Method for biosynthesis of human body structural material type-viii collagen

Abstract

Provided is a method for biosynthesis of a human body structural material type-VIII collagen. Also provided is a collagen, comprising one or more repeating units, wherein the repeating units are connected directly or by means of linkers, and each repeating unit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a variant thereof. The collagen can be used for biological dressings, human body bionic materials or plastic surgery materials. According to the method, the type-VIII collagen is produced by utilizing a genetic engineering technology, thereby overcoming the defects in the prior art.

Description

Method for biosynthesizing type VIII collagen, a structural material for the human body

[0001] This application claims the benefit of priority to Chinese invention patent application number 202310883772.3, filed July 18, 2023, entitled “Method for biosynthesizing type VIII collagen as a human structural material.” The contents of that application are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of collagen, and in particular to type VIII collagen, a structural material of the human body, and a biosynthesis method thereof. Background Art

[0003] Collagen is the most abundant and widely distributed protein in animals and is the primary component of connective tissue. Type VIII collagen is a short-chain, non-fibrillar collagen that is a major component of the retinal membrane and the corneal endothelial membrane (the basement membrane that separates the corneal endothelial cells from the corneal stroma). Type VIII collagen is produced by endothelial cells and forms a unique hexagonal lattice structure. It is found around chondrocytes in the heart, brain, liver, lungs, muscles, and cartilage. Furthermore, type VIII collagen is found around actively proliferating blood vessels in brain tumors and within the large, fibrotic vessels of hemangiomas.

[0004] Type VIII collagen is also known as short collagen or reticular collagen. Type VIII collagen contains two similar α chains, α1(VIII) and α2(VIII). Two homotrimeric isoforms, [α1(VIII)]3 and [α2(VIII)]3, are considered the major molecular species, but heterotrimers may also exist. The bovine Descemet's membrane exhibits a hexagonal reticular structure with thin type VIII collagen fibrils. Immunohistochemical analysis and electron microscopy indicate that type VIII collagen is the primary component of this structural framework. Furthermore, rotational shadowgraph analysis suggests that type VIII collagen may form a tetrahedral supramolecular structure that provides structural support and regulates cellular behavior.

[0005] Mice with double knockout of two type VIII collagen genes, Col8a1 and Col8a2, exhibit a pronounced anterior segment hypoplasia phenotype, with a bulbous anterior chamber and a bulbous cornea. The corneal stroma is diffusely thinned, similar to that seen in human corneas. Descemet's membrane is markedly thinned, and the anterior zonal region is absent. Corneal endothelial cells are enlarged and reduced in number. Finally, mutant corneal endothelial cells display reduced proliferation in response to various growth factors in vitro, suggesting that type VIII collagen may act as an enhancer of growth factor-induced cell proliferation.

[0006] Previously, type VIII collagen was described as a heterotrimer composed of two α1 chains and one α2 chain. However, in vitro studies have shown that homotrimers of either α1 or α2 form. These homotrimers are pepsin-resistant, and immunohistochemical studies have shown that they do not always colocalize in the cornea, optic nerve, aorta, and umbilical cord. The α1 chain is 744 amino acids long, with the first 27 amino acids at the N-terminus being a signal peptide.

[0007] The NC1 domain (aa572-744) of type VIII collagen is a protein called vastatin, which has been shown to be a potent angiogenesis inhibitor and has apoptosis-inducing activity in aortic endothelial cells. The α2 chain is 703 aa long, with a signal peptide at the N-terminus of α1-28. The α1 and α2 procollagen genes each contain four exons. A characteristic feature is that the largest exon encodes the entire triple-helical domain (COL1) and the C-terminal non-triple-helical domain (NC1). Type VIII collagen shares high sequence homology with type X collagen, with a similar intron-exon structure. This suggests that both collagens originate from the same precursor gene and belong to the same collagen subclass. Like type X collagen, type VIII collagen is able to form a hexagonal lattice structure.

[0008] Type VIII collagen is a glycoprotein that is very sensitive to neutrophil elastase and, unlike other vascular collagens such as types I, II, IV, and V, is completely degraded within 4 hours. Type VIII collagen is synthesized by aortic and corneal endothelial cells, pulmonary artery endothelial cells, and microvascular endothelial cells. Not all endothelial cells express type VIII collagen, as it is absent in both large and small vessels. Furthermore, monocytes and macrophages have been found to express type VIII collagen both in vitro and in vivo. Human mast cells have also been shown to produce type VIII collagen under normal and pathological conditions. It has been shown to contribute to angiogenesis, tissue remodeling, fibrosis, and cancer. Mast cells expressing type VIII collagen are found in the perivascular space. Type VIII collagen is also expressed in smooth muscle cells, where it stimulates cell migration.

[0009] The corneal endothelium secretes type VIII collagen, which assembles into a hexagonal lattice structure within the endothelial membrane through the interaction of α1 and α2 polypeptides. Mutations in the COL8A2 gene, but not COL8A1, are associated with early-stage Fuchs' endothelial corneal dystrophy. Type VIII collagen is thought to be involved in endothelial cell differentiation and organization. During cardiac development, type VIII collagen plays an important role in angiogenesis, immunolocalizing to the subendothelial layer of capillaries and arterioles. Type VIII collagen expression is upregulated in early atherosclerosis and is thought to be associated with thrombosis and mononuclear cell infiltration. It accumulates in atherosclerotic lesions, and its distribution pattern suggests a role in plaque stabilization. Type VIII collagen is present around actively proliferating brain tumor vessels and in the large fibrotic vessels of hemangiomas. Finally, type VIII collagen is expressed in human diabetic nephropathy but not in other renal diseases.

[0010] Until now, the vast majority of collagen used in various research studies has been derived from animal tissue and skin extracts. This animal-derived collagen has poor water solubility and poor processability, directly limiting its potential applications. However, collagen produced using genetic engineering techniques could effectively overcome these shortcomings.

[0011] Summary of the Invention

[0012] The present invention is based in part on the following discoveries: The inventors conducted a large-scale functional region screening of human type VIII collagen, obtaining target gene regions of various recombinant humanized type VIII collagens, some of which exhibited higher cell adhesion-promoting activity compared to the positive control (human type I collagen). The present invention also demonstrates that the collagen proteins or polypeptides of the present invention are suitable for expression and purification. Furthermore, the inventors discovered that compared to other collagens (C8b and C8d-j), C8a and C8c exhibited superior expression and purification, making them suitable for industrial production. C8a and C8c of the present invention also possess a triple-helical structure.

[0013] In one aspect, the present invention provides a collagen or polypeptide comprising one or more repeating units, wherein the repeating units are connected directly or through a linker, and the repeating units comprise an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence or (2) an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence.

[0014] In one embodiment, the plurality of repeat units is 2-50 repeat units, such as 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, or 2-8 repeat units.

[0015] In one embodiment, the linker comprises one or more amino acid residues, e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues.

[0016] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion.

[0017] In one embodiment, the substitutions are conservative amino acid substitutions.

[0018] In one embodiment, the collagen or polypeptide is recombinant collagen. In one embodiment, the collagen or polypeptide is recombinant type VIII collagen. In one embodiment, the collagen or polypeptide is human recombinant type VIII collagen.

[0019] In one embodiment, the collagen or polypeptide has cell adhesion activity or has a triple helical structure.

[0020] In one embodiment, the collagen or polypeptide comprises an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26 or 29, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence or (2) an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence.

[0021] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion. In one embodiment, the substitution is a conservative amino acid substitution.

[0022] In another aspect, a nucleic acid is provided that encodes a collagen protein or polypeptide as described herein. In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. In one embodiment, the nucleotide sequence is codon-optimized for expression in E. coli. In one embodiment, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30.

[0023] In another aspect, a vector is provided, which comprises a nucleic acid as described herein. In one embodiment, the vector comprises an expression control element, a purification tag nucleotide and / or a leader sequence nucleotide that is operably linked to the nucleic acid. In one embodiment, the expression control element is selected from a promoter, a terminator or an enhancer. In one embodiment, the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag. In one embodiment, the vector is an expression vector or a cloning vector, preferably pET-28a(+). pET-28a(+) may comprise an N-terminal His, Thrombin and T7 protein tag, and a C-terminal His tag. In this article, the N-terminus of the collagen or polypeptide may comprise an enzyme cleavage site for ease of purification, such as a TEV enzyme cleavage site.

[0024] In another aspect, a host cell is provided, comprising a nucleic acid or vector as described herein. In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. In one embodiment, the prokaryotic cell is an Escherichia coli cell, such as Escherichia coli BL21.

[0025] In another aspect, a composition is provided, comprising one or more of the collagen or polypeptide, nucleic acid, vector, and host cell described herein. In one embodiment, the composition is a kit. In one embodiment, the composition is a biological dressing, a human biomimetic material, a plastic surgery material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient, and a food additive. In one embodiment, the composition is an injectable composition or an oral composition.

[0026] In another aspect, provided herein are uses of the collagen or polypeptide, nucleic acid, vector, host cell and / or composition in one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0027] In another aspect, a method for promoting cell adhesion is provided, comprising contacting the collagen or polypeptide, nucleic acid, vector, host cell and / or composition herein with a cell. In one embodiment, the cell is an animal cell. The animal cell can be a mammalian cell or a human cell.

[0028] In another aspect, a method for performing cosmetic surgery, tissue injection filling, ophthalmic treatment, nerve repair, or vascular repair on a subject in need thereof is provided, comprising administering the collagen or polypeptide described herein to the subject. In one embodiment, the administration is oral administration or injection administration. In one embodiment, the subject has a disease or condition associated with type VIII collagen deficiency, such as anterior segment hypoplasia.

[0029] In another aspect, a method of producing the collagen or polypeptide described herein is provided, comprising:

[0030] (1) culturing the host cell described herein under appropriate culture conditions;

[0031] (2) harvesting host cells and / or culture medium containing the collagen or polypeptide; and

[0032] (3) Purification of collagen or polypeptide.

[0033] In one embodiment, the host cell is an E. coli cell, preferably an E. coli BL21 (DE3) cell.

[0034] In one embodiment, step (1) comprises culturing E. coli cells in LB medium and inducing expression by IPTG.

[0035] In one embodiment, step (2) comprises harvesting the E. coli cells, resuspending them in a balanced working solution, homogenizing the E. coli cells, preferably by high pressure homogenization, and separating the supernatant. In one embodiment, the balanced working solution comprises 100-500 mM sodium chloride, 10-50 mM Tris, 10-50 mM imidazole, pH 7-9.

[0036] In one embodiment, step (3) comprises crude purification, enzyme digestion, fine purification and / or inverse nickel column purification. In one embodiment, step (3) comprises crude purification, and one or more of the following: enzyme digestion, fine purification and / or inverse nickel column purification.

[0037] In one embodiment, the crude purification comprises subjecting the supernatant to Ni-agarose gel column purification to obtain an eluate containing the target protein, wherein the eluate comprises 100-500 mM sodium chloride, 10-50 mM Tris and 100-500 mM imidazole, preferably at pH 7-9.

[0038] In one embodiment, the purification comprises gradient elution of the enzymatically cleaved product using a strong anion exchange chromatography column; preferably, the gradient elution comprises 0-15% solution B for 1-5 minutes and then holding for 3 column volumes, 15-30% solution B for 1-5 minutes and then holding for 3 column volumes, 30-50% solution B for 1-5 minutes and then holding for 3 column volumes, and 50-100% solution B for 1-5 minutes and then holding for 3 column volumes; wherein solution B comprises 10-50 mM Tris, 0.5-5 M sodium chloride, pH 7-9.

[0039] In one embodiment, the nickel column purification comprises purifying the product after enzyme cleavage on a Ni-agarose gel column; preferably, the eluent comprises 10-50 mM Tris, 10-50 mM sodium chloride, 0.5-5 M imidazole, pH 7-9.

[0040] Herein, the enzymatic cleavage may be enzymatic cleavage by TEV enzyme.

[0041] Advantages of the present invention include:

[0042] 1. The collagen of the present invention (e.g., C8a-C8j) is derived from type VIII collagen, and is recombinant type VIII collagen; in particular, it is derived from human type VIII collagen, and is recombinant human type VIII collagen;

[0043] 2. The collagen of the present invention (e.g., C8a-C8j) is suitable for preparation in E. coli and can be isolated and purified;

[0044] 3. The collagen proteins of the present invention (e.g., C8a and C8c) have a large expression level and are suitable for subsequent purification; and

[0045] 4. The collagen of the present invention has cell adhesion activity. The collagen of the present invention (such as C8a and C8c) has higher cell adhesion activity than the positive control. The collagen of the present invention has a triple helical structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 shows the purification results of C8a. The purified C8a yield was high and the target protein was of good purity.

[0047] Figure 2 shows the purification results of C8b. The crude purified target protein of C8b had some impurities, and some target protein was present after 1M washing. The yield after fine purification was low, and the target protein was of good purity.

[0048] Figure 3 shows the purification results of C8c. The target protein of C8c was relatively pure, and a small amount of the target protein was not cleaved by the 20:1 enzyme digestion.

[0049] Figure 4 shows the purification results of C8d. The yield of C8d was high, and the 75 kDa impurity band was not removed after nickel inversion.

[0050] Figure 5 shows the purification results of C8e. The crude purified target protein of C8e contained impurity bands and had a low yield.

[0051] Figure 6 shows the purification results of C8f. The crude purified target protein of C8f contained many impurity bands.

[0052] Figure 7 shows the purification results of C8g. The crude yield of C8g was high, and the target protein was observed as two bands.

[0053] Figure 8 shows the purification results of C8h. The crude yield of C8h was low.

[0054] Figure 9 shows the results of C8i purification. For C8i, most of the protein remained intact after 20:1 digestion and 5:1 digestion.

[0055] The purification results of C8j are shown in Figure 10. The crude yield of C8j was low.

[0056] FIG11 shows the results of the adhesion activity assay of C8a and C8c cells.

[0057] FIG12 shows the results of circular dichroism spectroscopy analysis of C8a.

[0058] FIG13 shows the results of circular dichroism spectroscopy analysis of C8c. DETAILED DESCRIPTION

[0059] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0060] Recombinant collagen is a new type of biomaterial that is screened and prepared using cutting-edge structural biology, genetic engineering and other technologies, using the gene encoding the functional region of a specific type of human collagen as a template, to obtain the same or similar amino acid sequence as human collagen.

[0061] As used herein, "type VIII collagen" is referred to as short collagen or reticular collagen. Type VIII collagen has two similar α chains, α1(VIII) and α2(VIII). Two homotrimeric isoforms, [α1(VIII)]3 and [α2(VIII)]3, are considered the major molecular species, but heterotrimers may also exist. The bovine Descemet's membrane exhibits a hexagonal reticular structure with thin type VIII collagen fibrils. Immunohistochemical analysis and electron microscopy indicate that type VIII collagen is the primary component of this structural framework. Furthermore, rotational shadowgraph analysis suggests that type VIII collagen may form a tetrahedral supramolecular structure, which provides structural support and regulates cellular behavior.

[0062] As used herein, "polypeptide" refers to a plurality of amino acid residues linked by peptide bonds. Herein, a polypeptide comprises one or more repeating units. The repeating units may be derived from human type VIII collagen. Thus, the polypeptide may be recombinant human type VIII collagen. The multiple repeating units may be connected by a linker, which may be the natural amino acid residues of the repeating units on human type VIII collagen, for example, 1-50 amino acid residues. The repeating units may be SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28. The polypeptide may be SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29.

[0063] As used herein, "human recombinant type VIII collagen" refers to a recombinant protein consisting of or consisting essentially of a sequence derived from human type VIII collagen. Herein, human recombinant type VIII collagen can consist of or consist essentially of a fragment or multiple repeats of a fragment derived from human type VIII collagen. Herein, recombinant collagen, human recombinant type VIII collagen, collagen, or polypeptide are used interchangeably.

[0064] As used herein, the term "variant" means a collagen or polypeptide having cell adhesion activity that includes a change (i.e., substitution, addition, insertion and / or deletion) at one or more positions. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. Addition refers to the addition of one or more amino acid residues at the C-terminus and / or N-terminus of the amino acid sequence. The substitution may be a conservative substitution. The variant of the repeating unit may be a sequence after one or more amino acid residues are changed or mutated (i.e., substituted, added, inserted and / or deleted) in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28. The variant of the collagen or polypeptide may be a sequence after one or more amino acid residues are changed or mutated (i.e., substituted, added, inserted and / or deleted) in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26 or 29.

[0065] In the context of the present invention, conservative substitutions may be defined by substitutions within the amino acid classes reflected in one or more of the following tables:

[0066] Conservative classes of amino acid residues: acidic residues D and E basic residues K, R, and H hydrophilic uncharged residues S, T, N, and Q aliphatic uncharged residues G, A, V, L, and I nonpolar uncharged residues C, M, and P aromatic residues F, Y, and W.

[0067] Physical and functional classification of alternative amino acid residues: Alcohol-containing residues S and T Aliphatic residues I, L, V and M Cycloalkenyl-related residues F, H, W and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S and T Positively charged residues H, K and R Small residues A, C, D, G, N, P, S, T and V Very small residues A, G and S Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible residues Q, T, K, S, G, P, D, E and R.

[0068] As used herein, "cell adhesion" refers to the adhesion between cells and collagen. Collagen (such as the polypeptides described herein) can promote the adhesion between cells and the container in which the cells are cultured.

[0069] As used herein, the term "expression" includes any step involved in the production of collagen or polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0070] As used herein, the term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a collagen protein or polypeptide and is operably linked to control sequences that provide for its expression.

[0071] As used herein, the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0072] As used herein, the term "nucleic acid" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified into a nucleic acid-containing segment in a manner not originally found in nature, or is synthetic, and may contain one or more control sequences. The nucleic acid may be SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30. The nucleic acid may be codon-optimized, for example, a nucleic acid that is codon-optimized for expression in E. coli cells.

[0073] The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0074] The degree of association between two amino acid sequences or between two nucleotide sequences is described by parameter " sequence identity ".For purposes of the present invention, use as in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, people such as Rice, 2000, Trends Genet. [genetics trend] 16:276-277) (preferred 5.0.0 version or updated version) Ni Deer program implemented Ni Deerman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [J.Molecular Biology] 48:443-453) determine the sequence identity between two amino acid sequences.The parameter used is gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (using non-simplified option to obtain) of Ni Deer, who is labeled as " longest identity ", is used as identity percentage and is calculated as follows:

[0075] (number of identical residues × 100) / (length of alignment - total number of gaps in the alignment)

[0076] For purposes of the present invention, the sequence identity between two deoxynucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented by the Needleman program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferred 5.0.0 version or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needleman, labeled "longest identity," (obtained using the non-simplified option), which is labeled "longest identity," is used as percent identity and is calculated as follows:

[0077] (number of identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in the alignment)

[0078] collagen

[0079] The present invention provides a collagen protein or polypeptide comprising one or more repeating units, wherein the repeating units are connected directly or through a linker, and the repeating units comprise an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28. The variant can be (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28. For the collagen or polypeptide described herein, the mutation may be selected from substitution, addition, insertion or deletion. Preferably, the substitution is a conservative amino acid substitution.

[0080] The collagen or polypeptide described herein can comprise a plurality of repeating units, for example 2-50 repeating units, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 repeating units.

[0081] The linker in the collagen or polypeptide described herein may comprise one or more amino acid residues, for example 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues.

[0082] The collagen or polypeptide described herein is a recombinant collagen, in particular a recombinant type VIII collagen, preferably having cell adhesion activity. The collagen or polypeptide described herein may be derived from human, and thus is human recombinant type VIII collagen.

[0083] The collagen or polypeptide described herein may also comprise an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26 or 29, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence or (2) an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence.

[0084] The collagen described herein may have a triple helical structure or three identical chains (ie, a trimer). The sequence of each chain may be the sequence of the collagen or polypeptide described herein.

[0085] Nucleic acid constructs

[0086] The present invention also relates to nucleic acid constructs comprising a nucleic acid of the present invention operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. A vector may comprise a nucleic acid construct.

[0087] Nucleic acids can be manipulated in a variety of ways to provide expression of collagen or polypeptides. Depending on the expression vector, it may be desirable or necessary to manipulate the nucleic acid prior to its insertion into the vector. Techniques for modifying nucleic acids using recombinant DNA methods are well known in the art.

[0088] The control sequence can be a promoter, i.e., a polynucleotide that is recognized by the host cell for expression of the collagen protein or polypeptide encoding the present invention. The promoter comprises transcriptional control sequences that mediate the expression of the collagen protein or polypeptide. The promoter can be any nucleic acid that exhibits transcriptional activity in the host cell, including variants, truncated forms, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular collagen protein or polypeptide homologous or heterologous to the host cell.

[0089] Examples of suitable promoters for directing transcription of the vectors or nucleic acid constructs of the present invention in bacterial host cells are those obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltoamylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the Bacillus thuringiensis cryIIIA gene, the Escherichia coli lac operon, the Escherichia coli trc promoter.

[0090] In yeast hosts, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.

[0091] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the collagen protein or polypeptide. Any terminator that is functional in the host cell can be used in the present invention.

[0092] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0093] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al. (1992, supra).

[0094] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0095] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0096] The control sequence may also be a leader sequence, which is a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the collagen protein or polypeptide. Any leader sequence that is functional in the host cell may be used.

[0097] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0098] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0099] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0100] The control sequence can also be a signal peptide coding region that encodes a signal peptide connected to the N-terminus of a collagen protein or polypeptide and instructs the collagen protein or polypeptide to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide itself can contain a signal peptide coding sequence that is naturally connected to the coding sequence segment of the coding collagen protein or polypeptide in the translation reading frame. Alternatively, the 5'-end of the coding sequence can contain a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the collagen protein or polypeptide. However, any signal peptide coding sequence that instructs the expressed collagen protein or polypeptide to enter the secretory pathway of the host cell can be used.

[0101] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prs A. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0102] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al. (1992, supra).

[0103] expression vector

[0104] The present invention further relates to a recombinant expression vector comprising nucleic acid of the present invention, promoter and transcription and translation termination signals. Nucleic acid and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to insert or replace the polynucleotide encoding the collagen protein or polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting a nucleic acid or a nucleic acid construct comprising the nucleic acid into a suitable vector for expression. When producing an expression vector, the coding sequence is located in the vector so that the coding sequence is operably connected to the suitable control sequence for expression.

[0105] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be readily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0106] The carrier can be an autonomous replicating vector, i.e. a carrier existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The carrier can contain any means for ensuring self-replication. Alternatively, the carrier can be a carrier that is integrated into the genome and replicates with one or more chromosomes into which it has been integrated when it is introduced into the host cell. Moreover, a single carrier or plasmid or two or more carriers or plasmids can be used that contain the total DNA to be introduced into the host cell genome together, or a transposon can be used.

[0107] The vector preferably contains one or more selectable markers that allow for easy selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene whose product confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0108] Examples of bacterial selectable markers are the dal genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0109] The selectable marker may be a dual selectable marker system as described in WO 2010 / 039889. In one aspect, the dual selectable marker is the hph-tk dual selectable marker system.

[0110] The vector may contain elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0111] For being integrated into the host cell genome, the vector can rely on the polynucleotide sequence encoding the collagen or polypeptide or any other element of the vector for being integrated into the genome by homologous or non-homologous recombination. Alternatively, the vector can contain other polynucleotides at the precise position for guiding the integration of the chromosome in the host cell genome by homologous recombination. In order to improve the possibility of integration at the precise position, the integration element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs and 800 to 10,000 base pairs, and these nucleic acids have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integration element can be any sequence homologous to the target sequence in the host cell genome. Moreover, the integration element can be a non-coding or coded polynucleotide. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0112] For autonomous replication, the vector may further comprise an origin of replication that enables autonomous replication of the vector in the host cell in question. The origin of replication may be any plasmid replicator that mediates autonomous replication and functions in the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0113] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

[0114] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0115] More than one copy of a polynucleotide of the present invention can be inserted into a host cell to increase production of collagen or polypeptides. Increased copy numbers of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene and thus additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective agent.

[0116] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989).

[0117] host cells

[0118] The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of the collagen or polypeptide of the present invention. A construct or vector comprising the polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will depend largely on the gene encoding the collagen or polypeptide and its source.

[0119] The host cell can be any cell useful in the recombinant production of the collagen or polypeptides of the invention, eg, a prokaryotic or eukaryotic cell.

[0120] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0121] The host cell may also be a eukaryotic organism, such as a mammalian, insect, plant, or fungal cell.

[0122] The host cell can be a fungal cell, such as Basidiomycota, Chytridiomycota, Zygomycota, and Oomycota, etc. The fungal host cell can be a yeast cell, including ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0123] Production method

[0124] The present invention also relates to a method for producing the collagen or polypeptide described herein, comprising:

[0125] (1) culturing the host cell described herein under appropriate culture conditions;

[0126] (2) harvesting host cells and / or culture medium containing the collagen or polypeptide; and

[0127] (3) Purification of collagen or polypeptide.

[0128] Host cell is cultivated in the nutrient medium of suitable production collagen protein or polypeptide using methods known in the art.For example, can be by shake flask culture or in laboratory or industrial fermenter small-scale or large-scale fermentation (comprising continuous, batch, fed-batch or solid-state fermentation) culture cell, this culture is in suitable medium and is carried out under the condition that collagen protein or polypeptide is expressed and / or separated.Using program as known in the art, cultivation occurs in the suitable nutrient medium comprising carbon and nitrogen source and inorganic salt.Suitable culture medium can be obtained from commercial supplier or can be prepared according to disclosed composition (for example, in the catalogue of American Type Culture Collection).If collagen protein or polypeptide are secreted in this nutrient medium, then this collagen protein or polypeptide can be directly recovered from this culture medium.If collagen protein or polypeptide are not secreted, then it can be recovered from cell lysate.

[0129] Collagen or polypeptides can be detected using methods known in the art that are specific for the collagen or polypeptide. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, enzymatic assays can be used to determine the activity of the collagen or polypeptide.

[0130] The collagen or polypeptide can be recovered using methods known in the art. For example, the collagen or polypeptide can be recovered from the nutrient medium by conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the fermentation broth containing the collagen or polypeptide is recovered.

[0131] Collagen or polypeptides can be purified by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubilization (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction, in order to obtain substantially pure collagen or polypeptides.

[0132] Step (1) may include one or more of the following steps: constructing an expression plasmid, for example, inserting the coding nucleotide sequence into the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid. The successfully constructed expression plasmid can be transformed into Escherichia coli cells (for example, Escherichia coli competent cells BL21 (DE3)). The specific process can be: (1) taking the plasmid to be transformed and adding it to the Escherichia coli competent cells BL21 (DE3); (2) placing the mixture in an ice bath (for example, 10-60 minutes, for example, 30 minutes), then heat shocking it in a water bath (for example, at 40-50°C, for example, 42°C, for 45-90 seconds), taking it out and placing it in an ice bath (for example, 1-5 minutes, for example, 2 minutes); (3) adding liquid LB culture medium, and then culturing (for example, at 35-40°C, for example, 37°C, 150-300 rpm, for example, 220 rpm, for 40-80 minutes, for example, 60 minutes); (4) applying the bacterial solution and selecting single colonies. For example, take the bacterial solution and spread it evenly on an LB plate containing ampicillin sodium, and culture the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow.

[0133] Step (2) may include culturing a single colony in LB medium containing an antibiotic stock solution (e.g., at 150-300 rpm, e.g., 220 rpm, 35-40° C., e.g., 37° C., in a constant temperature shaker for 5-10 h, e.g., 7 h). The cultured shake flask is then cooled to 10-20° C., e.g., 16° C., and IPTG is added to induce expression for a period of time before collecting the cells (e.g., by centrifugation).

[0134] Step (3) may include resuspending the cells in a balanced working solution, cooling the solution to ≤15°C, homogenizing (e.g., high pressure homogenization, e.g., 1-5 times, e.g., 2 times), and separating the homogenized solution to obtain a supernatant. The balanced working solution may comprise 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole, with a pH of 7-9. For example, the concentration of sodium chloride can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0135] Step (3) may include purifying and enzymatically cleaving the collagen or polypeptide. The purification may be crude purification, including subjecting the supernatant to Ni-agarose gel column purification to obtain an eluate containing the target protein. Crude purification may include washing the column material with water, for example, 2-10 column volumes (CV), for example, 5 CV. The column material may be balanced with an equilibrium solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), for example, 2-10 CV, for example, 5 CV. The equilibrium solution may comprise 100-500 mM sodium chloride, 10-50 mM Tris and 10-50 mM imidazole, pH 7-9. For example, the concentration of sodium chloride can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0136] Step (3) may include adding the supernatant to the column material and washing the impurities with a washing solution. The washing solution may comprise 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole, at a pH of 7-9. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH can be 7, 7.5, 8, 8.5, or 9. Then, an eluent can be added and the flow-through can be collected. The eluent can comprise 100-500 mM sodium chloride, 10-50 mM Tris, 100-500 mM imidazole, pH 8.0. For example, the concentration of sodium chloride can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The pH can be 7, 7.5, 8, 8.5, or 9.

[0137] Enzymatic digestion can include adding TEV enzyme for digestion (at a ratio of 10-100:1, e.g., 50:1, to the total amount of protein and the total amount of TEV enzyme, and digestion at 10-20° C., e.g., 16° C., for 2-8 hours, e.g., 4 hours). The protein solution after digestion is dialyzed, e.g., placed in a dialysis bag, dialyzed at 1-6° C., e.g., 4° C., for 1-8 hours, e.g., 2 hours, and then transferred to new dialysate for overnight dialyzation at 1-6° C., e.g., 4° C.

[0138] Purification may include polishing (e.g., protein isoelectric point > 8.0). Preferably, polishing comprises gradient elution of the eluate containing the target protein or the product after enzymatic digestion (e.g., the product after enzymatic digestion and dialysis) using a strong anion exchange chromatography column (e.g., pH 7, 7.5, 8, 8.5, or 9). The gradient elution comprises 0-15% B solution for 1-5 minutes and then holding for 1-5, e.g., 3 column volumes, 15-30% B solution for 1-5 minutes and then holding for 1-5, e.g., 3 column volumes, 30-50% B solution for 1-5 minutes and then holding for 1-5, e.g., 3 column volumes, 50-100% B solution for 1-5 minutes and then holding for 1-5, e.g., 3 column volumes. Solution B may comprise 10-50 mM Tris, 0.5-5 M sodium chloride, pH 7-9. For example, the concentration of Tris is 15, 20, 25, 30, 35, 40, or 45 mM. The concentration of sodium chloride is 1, 2, 3, or 4 M. The pH can be 7, 7.5, 8, 8.5, or 9. Purification can include equilibrating the column with Solution A and loading the sample, followed by gradient elution. Solution A can comprise 10-50 mM Tris, 10-50 mM sodium chloride, and a pH of 7-9. For example, the concentration of Tris is 15, 20, 25, 30, 35, 40, or 45 mM. The concentration of sodium chloride is 15, 20, 25, 30, 35, 40, or 45 mM. The pH can be 7, 7.5, 8, 8.5, or 9.

[0139] Purification can include reverse hanging nickel column purification (e.g., protein isoelectric point <8.0). Reverse hanging nickel column purification can include Ni-agarose gel column purification of the product after enzyme cleavage (e.g., product after dialysis). The eluent can contain 10-50mM (e.g., 15, 20, 25, 30, 35, 40, or 45mM) Tris, 10-50mM (e.g., 15, 20, 25, 30, 35, 40, or 45mM) sodium chloride, 0.5-5M (e.g., 1, 2, 3, or 4M) imidazole, pH 7-9 (e.g., 7, 7.5, 8, 8.5, or 9).

[0140] The following examples are provided to further illustrate the present invention.

[0141] Example

[0142] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims. In conjunction with this specification and common knowledge in the art, a person of ordinary skill in the art will clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0143] Example 1: Construction, expression and screening of type VIII collagen fragments

[0144] 1. Conduct large-scale functional region screening to obtain the following different target gene functional regions of recombinant humanized type VIII collagen.

[0145] 1) C8a amino acid sequence:

[0146] (The amino acid sequence of the repeating unit of C8a is shown in SEQ ID NO: 1, with a repeat number of 6; the amino acid sequence of C8a is shown in SEQ ID NO: 2)

[0147] Base sequence:

[0148] 2) C8b amino acid sequence:

[0149] (The amino acid sequence of the repeating unit of C8b is shown in SEQ ID NO: 7, with a repeat number of 6; the amino acid sequence of C8b is shown in SEQ ID NO: 8)

[0150] Base sequence:

[0151] 3) C8c amino acid sequence:

[0152] (The amino acid sequence of the repeating unit of C8c is shown in SEQ ID NO: 4, with a repeat number of 6; the amino acid sequence of C8c is shown in SEQ ID NO: 5)

[0153] Base sequence:

[0154] 4) C8d amino acid sequence:

[0155] (The amino acid sequence of the repeating unit of C8d is shown in SEQ ID NO: 10, with a repeat number of 6; the amino acid sequence of C8d is shown in SEQ ID NO: 11)

[0156] Base sequence:

[0157] 5) C8e amino acid sequence:

[0158] (The amino acid sequence of the repeating unit of C8e is shown in SEQ ID NO: 13, with a repeat number of 2; the amino acid sequence of C8e is shown in SEQ ID NO: 14)

[0159] Base sequence:

[0160] 6) C8f amino acid sequence:

[0161]

[0162] (The amino acid sequence of the repeating unit of C8f is shown in SEQ ID NO: 16, with a repeat number of 2; the amino acid sequence of C8f is shown in SEQ ID NO: 17)

[0163] Base sequence:

[0164] 7) C8g amino acid sequence:

[0165]

[0166] (The amino acid sequence of the repeating unit of C8g is shown in SEQ ID NO: 19, with a repeat number of 6; the amino acid sequence of C8g is shown in SEQ ID NO: 20)

[0167] Base sequence:

[0168] 8) C8h amino acid sequence:

[0169]

[0170] (The amino acid sequence of the repeating unit of C8h is shown in SEQ ID NO: 22, with a repeat number of 6; the amino acid sequence of C8h is shown in SEQ ID NO: 23)

[0171] Base sequence:

[0172] 9) C8i amino acid sequence:

[0173]

[0174] (The amino acid sequence of the repeating unit of C8i is shown in SEQ ID NO: 25, with a repeat number of 6; the amino acid sequence of C8i is shown in SEQ ID NO: 26)

[0175] Base sequence:

[0176] 10) C8j amino acid sequence:

[0177]

[0178] (The amino acid sequence of the repeating unit of C8j is shown in SEQ ID NO: 28, with a repeat number of 6; the amino acid sequence of C8j is shown in SEQ ID NO: 29)

[0179] Base sequence:

[0180] 2. Each of the above encoding nucleotide sequences was commercially synthesized. Each of the above encoding nucleotide sequences (with a collagenase cleavage site added at the 5' end, the amino acid sequence of the collagenase cleavage site is ENLYFQ, and the nucleotide sequence is GAAAACCTGTATTTCCAG) was inserted between the KpnI and XhoI restriction sites of the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid.

[0181] 3. Transform the successfully constructed expression plasmid into E. coli competent cells BL21 (DE3). The specific process is as follows: (1) Take out the E. coli competent cells BL21 (DE3) from the ultra-low temperature refrigerator and place them on ice. When they are half melted, take 2 μl of the plasmid to be transformed and add it to the E. coli competent cells BL21 (DE3) and mix it slightly 2-3 times. (2) Place the mixture on ice for 30 minutes, then heat shock it in a 42°C water bath for 45-90 seconds, take it out and place it on ice for 2 minutes. (3) Transfer it to a biosafety cabinet and add 700 μl of liquid LB culture medium, then incubate it at 37°C and 220 rpm for 60 minutes. (4) Take 200 μl of the bacterial solution and evenly spread it on an LB plate containing ampicillin sodium. (5) Incubate the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow.

[0182] 4. Pick 5-6 single colonies from the transformed LB plate and place them in a shake flask containing LB medium containing antibiotic stock solution. Incubate in a constant temperature shaker at 220 rpm and 37°C for 7 hours. Then, cool the shake flask to 16°C. After inducing expression for a period of time, add IPTG. Then, divide the bacterial solution into centrifuge bottles and centrifuge at 8000 rpm and 4°C for 10 minutes. Collect the cells and record the cell weight. Take a sample (labeled "cell solution") for electrophoresis.

[0183] 5. Resuspend the collected cells in a balanced working solution (200mM NaCl, 25mM Tris, 20mM imidazole, pH 8.0). Cool the solution to ≤15°C and homogenize. High-pressure homogenize twice (take samples from each homogenization and label them as "homogeneous first" and "homogeneous second"). After completion, collect the solution. Aliquot the homogenized solution into centrifuge bottles and centrifuge at 17,000 rpm and 4°C for 30 minutes. Collect the supernatant and perform electrophoresis on the supernatant (labeled "supernatant") and the precipitate (labeled "precipitate").

[0184] 6. Recombinant humanized type VIII collagen was purified and enzymatically digested. The specific process was as follows: (1) Crude purification: a. Wash the column (Ni6FF, Cytiva) with water for 5 CV. b. Equilibrate the column with equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) for 5 CV. c. Loading: Add the supernatant after centrifugation to the column until the liquid has flowed through, and then take the flow-through for electrophoresis (marked as flow-through). d. Wash impurities: Add 25 mL of wash solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) until the liquid has flowed through, and then take the wash flow-through for electrophoresis (marked as wash impurities). e. Collect the target protein: add 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0) and collect the flow-through (marked as: elution), detect the protein concentration, calculate the protein amount, and perform electrophoresis detection. f. Wash the column with 1 M imidazole working solution (marked as 1 M wash). g. Wash the column with purified water. (2) Enzyme digestion: add TEV enzyme according to the ratio of total protein to total TEV enzyme of 50:1 (if not digested, consider increasing the enzyme concentration, for example, the total ratio is 20:1 or 5:1), digest at 16 ° C for 4 hours, take a sample (marked as after digestion) and perform electrophoresis detection. Place the protein solution after digestion in a dialysis bag, dialyze at 4 ° C for 2 hours, and then transfer to new dialysate for overnight dialysis at 4 ° C (marked as liquid change).

[0185] (3) Purification (protein isoelectric point > 8.0): a. Equilibrate the column (Capto Q, Cytiva): Use solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) to equilibrate the column at a flow rate of 10 ml / min. b. Load the sample: The flow rate is 5 ml / min, load the sample and collect the flow-through (marked as QFL), and perform electrophoresis detection. c. Gradient elution: Set 0-15% solution B (20 mM Tris, 1 M sodium chloride, pH 8.0) for 2 min (marked as 0-15% B wash) and then maintain for 3 CVs, 15-30% solution B for 2 min and then maintain for 3 CVs, 30-50% solution B for 2 min and then maintain for 3 CVs, 50-100% solution B for 2 min and then maintain for 3 CVs, collect the peaks and perform electrophoresis detection. d. Clean the column. Store the protein at 4°C.

[0186] (4) Nickel reverse mounting (Ni6FF, Cytiva) (protein isoelectric point <8.0): a. Equilibrate the column: Use solution A (20mM Tris, 20mM sodium chloride, 20mM imidazole, pH8.0) to equilibrate the column for 5 CVs. b. Load the sample: Add the protein after enzyme switching solution to the column, wait until the liquid has flowed out, and then take the flow-through for electrophoresis detection (marked as nickel reverse mounting). c. Wash the column with 1M imidazole working solution (20mM Tris, 20mM sodium chloride, 1M imidazole, pH8.0) (marked as 1M wash). d. Wash the column with purified water. Store the protein at 4°C.

[0187] 7. Concentration detection

[0188] Accurately measure an appropriate amount of sample and dilute 10-50 times with eluent. Stir thoroughly with a glass rod. Measure the absorbance at 280 nm using a UV-visible spectrophotometer. Calculate the protein concentration using the formula C (mg / ml) = A280 × Absorbance Coefficient × Dilution Factor (Note: The absorbance coefficient can be derived from the amino acid sequence; the absorbance value must be between 0.1 and 1).

[0189] The concentration test results are as follows:

[0190] The protein expression levels were C8a>C8i>C8b>C8g>C8d>C8c>C8f>C8e>C8j>C8h.

[0191] 8. Electrophoresis detection

[0192] The specific process is as follows: take 40 μl of sample protein solution, add 10 μl 5× protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), place it in boiling water at 100°C for 10 minutes, then add 10 μl per well to SDS-PAGE protein gel, run at 80 V for 2 hours, and then use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20 minutes, and then use protein decolorization solution (10% acetic acid, 5% ethanol) for decolorization.

[0193] The electrophoresis test results showed:

[0194] Figure 1 shows the purification results for C8a. C8a had a high yield after purification and good target protein purity. Figure 2 shows the purification results for C8b. The crude purified target protein from C8b showed some impurities, and some target protein remained after the 1M wash. The yield after purification was low, but the target protein purity was good. Figure 3 shows the purification results for C8c. The target protein purity from C8c was good, with a small amount of target protein remaining uncleaved after the 20:1 enzyme digestion. Figure 4 shows the purification results for C8d. C8d had a high yield, and the impurity band at 75 kDa was not removed after nickel inversion. Figure 5 shows the purification results for C8e. The crude purified target protein from C8e had impurities and a low yield. Figure 6 shows the purification results for C8f. The crude purified target protein from C8f had more impurities. Figure 7 shows the purification results for C8g. The crude purified target protein from C8g had a high yield, with the target protein appearing as two bands. Figure 8 shows the purification results for C8h. The crude purified target protein yield from C8h was low. Figure 9 shows the purification results for C8i. For C8i, the majority of the protein remained uncleaved after 20:1 digestion, and even after 5:1 digestion, no cleavage was observed. Figure 10 shows the purification results for C8j. The crude yield of C8j was low.

[0195] Electrophoresis results showed that: 1. The crude purified protein of C8e, C8h, and C8j had low yields and thin target protein bands; 2. The crude purified target protein of C8i had a distinct impurity band at 35 kDa, indicating that it was essentially undigested by the enzyme; 3. The target proteins of C8b and C8f had numerous impurity bands and low yields after purification; 4. The target protein of C8g had a high yield, but showed two bands, indicating low purity; 5. The target protein of C8d had a high yield, but after Ni column loading, it had numerous impurity bands and low purity; 6. The target protein of C8a and C8c had high yields and good purity. C8a and C8c were selected for subsequent testing.

[0196] Example 2: Mass spectrometry detection of recombinant humanized type VIII collagen

[0197] Experimental methods

[0198] After DTT reduction and iodoacetamide alkylation, the protein sample was digested with trypsin overnight. The resulting peptides were desalted using a C18 ZipTip and then spotted with the matrix α-cyano-4-hydroxycinnamic acid (CHCA). Finally, analysis was performed using a matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer (MALDI-TOF / TOF Ulraflextreme™, Brucker, Germany). (For peptide fingerprinting techniques, see Protein J. 2016;35:212-7.)

[0199] Data retrieval was performed using the MS / MS Ion Search page on the local masco website. Protein identification was based on the primary mass spectra of the peptides generated after enzymatic digestion. Detection parameters: Trypsin digestion with two missed cleavage sites. Alkylation of cysteine ​​was set as a fixed modification. Oxidation of methionine was set as a variable modification. The database used for identification was NCBprot.

[0200] Table 1: Molecular weights and corresponding peptides detected by mass spectrometry of recombinant type VIII humanized collagen C8a

[0201] The coverage of the detected polypeptide fragments compared with the theoretical sequence is 100%, and the detection results are very reliable.

[0202] Example 3: Bioactivity Detection of Recombinant Humanized Type VIII Collagen

[0203] The method for detecting collagen activity can be found in the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004). The specific implementation method is as follows:

[0204] (1) The concentration of the protein sample to be tested was detected by ultraviolet absorption method, including bovine type I collagen (China Food and Drug Inspection Institute, No.: 380002) and the recombinant proteins C8a and C8c provided by the present invention.

[0205] Specifically, the sample's UV absorbance at 215 nm and 225 nm is measured, and the protein concentration is calculated using the empirical formula C (μg / mL) = 144 × (A215 - A225). Note that the assay must be performed when A215 < 1.5. This method, which measures the characteristic absorption of peptide bonds under far-UV light, is unaffected by chromophore content, exhibits minimal interfering substances, and is simple to perform. It is suitable for detecting human collagen and its analogs, which do not develop color with Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.) After measuring the protein concentration, adjust the concentration of all tested proteins to 0.5 mg / mL with PBS.

[0206] (2) Sample preparation: Use the sample solution directly for the experiment; dilute the positive control human type I collagen (PC) to 1 mg / ml using D-PBS; the negative control is D-PBS buffer (NC).

[0207] (3) Coating: Add different concentrations of collagen and positive and negative controls to the ELISA plate, 100 μL per well, set up 5 replicate wells for each group, and incubate at 4°C overnight.

[0208] (4) Blocking: Discard the supernatant, add 100 μL of 1% BSA (heat-inactivated at 56°C for 30 min), and incubate at 37°C for 60 min. Discard the supernatant and wash three times with D-PBS solution.

[0209] (5) Cell inoculation: Add 10 5 Resuspend 3T3 / NIH cells in good culture in D-PBS and incubate at 37°C for 120 min. Wash each well three times with D-PBS solution.

[0210] (6) Detection: Use CCK8 detection kit (manufacturer: Beyotime, product catalog number C0038) to detect OD 450 The absorbance is measured in nm. The degree of cell adhesion is calculated using the following formula. The cell adhesion rate reflects the cell adhesion capacity of collagen. The higher the cell adhesion capacity, the more quickly the cells are provided with a high-quality external environment, helping them adhere.

[0211] Where:

[0212] P: relative cell adhesion ratio;

[0213] OD1: The average value of UV absorbance of each replicate well of the test collagen sample at 450nm;

[0214] OD2: average UV absorbance of each replicate well of the control collagen sample at 450 nm;

[0215] OD0: The average value of UV absorbance of each replicate well in the blank control group at 450 nm.

[0216] (7) Statistical analysis: Two-tailed t-test was used to analyze the statistical differences between the target recombinant humanized collagen and the negative control, where *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0217] The results are shown in FIG11 . Compared with the D-PBS group, the positive control group had a significant effect on promoting cell adhesion, and the recombinant humanized collagens C8a and C8c also promoted cell adhesion.

[0218] Example 4: UV-scanning analysis of recombinant humanized type VIII collagen by circular dichroism

[0219] Experimental methods

[0220] (1) Instrument parameter settings: Band width: 1.0 nm, Step: 1.0 nm, Measurement range: 190-260 nm (far-UV scan) / 250-340 nm (near-UV scan), Time per point: 0.5 s, Repeats: 3, Cell length: 10 mm | 0.5 mm, Temperature: Room temperature

[0221] (2) Far and near UV scanning of standard products

[0222] The scanning wavelength was set to 180-340 nm for background test and blank buffer test, and then the far- and near-UV circular dichroism absorption of 1 mg / mL CSA standard solution in the range of 180-340 nm was collected.

[0223] (3) Sample processing

[0224] The purified C8a and C8c protein samples were collected and concentrated to a protein concentration of 1 mg / ml using 10KD ultrafiltration concentrators (Millipore).

[0225] (4) Sample far-UV scanning

[0226] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry, collect the background first, then collect the blank buffer, then add an appropriate amount of test sample to the cuvette, perform far-UV scanning at 190-260nm according to the above parameters and collect data.

[0227] (5) Sample near-UV scanning

[0228] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry it. Collect the background first, then collect the blank buffer solution, then add an appropriate amount of test sample to the cuvette, perform near-UV scanning at 250-340nm according to the above parameters and collect data.

[0229] (6) Scanning spectrum processing

[0230] All scanned atlases were subjected to subtract baseline and smoothing processing using the software Pro-Data Viewer.

[0231] Experimental results and analysis

[0232] The results showed that both C8a and C8c had positive peaks at 221 nm, indicating that the proteins had a triple helical structure (ie, a common structural feature of active collagen). The results are shown in Figures 12 and 13 .

[0233] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A collagen comprising one or more repeating units, wherein the repeating units are connected directly or through a linker, wherein the repeating units comprise an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence; Preferably, the plurality of repeating units is 2-50 repeating units, such as 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10 or 2-8 repeating units; Preferably, wherein the linker comprises one or more amino acid residues, such as 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues; Preferably, wherein the mutation is selected from substitution, addition, insertion or deletion; Preferably, wherein the substitution is a conservative amino acid substitution; Preferably, the collagen is recombinant collagen; preferably recombinant type VIII collagen; preferably human recombinant type VIII collagen; Preferably, the collagen has cell adhesion activity, or has a triple helical structure or is in the form of a trimer.

2. The collagen according to claim 1, comprising an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26 or 29, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence; Preferably, wherein the mutation is selected from substitution, addition, insertion or deletion; Preferably, the substitution is a conservative amino acid substitution.

3. A nucleic acid encoding the collagen according to claim 1 or 2, Preferably, wherein the nucleic acid comprises a codon-optimized nucleotide sequence, Preferably, the nucleotide sequence is codon-optimized for E. coli expression; Preferably, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30.

4. A vector comprising the nucleic acid according to claim 3, Preferably, the vector comprises an expression control element, a purification marker, and a nucleic acid operably linked to the nucleic acid. The nucleotides of the signature and / or the nucleotides of the leader sequence, Preferably, wherein the expression control element is selected from a promoter, a terminator or an enhancer; Preferably, the purification tag is selected from a His tag, a GST tag, a MBP tag, a SUMO tag or a NusA tag; Preferably, the vector is an expression vector or a cloning vector, preferably pET-28a(+).

5. A host cell comprising a nucleic acid according to claim 3 or a vector according to claim 4; wherein preferably, the host cell is a eukaryotic cell or a prokaryotic cell; wherein preferably, the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell, and / or the prokaryotic cell is an Escherichia coli cell, such as Escherichia coli BL21.

6. A composition comprising one or more of the collagen according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4 and the host cell according to claim 5, preferably, the composition is a kit; preferably, the composition is one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives, preferably, the composition is an injectable composition or an oral composition.

7. Use of the collagen according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5 and / or the composition according to claim 6 in one or more of biological dressings, human bionic materials, plastic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

8. A method for promoting cell adhesion, comprising the step of contacting the collagen according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5 and / or the composition according to claim 6 with cells, preferably, the cells are animal cells, preferably mammalian cells, preferably human cells.

9. A method for performing cosmetic surgery, tissue injection filling, ophthalmic treatment, nerve repair or vascular repair on a subject in need thereof, comprising administering the collagen according to claim 1 or 2 to the subject, preferably orally or by injection; preferably, the subject has a disease or condition associated with type VIII collagen deficiency, such as anterior segment hypoplasia, and preferably, the subject is human.

10. A method for producing collagen according to claim 1 or 2, comprising: (1) culturing the host cell according to claim 5 under suitable culture conditions; (2) harvesting host cells and / or culture medium containing collagen; and (3) Purification of collagen; Preferably, the host cell is an Escherichia coli cell, preferably an Escherichia coli BL21 (DE3) cell; Preferably, step (1) comprises culturing E. coli cells in LB medium and inducing expression by IPTG; Preferably, step (2) comprises harvesting the E. coli cells, resuspending them in a balanced working solution, homogenizing the E. coli cells, preferably high pressure homogenization, and separating the supernatant; preferably, the balanced working solution comprises 100-500 mM sodium chloride, 10-50 mM Tris, 10-50 mM imidazole, pH 7-9; Preferably, step (3) comprises crude purification and one or more of the following: enzyme digestion, fine purification and reverse hanging nickel column purification; Preferably, the crude purification comprises subjecting the supernatant to Ni-agarose gel column purification to obtain an eluate containing the target protein, wherein the eluate comprises 100-500 mM sodium chloride, 10-50 mM Tris and 100-500 mM imidazole, preferably at pH 7-9; Preferably, the enzymatic digestion includes digestion with TEV enzyme, preferably with a ratio of total protein to total TEV enzyme of 10-100:1 for 2-8h; Preferably, the purification comprises gradient elution of the product after enzyme digestion with a strong anion exchange chromatography column; preferably, the gradient elution comprises 0-15% B solution for 1-5 minutes and then maintained for 3 column volumes, 15-30% B solution for 1-5 minutes and then maintained for 3 column volumes, 30-50% B solution for 1-5 minutes and then maintained for 3 column volumes, 50-100% B solution for 1-5 minutes and then maintained for 3 column volumes; wherein B solution contains 10-50mM Tris, 0.5-5M sodium chloride, pH7-9; Preferably, the inverted nickel column purification comprises purifying the product after enzyme cleavage on a Ni-agarose gel column; preferably, the eluent comprises 10-50 mM Tris, 10-50 mM sodium chloride, 0.5-5 M imidazole, pH 7-9.

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