[0144] 2.2 Plasmid extraction

[0145] The plasmid was extracted using the MN endotoxin-free plasmid extraction kit, and its concentration and A260 / A280 were determined using NanoDrop. The concentration was >200ug / ul, and A260 / A280 was 1.8.

[0146] 2.3 Recombinant protein expression

[0147] (1) Cell preparation before transfection

[0148] ① Cell status detection: Take out the cells to be transfected (HEK293 cells) from the incubator and perform counting and viability detection.

[0149] Status description: Cells are in good condition.

[0150] ② Cell treatment: One day before transfection, dilute the cells to a density of about 1×10 6 / mL for transfection the next day.

[0151] (2) Transfection and expression

[0152] Prepare high-quality plasmids and cells in advance. The general transfection plasmid concentration is 1µg / mL, and the mass ratio of plasmid and transfection reagent (PEI is 1:3. The transfection buffer is KPM, and the transfection buffer system is 1 / 10 of the expression volume.

[0153] The specific steps are as follows:

[0154] ① Ensure that the cell density for expression is about 2×10 6 / ml, cell viability as high as 95%.

[0155] ② Add 600µg plasmid and 1800µg transfection reagent (PEI) into 30 mL transfection buffer KPM, mix the transfection reagent and plasmid, let stand at room temperature for 10 min, and then add to 600 mL cells;

[0156] ③ 24 hours after transfection, add cell protein expression enhancer (0.6% for HEK293 cells) and 1× transient transfection nutrient additive, transfer to 32°C for culture, and measure cell viability and status every day for 7 days.

[0157] Example 3 Purification of recombinant protein (Ni column affinity purification)

[0158] 3.1. Sample preparation

[0159] (1) The cell culture medium was centrifuged at 6000 rpm for 20 min and the supernatant was collected and recorded as S;

[0160] (2) Filter the supernatant through a 0.45 µm filter to remove solid particles and transfer to a clean centrifuge tube.

[0161] 3.2 Nickel column affinity purification

[0162] Since the recombinant proteins expressed by the wild-type coding sequence (SEQ ID No: 1) and the optimized coding sequence (SEQ ID No: 2-4) were secreted and carried a 6His tag, a nickel column was used for affinity purification.

[0163] The purification was performed using the following buffers and steps:

[0164] Buffer A: 20mM PB, 150mM NaCl, pH7.4

[0165] Buffer B: 20mM PB, 150mM NaCl, pH7.4, 20mM Imidazole

[0166] Buffer C: 20mM PB, 150mM NaCl, pH7.4, 250mM Imidazole

[0167] (1) Take 5 mL of nickel column packing, add it to the gravity column, and rinse the packing with 10 CV of 0.5% Triton X-114;

[0168] (2) Wash the packing with 10CV water;

[0169] (3) Use 10CV Buffer A to equilibrate the packing;

[0170] (4) The sample is loaded onto a gravity column and the flow-through is recovered and recorded as FT;

[0171] (5) Use 10CV Buffer A to re-equilibrate the packing, collect the wash solution for subsequent analysis and record it as F0;

[0172] (6) Wash with 10 CV of Buffer A containing 0.1% Triton X-114, collect the wash solution for subsequent analysis and record it as 114;

[0173] (7) Use 10CV of Buffer A to wash away the residual Triton X-114 in the filler, and collect the washing solution for subsequent analysis and record it as W1;

[0174] (8) Elution with 10 CV of Buffer B, collecting the elution solution for subsequent analysis and recorded as W2;

[0175] (9) Elution was performed using 10 CV of Buffer C, with approximately 4 mL per tube, for a total of 7 tubes. The eluate was collected for subsequent analysis and recorded as E1-E7;

[0176] (10) Resuspend the filler with 1 CV of Buffer C and take out 20 µL as the column residue, recorded as R;

[0177] (11) Wash the filler with 5CV ultrapure water to remove the eluent in the filler;

[0178] (12) Wash the medium with 5 CV of 20% ethanol and store at 4°C.

[0179] (13) Take 20 µL of each sample (S, FT, F0, 114, W1, W2, Ex, R), add 5 µL of 5× Loading Buffer, mix well, and place in a 95°C metal bath for 5 min;

[0180] (14) Perform SDS-PAGE analysis.

[0181] Experimental Results

[0182] (a) The purification results using the wild-type coding sequence are as follows Fig.10 shown.

[0183] Combine W2, E250, and E500 to obtain a total of 80 ml with a concentration of 0.08 mg / ml and a protein yield of 6.4 mg.

[0184] (b) The purification result using the optimized sequence of SEQ ID No: 2, as shown in Figure 2 shown.

[0185] E1-E7 were combined to obtain a total of 35 ml, with a concentration of 0.53 mg / ml and a protein yield of 18.55 mg.

[0186] After merging, they were used in the next step (adding EK enzyme for digestion).

[0187] (c) The purification result using the optimized sequence of SEQ ID No: 3, as shown in Fig.11 shown.

[0188] Combine E250 and E500 to obtain a total of 100 ml, with a concentration of 0.1 mg / ml and a protein yield of 10 mg.

[0189] (d) The purification result using the optimized sequence of SEQ ID No: 4 is as follows: Fig.12 shown.

[0190] E250 was collected to obtain a total of 50 ml, with a concentration of 0.1 mg / ml and a protein yield of 5 mg.

[0191] The purification results of four different coding sequences are summarized in Table 1 below:

[0192] Table 1

[0193]

[0194] Result analysis: Comparing the expression and Ni column purification results of SEQ ID NO: 1 (native sequence), codon-optimized sequence SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, it can be seen that the expression level of SEQ ID NO: 2 is significantly better than that of the native sequence and the other two codon-optimized sequences. Therefore, the Ni column purification component of SEQ ID NO: 2 was selected to continue molecular exclusion chromatography purification.

[0195] Example 4 Enzymatic digestion and purification of recombinant protein

[0196] In this example, the fusion protein of the present invention (SEQ ID No. 6) contains an EK cleavage site, so an enzyme cleavage treatment is performed, and the C-terminal fragment ("impurity fragment") after the enzyme cleavage still contains a 6His tag, so a Ni column is used to remove the impurity fragment containing 6His. The method is as follows:

[0197] 4.1 Enzyme digestion

[0198] Dialysate: 20mM PB, 150mM NaCl, pH 7.4

[0199] (1) After combining Ni column E1-7 tubes, take 20 µL as the sample before enzyme digestion, recorded as 443B

[0200] (2) Add EK enzyme (1 unit of enzyme activity corresponds to 100 μg of protein to be digested) and dialyze overnight. Take 20 μL as the digested sample, recorded as 443A.

[0201] (3) Perform SDS PAGE analysis.

[0202] result

[0203] SDS-PAGE gel electrophoresis pattern Figure 3 As shown, the results show that after the recombinant fusion protein was cleaved by EK enzyme, the molecular weight of the released sRANKL protein became smaller, which was consistent with expectations. This indicates that the full-length fusion protein was completely cleaved.

[0204] 4.2 Nickel column affinity purification and impurity removal

[0205] BufferA:20mMPB,150mMNaCl,pH7.4

[0206] BufferB: 20mM PB, 150mM NaCl, pH7.4, 20mM imidazole

[0207] BufferC: 20mM PB, 150mM NaCl, pH7.4, 250mM imidazole

[0208] (1) Take 3 mL of nickel column packing, add it to the gravity column, and rinse the packing with 10 CV of 0.5% TritonX-114;

[0209] (2) Wash the packing with 10 CV of water;

[0210] (3) Equilibrate the medium with 10 CV Buffer A;

[0211] (4) The sample is loaded onto a gravity column and the flow-through is recovered and recorded as FT;

[0212] (5) Use 10 CV Buffer A to re-equilibrate the packing, collect the washing solution for subsequent analysis and record it as F0①-F0④;

[0213] (6) Elution with 10 CV of Buffer B was performed and the elution solution was collected for subsequent analysis and recorded as W1①-W1⑥ (containing the 6His short fragment);

[0214] (7) Elute with 10 CV of Buffer C and collect the elution solution for subsequent analysis and record it as W2;

[0215] (8) Use 1 CV of Buffer C to resuspend the filler and take out 20 µL as the column residue, recorded as R;

[0216] (9) Wash the packing with 5 CV of ultrapure water to remove the eluent from the packing;

[0217] (10) Wash the medium with 5 CV of 20% ethanol and store at 4°C.

[0218] (11) Take 20 µL of each sample (S, FT, F0, W1, W2), add 5 µL of 5× Loading Buffer, mix well, and place in a 95°C metal bath for 5 min;

[0219] (12) Perform SDS-PAGE analysis.

[0220] result

[0221] After purification, the results were Figure 4 As shown, where:

[0222] M: Marker; S: Ni column pre-loaded fraction; Ft: Ni column flow-through fraction; F0: Buffer A wash fraction and collected in 1-4; W1: Buffer B elution fraction and collected in 1-6; W2: Buffer C elution and collection of wash liquid; R: Buffer C resuspended filler, aspirated 20µL as column residue.

[0223] Combine Ft, F0①-F0③ and W1②-W1⑤ to obtain 60 mL, and the measured concentration is 0.25 mg / ml; the total mass is 15 mg.

[0224] Example 5 Purification of sRANKL protein

[0225] In this example, the sRANKL protein prepared in Example 3 was further purified by SEC to obtain a sRANKL protein with higher purity.

[0226] Sample: Sample purified by Ni column prepared in Example 4 (concentrated to 4 mL)

[0227] Buffer:20mMPB,150mMNaCl,pH7.4

[0228] Purification column: Superdex200pg

[0229] (1) Treat the column with 0.5 M NaOH and H2O and then equilibrate with buffer for 1 CV until UV280 and conductivity are stable;

[0230] (2) 1 mL / min sample loading;

[0231] (3) Elute 120 mL and collect 3 mL in each tube (starting from the 50th minute);

[0232] (4) Check the SDS-PAGE test results.

[0233] result

[0234] Superdex200pg chromatogram Figure 5 As shown, it indicates that the soluble sRANKL protein has been effectively separated and purified, and the protein structure has good homogeneity as judged by the main peak shape.

[0235] The results of SDS-PAGE gel electrophoresis are as follows Figure 6 A9-A12 were combined to obtain 2.4 mL, and the concentration was 5.7614 mg / ml; the total mass was 13.83 mg. It can be seen that the recombinant RANKL protein of the present invention has a significantly improved yield and good solubility.

[0236] Example 6 Purity detection

[0237] SDS-PAGE is a denaturing polyacrylamide gel electrophoresis method. The principle of this method for separating proteins is that most proteins can combine with anionic surfactant sodium dodecyl sulfate (by weight ratio to form a complex, so that the negative charge carried by the protein molecule far exceeds the net charge of the natural protein molecule, eliminating the charge effect of different protein molecules and separating the proteins by molecular size.

[0238] Test sample treatment: 2 μg of sample was transferred and reduced test sample buffer was added at a ratio of 3:1. The sample volume was mixed and heated in a metal bath at 100°C for 5 minutes. The mixture was cooled to room temperature and then loaded on SDS-PAGE.

[0239] Electrophoresis conditions: 12% SDS-PAGE was used for electrophoresis, constant voltage electrophoresis, initial voltage was 80V, adjusted to 200V when entering the separation gel, and electrophoresis was stopped when bromophenol blue migrated to the bottom of the gel.

[0240] Fixation and staining (Coomassie Brilliant Blue method): Take out the electrophoresis gel slice, place it in the fixative for 60 minutes, take out the film and place it in excess Coomassie Brilliant Blue staining solution for 1 to 2 hours, discard the staining solution, place it in excess destaining solution, change the destaining solution several times as needed, and store it in the preservation solution after destaining until the gel background is transparent.

[0241] After the gel is developed, it is photographed or scanned. Usually, a commercial gel scanning system with data analysis software is used to take pictures and analyze the gel to obtain molecular weight and purity information. Molecular weight analysis: Calibrate according to molecular weight standards. Purity analysis: Scan with a gel imager and calculate the results by peak area normalization.

[0242] The results are as follows Figure 7 As shown, M: Marker; R: reduction electrophoresis of the final purified component.

[0243] Purification results: The purity result was 99%, and the molecular weight was 28.81KDa. The theoretical molecular weight of RANKL protein is 20.5kd. Due to post-translational modification processes such as glycosylation, the apparent molecular weight of the protein electrophoresis is greater than the theoretical molecular weight. It can be seen that the recombinant protein of the present invention has other modifications, such as glycosylation, relative to the general RANKL protein.

[0244] Example 7 Activity detection of recombinant protein

[0245] 7.1 Cell culture

[0246] a) Cell line used for activity measurement: RAW264.7 cells

[0247] b) Cell culture medium: DMEM medium + 10% FBS + 1% P / S

[0248] 7.2 Formal Experiment

[0249] 7.2.1 Experimental procedures

[0250] (1) Cells were seeded in 24-well plates at a density of 50,000 cells / well;

[0251] (2) After the cells adhered to the plate, the culture medium was discarded and 500 μL of culture medium containing different concentrations of Human TNFSF11 protein was added to the 24-well plate so that the final concentration of Human TNFSF11 protein in each well was 1000, 500, 100, 50, 25, 10, 5, 2, 1, 0.5, 0.1, 0.01, and 0 ng / mL;

[0252] (3) Cells were cultured for 72 h.

[0253] (4) After the incubation time is reached, lyse the cells with 200 μL of cell lysis buffer (containing PMSF at a final concentration of 1 mM); after 2 min, collect the cell lysate, centrifuge, and take the supernatant.

[0254] (5) Use a 96-well plate to set up blank control wells, standard wells, and sample wells. The amounts of standard wells are 4, 8, 16, 24, 32, and 40 μL, respectively; Figure 8 As shown;

[0255] (6) Mix by gently pipetting and incubate at 37°C for 30 minutes.

[0256] (7) Add 160 µl of reaction stop solution to each well to terminate the reaction;

[0257] (8) Measure the absorbance at 405 nm.

[0258] 7.3 Data Processing

[0259] GraphPad Prism 9 software was used to draw the curve with OD405 nm value as the ordinate and protein concentration as the abscissa, and the ED50 value was calculated.

[0260] The results are as follows Fig. 9 shown.

[0261] ED50 = 19.69 ng / mL, calculated according to the enzyme activity converter: 5.079 × 10 5 U / mg.

[0262] discuss

[0263] The inventors' studies have shown that when the wild-type coding sequence of human RANKL is used and eukaryotic cells derived from humans are used as host cells for expression and production, although theoretically the human wild-type sequence matches the preferred codons of human eukaryotic cells, the expression level of human sRANKL is still very low.

[0264] To this end, the inventors have analyzed and optimized some other factors that may affect protein expression besides codon preference, including (but not limited to): eliminating sequences that destabilize mRNA, minimizing DNA secondary structure, adjusting the GC content of DNA, optimizing the gene translation start frame, optimizing the translation termination sequence frame, etc.

[0265] The results showed that the optimized nucleotide sequence of the present invention (SEQ ID No: 2) had a very low consistency with the original nucleotide sequence (only about 72.5%).

[0266] However, introducing the optimized coding sequence with very low consistency into plasmid pCDNA3.4 and transiently transfecting HEK293 cells can significantly increase the production of sRANKL protein in human host cells (increased by about 2 times), and the glycosylation pattern of the prepared sRANKL protein is more consistent with the glycosylation pattern of natural human RANKL.

[0267] In the present invention, when human cells such as HEK293 are used for production, the recombinant sRANKL protein will be secreted into the culture supernatant, which makes the subsequent purification very simple. After separation and purification, the purity of the recombinant protein reaches 99% or higher and is highly soluble (concentration is about 5.8 mg / mL), achieving unexpected technical effects.

[0268] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An optimized polynucleotide encoding a soluble RANKL (sRANKL) recombinant protein, characterized in that: The nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 2 or 7.

2. The polynucleotide according to claim 1, wherein The polynucleotide encodes a soluble sRANKL protein having an amino acid sequence as shown in SEQ ID NO: 5; Or a sRANKL precursor protein with an amino acid sequence such as SEQ ID No:

6.

3. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 1.

4. A host cell, characterized in that The host cell contains the vector of claim 3, or the polynucleotide of claim 1 is integrated into its genome; Wherein, the host cell is HEK293 cell.

5. The host cell according to claim 4, characterized in that The host cell is a somatic cell derived from human.

6. A method for preparing soluble sRANKL protein, characterized in that: The method comprises the steps of: (a) culturing the host cell of claim 4 under conditions suitable for culture to obtain a culture; and (b) isolating and purifying soluble sRANKL protein from the culture.

7. The method according to claim 6, characterized in that The step (b) comprises: (b1) isolating sRANKL precursor protein from the culture supernatant, wherein the sequence of the sRANKL precursor protein is shown in SEQ ID No: 6; (b2) performing an enzyme digestion treatment on the sRANKL precursor protein to obtain an enzyme digestion mixture containing the sRANKL protein, wherein the amino acid sequence of the sRANKL protein is shown in SEQ ID No: 5; (b3) separating the sRANKL protein from the enzyme cleavage mixture containing the sRANKL protein.

8. The method according to claim 7, characterized in that In step (b1), the sRANKL precursor protein is separated from the culture supernatant by a nickel column affinity purification method.

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