Method for biosynthesis of human structural material type vi collagen

Abstract

Provided are biosynthetic human structural material type VI collagen and a preparation method therefor. The collagen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 21, or a variant thereof, and can be used as a biological dressing, a human bionic material, a plastic surgery material or the like.

Description

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

[0001] This application claims priority to Chinese invention patent application No. 202310883890.4, filed July 18, 2023, entitled “Method for biosynthesizing type VI collagen as a human structural material.” The aforementioned application is incorporated herein by reference. Technical Field

[0002] The present application relates to the field of biosynthesis, and in particular to a method for preparing type VI collagen, a biosynthetic human structural material. Background Art

[0003] Collagen is a crucial component of the extracellular matrix in animals, playing a vital role in cell migration, cell metabolism, cell signaling pathway responses, platelet aggregation, and the maintenance, regulation, and repair of normal physiological functions of cells, tissues, and organs. As a key natural biological protein, collagen possesses excellent biocompatibility, bioactivity, and biodegradability, and is widely used in numerous fields, including the chemical industry, pharmaceuticals, food, and cosmetics.

[0004] Currently, collagen is primarily obtained through the use of acid, alkali, and enzymatic hydrolysis to obtain collagen extracts from animal tissues. While the technology for extracting collagen from animal tissues is relatively mature, this method produces uneven properties of collagen peptides, with large batch-to-batch variability and the potential for viral infection. Furthermore, the amino acid sequence of animal-derived collagen differs significantly from that of human collagen, which can easily lead to immunogenicity and allergic reactions. Furthermore, collagen is expressed through genetic engineering techniques, primarily in Escherichia coli, Pichia pastoris, mammalian cells, insect cells, and plants. These expression systems also have drawbacks, such as the difficulty in purifying the product expressed in E. coli; the high cost and low yield of mammalian cell expression; the high cost and low yield of insect cell expression systems, and the significant differences in post-translation expression from human cells; and the long plant expression cycle, making it unsuitable for industrial production. While the Pichia pastoris expression system offers advantages for large-scale industrial production, such as high-density fermentation production, extremely low culture costs, short cycle times, and high expression, it is not certain that it is suitable for all types of collagen expression.

[0005] Currently, the most studied recombinant human collagens are primarily types I, II, and III, with little research on type VI collagen. Human type VI collagen is a type of collagen present in all extracellular matrices (ECMs). It can bind to various ECM substances, thereby bridging cells to surrounding connective tissue and organizing the three-dimensional tissue structure of skeletal muscle, tendon, bone, and cartilage. By binding to type IV collagen and other pearl albumins in the basal layer, type VI collagen can establish a close connection between muscle cells and the ECM. As a major component of the extracellular matrix, type VI collagen can promote the adhesion of fibroblasts during wound repair and enhance the regeneration of chondrocytes, playing a vital role in wound healing.

[0006] There is a need in the art for methods of biosynthesizing type VI collagen, a structural material of the human body.

[0007] Summary of the Invention

[0008] The inventors conducted a large-scale screening of human type VI collagen and selected 11 types of human recombinant type VI collagen. These recombinant type VI collagens are suitable for separation and purification. After performing activity assays on these human recombinant type VI collagens, the inventors found that recombinant type VI collagens have cell adhesion activity and can be used in industry. The inventors also found that some recombinant type VI collagens (C6c, C6g, C6i, and C6k proteins) have higher yields, yields, and / or purities than other recombinant type VI collagens. The present invention provides a method for efficiently obtaining type VI collagen, a structural material for the human body.

[0009] 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, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21, 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.

[0010] The repeating sequence is as follows:

[0011] In one embodiment, the plurality of repeat units is 2-80 repeat units, such as 2-60, 2-70, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15 or 2-10 repeat units, such as 2, 3, 4, 5, 6, 7, 8, 9.

[0012] In one embodiment, the linker comprises one or more amino acid residues, e.g., 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues.

[0013] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion.

[0014] In one embodiment, the substitutions are conservative amino acid substitutions.

[0015] In one embodiment, the collagen or polypeptide is recombinant collagen. In one embodiment, the collagen or polypeptide is recombinant type VI collagen or human recombinant type VI collagen.

[0016] In one embodiment, the collagen or polypeptide has cell adhesion activity.

[0017] 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, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21, 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.

[0018] 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.

[0019] 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 NOs: 22-32.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] In another aspect, a method for promoting cell adhesion is provided, comprising the step of contacting the collagen or polypeptide, nucleic acid, vector, host cell and / or composition herein with a cell (eg, an animal cell, a mammalian cell or a human cell).

[0025] 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.

[0026] In another aspect, there is provided a method for producing a collagen protein or polypeptide as described herein, comprising:

[0027] (1) culturing the host cell described herein under appropriate culture conditions;

[0028] (2) harvesting host cells and / or culture medium containing the collagen or polypeptide; and

[0029] (3) Purification of collagen or peptides.

[0030] In one embodiment, the host cell is an E. coli cell, preferably an E. coli BL21 (DE3) cell.

[0031] In one embodiment, step (1) comprises culturing E. coli cells in LB medium and inducing expression by IPTG.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In one embodiment, the purification comprises gradient elution of the eluate containing the target protein or the product after enzymatic cleavage 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.

[0036] 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.

[0037] Herein, the enzymatic cleavage may be enzymatic cleavage by TEV enzyme.

[0038] The advantages of the present invention include: (1) providing human recombinant type VI collagen, which has cell adhesion activity and can be used in industry; (2) providing a method for preparing human recombinant type VI collagen with high yield, yield and / or purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows the electropherogram of C6a.

[0040] Figure 2 shows the electropherogram of C6d.

[0041] FIG3 shows the electropherogram of C6e.

[0042] FIG4 shows the electropherogram of C6h.

[0043] FIG5 shows the electropherogram of C6j.

[0044] FIG6 shows the electropherogram of C6f.

[0045] FIG7 shows the electropherogram of C6b.

[0046] FIG8 shows the electropherogram of C6c.

[0047] FIG9 shows the electropherogram of C6i.

[0048] FIG10 shows the electropherogram of C6g.

[0049] FIG11 shows the electropherogram of C6k.

[0050] FIG12 shows the cell adhesion results. DETAILED DESCRIPTION

[0051] 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.

[0052] As used herein, "type VI collagen" is a type of collagen present in all extracellular matrices (ECMs), capable of binding different substances of the ECM, thereby bridging cells to surrounding connective tissue and organizing the three-dimensional tissue structure of skeletal muscle, tendon, bone, and cartilage.

[0053] As used herein, "polypeptide" refers to a plurality of amino acid residues linked by peptide bonds. In this article, collagen or polypeptide comprises one or more repeating units. The repeating unit may be derived from human type VI collagen. Therefore, the collagen or polypeptide may be human recombinant type VI collagen. The multiple repeating units may be connected by a linker, which may be a natural amino acid residue of the repeating unit on human type VI collagen, such as 1-80 amino acid residues, such as 1, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80. The repeating unit may be SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21. The collagen or polypeptide may be SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 21.

[0054] As used herein, "human recombinant type VI collagen" refers to a recombinant protein consisting of, or consisting essentially of, a sequence derived from human type VI collagen. Herein, human recombinant type VI collagen can consist of, or consist essentially of, a fragment or multiple repeats of a fragment derived from human type VI collagen. Herein, collagen, recombinant collagen, human recombinant type VI collagen, or polypeptide are used interchangeably.

[0055] 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, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. 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, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21.

[0056] 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:

[0057] 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.

[0058] 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.

[0059] As used herein, "cell adhesion" refers to the adhesion between cells and collagen. Collagen (such as the collagen or polypeptide described herein) can promote the adhesion between cells and the container in which the cells are cultured.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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 present in nature, or is synthetic, and may contain one or more control sequences. The nucleic acid may be SEQ ID NO: 22-32. The nucleic acid may be a codon-optimized nucleic acid, such as a nucleic acid codon-optimized for expression in E. coli cells.

[0064] 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.

[0065] 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:

[0066] (number of identical residues × 100) / (length of alignment - total number of gaps in the alignment)

[0067] 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:

[0068] (number of identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in the alignment)

[0069] collagen

[0070] The present invention provides collagen or polypeptides 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, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. 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, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21, 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, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. 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.

[0071] The collagen or polypeptide described herein can comprise a plurality of repeating units, for example 2-80 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.

[0072] 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 amino acid residues.

[0073] The collagen or polypeptide described herein is a recombinant collagen, in particular a recombinant type VI collagen, preferably having cell adhesion activity. In this context, the recombinant type VI collagen is a human recombinant type VI collagen.

[0074] 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, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21, 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.

[0075] Nucleic acid constructs

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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).

[0083] 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).

[0084] 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.

[0085] 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).

[0086] 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.

[0087] 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).

[0088] 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.

[0089] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0090] 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.

[0091] 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.

[0092] 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).

[0093] Type VI collagen

[0094] Collagen plays a crucial role in maintaining the structure and function of the cell matrix. Type VI collagen forms a delicate reticular structure, distributed between type I, III, and V collagen fibrils. It connects type I, III, and V collagen to the basement membrane, serves as an anchor point between collagens and a site of cell attachment within the matrix, and plays a crucial role in the matrix's internal stability. Under an electron microscope, type VI collagen has a dumbbell-like shape, with a central rod approximately 150 nm and two spherical wings. Type VI collagen is composed of three distinct peptide chains: α1, α2, and α3. The genes for the human type VI collagen α1 and α2 chains are both located at locus 21g 22.3, are 36 kb in length, and consist of 30 exons. The gene for α3 collagen is located at locus 2q 37. Each peptide chain consists of a helical region and large globular regions at both the N- and C-termini. These globular regions primarily contain von Willebrand factor A-type repeats. These repeats have a large molecular weight of approximately 21 kDa, and the number of these repeats at the N-termini varies between α1, α2, and α3. Studies have shown that the center of the A region is a β-sheet flanked by three α-helices. Type VI collagen contains 18 non-helical von Willebrand factor A-type repeats, each with a repeating sequence of 200 amino acid residues. α3 contains 12 repeats, while α1 and α2 each contain three. Within microfibrils, discontinuous Gly-XY residues divide the helical structure into multiple segments. Type VI collagen forms monomers, dimers, and tetramers through the action of disulfide bonds. Dimers are formed by two monomers arranged in anti-parallel configurations, with an overlap of 75 mm. The globular regions remain at either end, and the C-termini are stabilized by disulfide bonds with adjacent helices. Two dimers form a tetramer in parallel. Recent studies have shown that the C-terminal region, although a non-helical region of type VI collagen, plays a crucial role and is required for both dimer and tetramer formation. Extracellularly, the termini of tetramers connect to form microfibrils, with tetramers bound to each other by non-covalent bonds, hypothesized to be interactions within the A region. Type II collagen and aggrecan bind to type VI collagen via a complex of leucine-rich repeat-like proteoglycans. Through this association, type VI collagen plays a dominant role in tissue formation, organization, and self-stabilization during the early stages of tissue formation or during repair of trauma or fractures. Type VI collagen also interweaves with type V collagen between fibrils, collaborating with mucopolysaccharides to maintain the structure of the epidermal matrix. In addition to the above collagens, type VI collagen can also bind to other macromolecules, such as type IV collagen, type XIV collagen, decorin, microfibril-associated glycoprotein 1, hyaluronic acid, 1B2 and ot2pl binding proteins, and cell surface proteoglycan NG. Collagen VI stabilizes the structure of the extracellular matrix by binding to these macromolecules.By maintaining the structure and function of the extracellular matrix, type VI collagen helps maintain the integrity of tissues such as blood vessels, lungs, cartilage, muscle, and skin. Mutations in the gene encoding type VI collagen can lead to Bethlem myopathy and Ullrich syndrome, resulting in muscle weakness and wasting. Type VI collagen deficiency can also lead to loss of mitochondrial function and cell apoptosis. On the other hand, increased or accumulated type VI collagen can also contribute to diseases such as superficial fibromas, neurofibromas, keloids, pulmonary fibrosis, liver fibrosis, diabetic kidney damage, and rheumatoid arthritis. Type VI collagen also affects cell differentiation, adhesion, migration, proliferation, and survival. Cardiac fibroblasts cultured in a matrix containing type VI collagen have been shown to differentiate into myofibroblasts, a finding confirmed by in vivo experiments. Studies of type VI collagen's function have shown that it strongly adheres to various committed hematopoietic stem cells, with this adhesion being localized within the helical region of the three peptide chains. Collagen VI also promotes the proliferation of various cells, a phenomenon that can be blocked by the peptide chains of type VI collagen alone. The enhancement of fibroblast proliferation and migration is achieved by combining type VI collagen with cell surface proteoglycans NG, decorin, syndecan, hyaluronic acid, and other types of collagen.

[0095] expression vector

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance (e.g., 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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).

[0109] host cells

[0110] 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.

[0111] 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.

[0112] 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.

[0113] Host cells can also be eukaryotic, such as mammalian, insect, plant or fungal cells. Plant cells herein do not include plant cells that can be regenerated into plants. Animal cells also do not include cells that can produce animal bodies.

[0114] 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.

[0115] Production method

[0116] The present invention also relates to a method for producing the collagen or polypeptide described herein, comprising:

[0117] (1) culturing the host cell described herein under appropriate culture conditions;

[0118] (2) harvesting host cells and / or culture medium containing the collagen or polypeptide; and

[0119] (3) Purification of collagen or peptides.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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).

[0126] 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.

[0127] 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.

[0128] Step (3) may include adding the supernatant to the column material and washing the impurity protein with a washing solution. The washing 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 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. 440, 450, 460, 470, 480, or 490 nM. The pH may be 7, 7.5, 8, 8.5, or 9.

[0129] 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.

[0130] 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.

[0131] 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).

[0132] Advantages of the present invention: 1. The collagen or polypeptide of the present invention is derived from type VI collagen and is recombinant type VI collagen; 2. The collagen or polypeptide of the present invention is suitable for preparation in Escherichia coli and can be separated and purified; 3. The collagen or polypeptide of the present invention has a large expression amount and is suitable for subsequent purification.

[0133] The following examples are provided to further illustrate the present invention.

[0134] Example

[0135] 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.

[0136] Example 1: Construction, expression and screening of type VI collagen fragments

[0137] 1. Perform large-scale functional region screening to obtain the following different target gene functional regions of recombinant humanized type VI collagen

[0138] Amino acid sequence

[0139] (1)C6a

[0140] (2)C6b

[0141] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDLGPVGYQGMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEMGYPGLPGCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRPGSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP (the amino acid sequence of the repeating unit of C6b is SEQ ID NO: 9, the number of repeating units is 2, and the amino acid sequence of C6b is SEQ ID NO: 10)

[0142] (3)C6c

[0143] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGREGPVGVPGDPGEAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGEDGPA

[0144] (The amino acid sequence of the repeating unit of C6c is SEQ ID NO: 1, the number of repeating units is 3, and the amino acid sequence of C6c is SEQ ID NO: 2)

[0145] (4)C6d

[0146] GIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRPGSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAPGERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGREGPVGVPGDPGEAGPIGPKGYRGDEGPP (the amino acid sequence of the repeating unit of C6d is SEQ ID NO: 11, the number of repeating units is 2, and the amino acid sequence of C6d is SEQ ID NO: 12)

[0147] (5)C6e

[0148] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDL (the amino acid sequence of the repeating unit of C6d is SEQ ID NO: 13, the number of repeating units is 6, and the amino acid sequence of C6d is SEQ ID NO: 14)

[0149] (6)C6f

[0150] GMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEM (the amino acid sequence of the repeating unit of C6f is SEQ ID NO: 15, the number of repeating units is 6, and the amino acid sequence of C6f is SEQ ID NO: 16)

[0151] (7)C6g

[0152] GCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRP (the amino acid sequence of the repeating unit of C6g is SEQ ID NO: 3, the number of repeating units is 6, and the amino acid sequence of C6c is SEQ ID NO: 4)

[0153] (8)C6h

[0154] GSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP (the amino acid sequence of the repeating unit of C6h is SEQ ID NO: 17, the number of repeating units is 6, and the amino acid sequence of C6h is SEQ ID NO: 18)

[0155] (9)C6i

[0156] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGRE (the amino acid sequence of the repeating unit of C6i is SEQ ID NO: 5, the number of repeating units is 6, and the amino acid sequence of C6i is SEQ ID NO: 6)

[0157] (10)C6j

[0158] GDPGEAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGEDGPA (the amino acid sequence of the repeating unit of C6j is SEQ ID NO: 19, the number of repeating units is 4, and the amino acid sequence of C6j is SEQ ID NO: 20)

[0159] (11)C6k

[0160] GTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDD (the amino acid sequence of the repeating unit of C6k is SEQ ID NO: 7, the number of repeating units is 6, and the amino acid sequence of C6k is SEQ ID NO: 8)

[0161] Nucleotide sequence

[0162] 1.C6a

[0163] 2.C6b

[0164] 3.C6c

[0165] 4.C6d

[0166] 5.C6e

[0167] 6.C6f

[0168] 7.C6g

[0169] 8.C6h

[0170] 9.C6i

[0171] 10. C6j

[0172] 11. C6k

[0173] Plasmid cloning

[0174] 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 being ENLYFQ and the nucleotide sequence being GAAAACCTGTATTTCCAG) was inserted between the KpnI and XhoI restriction sites of the pET-28a-Trx-His expression vector to generate a recombinant expression plasmid.

[0175] Host cell transformation

[0176] The successfully constructed expression plasmid was transformed 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. 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.

[0177] Collagen or peptide separation and purification

[0178] 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 twice using high pressure homogenization. Collect the resulting 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.

[0179] Recombinant humanized type VI 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 at a ratio of 50:1 between the total amount of protein and the total amount of TEV enzyme, digest at 16°C for 4 hours, and take a sample for electrophoresis detection (after digestion). Place the protein solution after enzyme digestion in a dialysis bag, dialyze at 4°C for 2 hours, and then transfer to new dialysis solution for overnight dialysis at 4°C (marked as changing A solution).

[0180] (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 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 (marked as B wash). d. Wash the column. Store the protein at 4°C.

[0181] (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.

[0182] Concentration detection

[0183] 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-Vis 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).

[0184] The concentration test results are as follows:

[0185] Protein expression: C6f>C6g>C6i>C6b>C6j>C6k>C6h>C6e>C6c>C6a>C6d

[0186] Electrophoresis detection

[0187] The specific process is as follows: take 40 μl of sample liquid, add 10 μl of 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 to each well of 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.

[0188] Figure 1 shows the electrophoretogram of C6a. Figure 2 shows the electrophoretogram of C6d. Figure 3 shows the electrophoretogram of C6e. Figure 4 shows the electrophoretogram of C6h. Figure 5 shows the electrophoretogram of C6j. Figure 6 shows the electrophoretogram of C6f. Figure 7 shows the electrophoretogram of C6b. Figure 8 shows the electrophoretogram of C6c. Figure 9 shows the electrophoretogram of C6i. Figure 10 shows the electrophoretogram of C6g. Figure 11 shows the electrophoretogram of C6k.

[0189] Electrophoresis detection results showed that the crude purification yields of C6a, C6d, and C6h were low (Figures 1, 2, and 4); the crude purification yield of C6e was low and the enzymatic digestion effect was poor (Figure 3); the crude purification yield of C6j was low and there were more impurities (Figure 5); C6f had some impurity bands after elution, and the amount of protein after enzymatic digestion and purification was small (Figure 6); C6b had some impurity bands after elution, and the enzymatic digestion was incomplete (Figure 7); C6c, C6g, C6i, and C6k had high crude purification yields, and the purity of the target protein was high after nickel reverse mounting (Figures 8, 9, 10, and 11).

[0190] According to the purification results, the recombinant type VI humanized collagen prepared by the two plasmids C6c, C6g, C6i, and C6k was better, so mass spectrometry detection was performed on the C6c, C6g, C6i, and C6k proteins.

[0191] Example 2: Mass spectrometry detection of recombinant humanized type VI collagen

[0192] Experimental methods

[0193] 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.)

[0194] Data retrieval was performed using the MS / MSIon Search page on the local masco website. Protein identification was based on the primary mass spectra of 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.

[0195] Table 1: Molecular weight and corresponding peptides detected by mass spectrometry of recombinant humanized type VI collagen C6c

[0196] The coverage of the detected peptide fragments compared with the theoretical sequence is 100%, and the detection results are very reliable. Table 2: Mass spectrometry detection molecular weight of recombinant type VI humanized collagen C6g and corresponding peptides

[0197] The coverage of the detected polypeptide fragments compared with the theoretical sequence is 100%, and the detection results are very reliable.

[0198] Table 3: Molecular weights and corresponding peptides detected by mass spectrometry of recombinant humanized type VI collagen C6i

[0199] The coverage of the detected polypeptide fragments compared with the theoretical sequence is 100%, and the detection results are very reliable.

[0200] Table 4: Molecular weight and corresponding peptides detected by mass spectrometry of recombinant humanized type VI collagen C6k

[0201] The coverage of the detected polypeptide fragments compared with the theoretical sequence was 78.57%, and the detection results were very reliable.

[0202] Example 3: Bioactivity Detection of Recombinant Humanized Type VI 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 samples to be tested was detected by ultraviolet absorption method, including bovine type I collagen (PC, China Food and Drug Inspection Institute, No.: 380002) and the recombinant type VI humanized collagen C6c, C6g, C6i, and C6k provided by the present invention.

[0205] Specifically, the sample's UV absorbance at 215nm and 225nm 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 proteins to 1 mg / mL with PBS.

[0206] (2) Different concentrations of collagen and positive and negative controls were added to the ELISA plate, 100 μL per well, 5 replicate wells per group, and incubated at 4°C overnight.

[0207] (3) 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 (NC).

[0208] (4) Add 10 5 Resuspend well-cultured 3T3 / NIH cells in D-PBS and incubate at 37°C for 120 min. Wash each well three times with D-PBS solution.

[0209] (5) Detect OD using CCK8 detection kit 450 The absorbance is measured in nm. Based on the blank control value, the cell adhesion rate can be calculated. The calculation formula is as follows: The cell adhesion rate reflects the activity of collagen. The higher the activity of the protein, the better the external environment it can provide to the cells in a short time, helping them to adhere to the wall.

[0210] The results are shown in Figure 12. Compared with the D-PBS group, the positive control had a significant effect on promoting cell adhesion. Recombinant humanized collagen also promoted cell adhesion at the experimental concentration (the vertical axis is the percentage of cell adhesion activity relative to the D-PBS group). The results were statistically significant, where *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0211] The above embodiments are preferred implementation modes of the present invention, but the implementation modes 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 as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A collagen protein 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, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21, 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, 2-8 or 2-6 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 VI collagen; preferably human recombinant type VI collagen; Preferably, the collagen has cell adhesion activity.

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, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21, 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: 22-32.

4. A vector comprising the nucleic acid according to claim 3, Preferably, the vector comprises an expression control element, a purification tag nucleotide and / or a leader sequence nucleotide operably linked to the nucleic acid, 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 is a 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) purifying the collagen and optionally removing the tag; 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, enzyme digestion, fine purification and / or 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 eluate containing the target protein or the product after enzyme cleavage 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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