Type XX collagen, a structural material for the human body, and its biosynthesis method

Abstract

This application provides a human structural material, type XX collagen, and a method for its biosynthesis. The collagen of this application comprises the amino acid sequence shown in SEQ ID NO:2 or a variant amino acid sequence thereof, wherein the variant amino acid sequence retains the function of the amino acid sequence shown in SEQ ID NO:2. The collagen of this application can promote cell adhesion and has a triple helix structure.

Description

[0001] This application claims the priority of Chinese Invention Patent Application with the application date of April 29, 2024, the application number of 202410532686.2, and the invention name of “Human Structural Material XX Type Collagen Protein and Its Biosynthesis Method”. TECHNICAL FIELD

[0002] The present application relates to the field of proteins or polypeptides, in particular to collagen proteins, methods of making and uses thereof. BACKGROUND

[0003] Collagen is a kind of protein widely distributed in human connective tissue, and is also the most abundant protein in the human body, which can account for 25%~35% of the total amount of protein. Its main functions are reflected in maintaining the extracellular environment, maintaining the normal physiological functions of tissues and organs, repairing body damage, etc. Collagen is a natural biological resource, which has biological tissue compatibility that other high molecular materials cannot match, cell support elasticity and degradability, therefore, collagen can be widely used in the pharmaceutical and cosmetic industries, etc.

[0004] Natural collagen molecules can form a special superhelix structure, which is a left-handed helix with 3 amino acid residues as the basic repeating unit. The three amino acid residues are usually Gly-X-Y. Gly is essential for the formation of hydrogen bonds in collagen. It has no side chain, which makes collagen tightly packed and can maintain skin tension and elasticity.

[0005] In recent years, with the wide application of genetic engineering technology, researchers have created various types of recombinant collagen proteins. For example, a recombinant collagen protein can be constructed by selecting a short amino acid sequence from natural human collagen. The recombinant collagen protein constructed in this way has advantages such as low immunogenicity, high biological activity, and good stability. In theory, the shorter the amino acid sequence of this recombinant collagen protein, the better the transdermal absorption performance. However, the shorter the amino acid sequence is not necessarily better. How to design a short amino acid sequence to make the constructed recombinant collagen protein have better transdermal absorption performance, there is no theory in the prior art to guide.

[0006] XX type collagen protein (COL20) is a collagen protein that has not been developed. According to its structural characteristics, XX type collagen is part of the fibril-associated collagen with interrupted triple helices (FACIT) family. COL20A1 RNA expression is enriched in the human brain, and RNA expression in testicular and spleen tissues is slightly increased. At present, the research on XX type collagen protein is relatively lacking, and there is a need for XX type collagen protein and its preparation method in the art. SUMMARY

[0007] To meet the current needs, the inventors provide a new collagen XX. The collagen XX of the present document has a cell adhesion promoting effect and has a triple helix structure. The present invention also provides a method of biosynthesizing a human structural material collagen XX.

[0008] In one aspect, a collagen is provided, comprising one or more repeating units, wherein the repeating unit comprises:

[0009] (1) an amino acid sequence set forth in SEQ ID NO: 9; or

[0010] (2) an amino acid sequence in which one or more amino acid residues in the amino acid sequence set forth in SEQ ID NO: 9 are mutated; or

[0011] (3) an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 9.

[0012] In one embodiment, the number of repeating units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; each repeating unit is directly connected by a peptide bond or connected by a linker of one amino acid or a plurality of amino acids.

[0013] In one embodiment, the mutation is a substitution, an insertion, a deletion, or an addition; preferably, the substitution is a conservative amino acid substitution.

[0014] In one embodiment, the collagen is derived from a human.

[0015] In one embodiment, the collagen has a cell adhesion promoting effect.

[0016] In one embodiment, the collagen is a recombinant collagen, a recombinant humanized collagen, or a recombinant humanized collagen XX.

[0017] In one embodiment, the collagen is a collagen in a single chain form.

[0018] In one embodiment, the addition is an addition of (Gly-X-Y)m to the N-terminus and / or C-terminus of the repeating unit, wherein X and Y are any amino acid residues, and m is 1-5.

[0019] In one embodiment, Gly-X-Y is GER, GEK, GEP, or GTS.

[0020] In one embodiment, the repeat sequence comprises the amino acid sequence of:

[0021] (1) the amino acid sequence set forth in SEQ ID NO: 1 or 8;

[0022] (2) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or 8; or

[0023] (3) an amino acid sequence that is mutated at one or more amino acid residues from the amino acid sequence set forth in SEQ ID NO: 1 or 8; preferably, the mutation is a substitution, an insertion, a deletion, or an addition; preferably, the substitution is a conservative amino acid substitution.

[0024] In one embodiment, the collagen comprises the amino acid sequence of any one of SEQ ID NOs: 1, 2, 5-9 or an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs: 1, 2, 5-9.

[0025] In one aspect, there is provided a collagen comprising 1 or more repeat units, the repeat unit comprising the amino acid sequence set forth in SEQ ID NO: 1 or 8 or an amino acid sequence that is mutated at one or more amino acid residues from the amino acid sequence set forth in SEQ ID NO: 1 or 8 or an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, or at least 99% identity with the amino acid sequence set forth in SEQ ID NO: 1 or 8; the number of repeat units is 2-20, 3-16, or 4-8.

[0026] In one embodiment, the number of repeat units is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0027] In one embodiment, each repeat unit is directly linked by a peptide bond or linked by a linker of one amino acid or a plurality of amino acid residues. In one embodiment, the linker comprises 2, 3, 4, 5, 6, 7, or 8 amino acid residues.

[0028] In one embodiment, the mutation is a substitution, an insertion, a deletion, or an addition. In one embodiment, the substitution is a conservative amino acid substitution.

[0029] In one embodiment, the collagen is derived from human. In one embodiment, the collagen has a triple helix structure. In one embodiment, the triple helix structure is formed by three identical single strands of collagen. In one embodiment, the collagen has cell adhesion efficacy. In one embodiment, the collagen is recombinant collagen, recombinant humanized collagen, or recombinant humanized collagen type XX.

[0030] In one embodiment, the collagen comprises the following amino acid sequence:

[0031] (1) the amino acid sequence set forth in SEQ ID NO: 2;

[0032] (2) an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; or

[0033] (3) an amino acid sequence that is mutated at one or more amino acid residues in the amino acid sequence set forth in SEQ ID NO: 2.

[0034] In one embodiment, the mutation is a substitution, insertion, deletion, or addition. In one embodiment, the substitution is a conservative amino acid substitution.

[0035] In one aspect, a fusion protein comprising the collagen herein and a protein for facilitating secretion, isolation, and / or purification of the collagen is provided.

[0036] In one embodiment, the protein is selected from the group consisting of an enzyme cleavage site sequence, a signal peptide, and a purification tag sequence.

[0037] In one embodiment, the enzyme cleavage site sequence is a TEV protease cleavage site sequence.

[0038] In one embodiment, the protein tag sequence is a His tag, a GST tag, a MBP tag, a SUMO tag, a Cytiva Protein Select tag, or a NusA tag.

[0039] In one embodiment, the collagen is directly linked to the protein or linked through a linker, the linker being a flexible linker such as (G) a or (GGGGS) b wherein a and b are each independently an integer from 1-10 or 1-5 or 1-3.

[0040] In an aspect, a nucleic acid is provided, which encodes a collagen or fusion protein described herein.

[0041] In one embodiment, the nucleic acid has a nucleotide sequence set forth in SEQ ID NO: 3.

[0042] In an aspect, a vector is provided, which comprises a nucleic acid described herein. In one embodiment, the vector comprises a nucleotide encoding a purification tag, a nucleotide encoding a leader, and / or a regulatory element.

[0043] In one embodiment, the purification tag is selected from a His tag, a GST tag, a MBP tag, a SUMO tag, a Cytiva Protein Select tag tag, or a NusA tag.

[0044] In one embodiment, the regulatory element is selected from a promoter, a terminator, and / or an enhancer.

[0045] In an aspect, a host cell is provided, which comprises a nucleic acid described herein or a vector described herein.

[0046] 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, and / or the prokaryotic cell is an E. coli cell, for example, E. coli BL21.

[0047] In an aspect, a collagen with a triple helix structure is provided, which comprises three identical collagen of claim 1 or 2 in single-stranded form.

[0048] In an aspect, a method of producing a collagen or fusion protein is provided, which comprises:

[0049] (1) culturing a host cell described herein under suitable culture conditions;

[0050] (2) harvesting the host cell and / or the culture medium comprising the collagen or fusion protein; and

[0051] (3) purifying the collagen or fusion protein, for example, comprising (1) crude purification of the collagen on a Ni affinity column; (2) enzymatic cleavage; and / or (3) ion exchange column fine purification of the collagen.

[0052] In an aspect, a composition is provided, which comprises a collagen, a nucleic acid, a vector, a collagen with a triple helix structure, and / or a host cell described herein.

[0053] In one embodiment, the composition is a pharmaceutical composition or a cosmetic composition.

[0054] In one embodiment, the composition is one or more of a biological dressing, a human bionic material, a plastic and cosmetic 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 blood vessel repair and regeneration material, a 3D printing artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient, and a food additive.

[0055] In one embodiment, the composition comprises a pharmaceutically and / or cosmetically acceptable carrier.

[0056] In one embodiment, the composition is a solid, liquid or gel composition.

[0057] In one embodiment, the composition is an orally and / or topically administered composition.

[0058] In one embodiment, the composition is a kit.

[0059] In one embodiment, the composition is a liquid formulation comprising the collagen or collagen having a triple helix structure described herein and a pharmaceutically and / or cosmetically acceptable carrier.

[0060] In one embodiment, the carrier is a buffer, such as a D-PBS buffer or a PBS buffer.

[0061] In one aspect, there is provided a method of promoting cell adhesion or attachment, the method comprising contacting a cell with the collagen and / or composition herein.

[0062] In one aspect, there is provided use of the collagen, nucleic acid, carrier, host cell, collagen having a triple helix structure and / or composition described herein in one or more of a biological dressing, a human bionic material, a plastic and cosmetic 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 blood vessel repair and regeneration material, and a 3D printing artificial organ biomaterial.

[0063] In one embodiment, the biological dressing is a collagen biological dressing.

[0064] In one aspect, there is provided use of the collagen, collagen having a triple helix structure and / or composition described herein in the manufacture of a medicament or kit for promoting cell adhesion or attachment.

[0065] In another aspect, there is provided a method of forming collagen in a triple helix structure form, comprising the step of dissolving the collagen described herein in a single chain form in water or a buffer, such as a D-PBS buffer or a PBS buffer.

[0066] In another aspect, there is provided a method of screening for a collagen, comprising: mutating one or more amino acid residues of the amino acid sequence of SEQ ID NO: 9 to obtain a collagen library;

[0067] determining cell adhesion activity for each collagen in the collagen library;

[0068] selecting a collagen having cell adhesion activity.

[0069] In one embodiment, the mutation is a substitution, insertion, deletion or addition.

[0070] In one embodiment, the substitution is a conservative amino acid substitution.

[0071] In one embodiment, the substitution, insertion or deletion is a substitution, insertion or deletion of 1-6 amino acid residues.

[0072] In one embodiment, the addition is an addition of 1-15 amino acid residues.

[0073] In one embodiment, the linker is a linker of 2, 3, 4, 5, 6, 7 or 8 amino acid residues.

[0074] In one embodiment, the addition is an addition of (Gly-X-Y)m of the N- and / or C-terminus of the repeat unit, wherein X and Y are any amino acid residues and m is 1-5.

[0075] In one embodiment, the Gly-X-Y is GER, GEK, GEP or GTS.

[0076] In one embodiment, the selected collagen comprises the amino acid sequence of SEQ ID NO: 1 or 8.

[0077] In one embodiment, the method further comprises tandem repeating the collagen of SEQ ID NO: 9 or the selected collagen as a repeat unit 2 or more times, for example 2-30.

[0078] In one embodiment, the method comprises determining cell adhesion activity for a collagen comprising 2 or more repeat units; and

[0079] selecting a collagen comprising 2 or more repeat units having cell adhesion activity.

[0080] In one embodiment, the number of repeat units of the selected collagen comprising 2 or more repeat units is 2-12.

[0081] Advantages of the present application include that the collagen described herein is a new humanized type XX collagen, which has the efficacy of promoting cell adhesion and has a triple helix structure; the recombinant humanized collagen C20 herein has a higher (e.g., about 2 times) cell adhesion activity than bovine type I collagen, achieving an unexpected technical effect. BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1 An electropherogram of C20 collagen (which can also be referred to as recombinant humanized type XX collagen C20) is shown.

[0083] Figure 2 A cell adhesion effect of C20 collagen is shown.

[0084] Figure 3 A circular dichroism spectrum of C20 collagen is shown.

[0085] Figure 4 A graph showing the relative cell adhesion ratio of C1, C2, and C3 collagens is shown.

[0086] Figure 5 An electropherogram of C20T6 and C20T9 is shown.

[0087] Figure 6 A graph showing the relative cell adhesion ratio of C20T6 and C20T9 is shown. DETAILED DESCRIPTION

[0088] To make the objectives, technical solutions, and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described below in conjunction with embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0089] As used herein, a recombinant humanized collagen is a full-length or partial amino acid sequence fragment of a human collagen specific gender gene encoded by DNA recombination technology, or a combination containing a functional fragment of a human collagen. In this document, the recombinant humanized collagen is a recombinant humanized type XX collagen, which is a peptide or polypeptide of multiple amino acid residues connected by a peptide bond.

[0090] As used herein, "collagen" refers to a protein of multiple repeating units connected by a peptide bond. As used herein, "C-terminal" and "N-terminal" with respect to a collagen or a specific amino acid sequence refer to the position relative to the collagen or the specific amino acid sequence, specifically referring to the carboxyl terminal or amino terminal direction of the collagen or the specific amino acid sequence.

[0091] In the present context, the collagen can comprise an N-terminal sequence and a C-terminal sequence, from the N-terminus to the C-terminus. The N-terminal sequence can comprise one or more repeating units comprising the amino acid sequence set forth in SEQ ID NO. 9, 1 or 8 or an amino acid sequence obtained by mutation (substitution, addition, insertion or deletion) of one or more amino acid residues from the amino acid sequence. The number of repeating units can be 1-20. For example, the number of repeating units is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In particular, the mutation can be a substitution, for example, a conservative amino acid substitution. The amino acid sequence of SEQ ID NO. 1 is GTSGERGPPGTVGPTGLPGPKGERGEKGEP, and the resulting collagen retains the functions of the present application, such as cell adhesion, the ability to form a gel by itself, etc., in the case where the collagen sequence is mutated or a spacer sequence is present.

[0092] In the present context, the collagen can be a single-chain form of collagen or a triple-helix form of collagen. The triple-helix form of collagen can be formed by or comprise three identical single-chain forms of collagen.

[0093] As used herein, "repeating unit" refers to an amino acid sequence that is a basic sequence in the collagen. The repeating unit can obtain the collagen of the present context by repeating in series multiple times. In the present context, the collagen can comprise one repeating unit or a series of repeating units comprising multiple repeating units. The repeating unit comprises the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence obtained by mutation (substitution, addition, insertion or deletion) of one or more amino acid residues from the amino acid sequence. The number of repeating units can be 1-30. For example, the number of repeating units is 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 or 30. In particular, the mutation can be a substitution, for example, a conservative amino acid substitution. Those skilled in the art can obtain a collagen that retains the functions of the collagen of the present application, such as cell adhesion, the ability to form a gel by itself, etc., in the case where the collagen sequence is appropriately mutated or an appropriate spacer sequence is present.

[0094] As used herein, a “fusion protein” refers to a fusion of a collagen protein and another functional part of a protein. In some embodiments, the other functional part of a protein is a protein used to facilitate secretion, isolation, and / or purification of the collagen protein. Proteins used to facilitate secretion, isolation, and / or purification of the collagen protein are well known to those skilled in the art and include, but are not limited to, a cleavage site sequence, a signal peptide, and a purification tag sequence. In some embodiments, the cleavage site sequence is a TEV protease cleavage site sequence. In some embodiments, the protein tag sequence is a His tag, a GST tag, a MBP tag, a SUMO tag, a Cytiva Protein Select tag, or a NusA tag. The collagen protein can be directly linked to the protein or can be linked through a linker. Selection of a linker is routine to those skilled in the art, and the linker can generally be a flexible linker, such as (G) a or (GGGGS) b where a and b are each independently an integer from 1-10 or 1-5 or 1-3.

[0095] As used herein, a “nucleic acid” refers to a plurality of nucleotides linked by internucleotide linkages. The internucleotide linkages can be, for example, phosphodiester bonds. The nucleic acids herein can comprise a polynucleotide encoding a collagen protein of the present application. To facilitate subsequent processing of the collagen protein, the nucleic acids of the present application can also comprise nucleotide sequences encoding a purification tag, such as a His tag, a GST tag, a MBP tag, a SUMO tag, a Cytiva Protein Select tag, or a NusA tag, and, when needed, a nucleotide sequence encoding a leader sequence.

[0096] As used herein, the term “vector” is a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of mediating expression of a polynucleotide inserted therein, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Vectors can contain various elements to control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain a replication origin. A vector can comprise a nucleic acid of the present application to facilitate introduction into a cell for expression. A vector can comprise expression control elements, such as promoters, terminators, and / or enhancers, operably linked to the nucleic acid.

[0097] As used herein, the term "host cell" is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. The host cell can be a eukaryotic cell or a prokaryotic cell. For example, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. The prokaryotic cell can be an E. coli cell.

[0098] As used herein, the degree of identity between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For the purposes of the present application, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are a gap open penalty of 10 and a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -l option) is used as the percent identity and is calculated as follows:

[0099] (identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0100] For the purposes of the present application, the sequence identity between two deoxynucleotide sequences is determined using the Needleman-Wunsch algorithm as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are a gap open penalty of 10 and a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -l option) is used as the percent identity and is calculated as follows:

[0101] (identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in the alignment)

[0102] In the context of the present application, a conservative amino acid substitution or conservative substitution can be defined by a substitution within an amino acid class reflected in one or more of the following tables:

[0103] Conserved classes of amino acid residues:

[0104]

[0105] Alternative physical and functional classification of amino acid residues:

[0106]

[0107] A "collagen biological dressing" is a film-like or sponge-like biological dressing formed by cross-linking collagen extract through physical or chemical methods, which can be applied to cover and close skin wounds.

[0108] "Biomimetic materials" refer to materials developed by imitating various characteristics or properties of organisms. Artificial materials designed and manufactured by imitating the operation mode of life systems and the structural rules of biological materials are generally referred to as biomimetic materials. Human biomimetic materials are artificial materials designed and manufactured by imitating the operation mode of life systems and the structural rules of biological materials. The collagen protein in this paper has the effect of promoting cell adhesion, and therefore can be used for human biomimetic materials.

[0109] "Plastic and cosmetic materials" refer to materials used in cosmetology. It is known to those skilled in the art that collagen protein can be applied to the human body in cosmetology.

[0110] "Organoid culture materials" refer to materials used for culturing organoids. The collagen protein in this paper has the effect of promoting cell adhesion, and therefore can be used for organoid culture.

[0111] "Cardiovascular stent materials" refer to materials used in the preparation of cardiovascular stents. The collagen protein in this paper can be coated on the surface of cardiovascular stents.

[0112] "Materials for tissue injection and filling" refer to materials that can be used for injection into the human body and as filling materials. The collagen protein in this paper can be used as a material for tissue injection and filling.

[0113] Recombinant humanized collagen type XX

[0114] In this paper, the collagen protein can be a recombinant humanized collagen type XX. Unless otherwise specified, the collagen protein can encompass collagen protein in single chain form and / or triple helix form. The collagen protein in this paper is also sometimes referred to as C20 collagen protein.

[0115] The recombinant humanized collagen XX of the present application can have certain mutations. For example, the amino acid sequence of one or more of these portions can have substitutions, deletions, additions, or insertions of amino acid residues. That is, the present application can use variants so long as the variants retain the activity of promoting cell adhesion and / or proliferation. Specifically, the variants can have a certain percentage identity to the specified sequence, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity. The specified sequence can be any sequence of the present application, such as SEQ ID NO: 1 or 2, but preferably these variants retain the function of the recombinant humanized collagen XX of the present application. The recombinant humanized collagen XX of the present application can have good cell adhesion efficacy. The recombinant humanized collagen XX of the present application can have a triple helix structure (i.e., collagen protein in the form of triple helix).

[0116] The recombinant humanized collagen XX of the present application can be prepared by any suitable means, such as can be prepared by synthesis. Preferably, the recombinant humanized collagen XX of the present application can be prepared by recombinant means.

[0117] The collagen protein of the present application can have a triple helix structure region, i.e., collagen protein in the form of triple helix structure, i.e., having three identical collagen protein chains. For example, the collagen protein has a flexible triple helix structure region. The collagen protein of the present application can form a triple helix structure. It has been determined that the recombinant humanized collagen XX of the present application (C20 collagen protein) can form a triple helix structure.

[0118] Repeating unit

[0119] Provided herein are multiple repeat units, wherein the smallest repeat unit comprises the amino acid sequence of SEQ ID NO: 9 and variant amino acid sequences of SEQ ID NO: 9. In some embodiments, the variant amino acid sequence of SEQ ID NO: 9 comprises an amino acid sequence that is at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the variant amino acid sequence of SEQ ID NO: 9 comprises the amino acid sequence of SEQ ID NO: 9 after mutation of 1-15 amino acid residues. The mutation can be a substitution, insertion, deletion, or addition. For example, one of skill in the art can substitute, insert, delete, or add one or more (e.g., 1-15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid residues in the amino acid sequence of SEQ ID NO: 9 while retaining the function of the original repeat unit. In some embodiments, one or more (e.g., 1-15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid residues can be added N-terminally and / or C-terminally to the amino acid sequence of SEQ ID NO: 9 while retaining the function of the original repeat unit. In some embodiments, the addition is an addition of the motif (Gly-X-Y)m to the N-terminus and / or C-terminus of the repeat unit, where X and Y are any amino acid residues, and m is 1-5. In some embodiments, Gly-X-Y is GER, GEK, GEP, or GTS.

[0120] The prior art Diane et al. (THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 275, No. 19, Issue of May 12, pp. 14532-14536, 2000) demonstrated that the tripeptide unit GER is generally present in collagen sequences, can form a triple helix structure in the absence of a typical collagen-like tripeptide unit, and functions in the stabilization of the triple helix structure. In the present invention, by adding a Gly-X-Y sequence to the N- and C-terminus of SEQ ID NO: 1, SEQ ID NO: 1 or SEQ ID NO: 8 was obtained. The extended collagen that was envisioned to have biological activity prior to the performance of the biological activity likewise possessed cell adhesion activity. In the examples, after being verified experimentally, in accordance with the initial assumption, the collagen of SEQ ID NO: 1 or SEQ ID NO: 8 did indeed retain the cell adhesion function of SEQ ID NO: 9.

[0121] In some embodiments, the repeat unit comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8 or an amino acid sequence that is at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1 or 8. In some embodiments, the variant amino acid sequence of SEQ ID NO: 1 or 8 comprises an amino acid sequence that is the amino acid sequence of SEQ ID NO: 1 or 8 with 1-15 amino acid residues mutated. The mutation can be a substitution, an insertion, a deletion, or an addition. For example, one of skill in the art can substitute, insert, delete, or add one or more (e.g., 1-15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid residues in the amino acid sequence of SEQ ID NO: 1 or 8 while retaining the function of the original repeat unit.

[0122] Tandem repeat strategy for repeat units of collagen

[0123] Prior art documents report that recombinant collagens can be constructed by tandem repeats of repeating units. The number of repeating units can vary. For example, Chen Hua et al. Biochemical and Biophysical Research Communications 508 (2019) 1018e1023 discloses that both the repeating unit GERGAPGFRGPAGPNGIPGEKGPAGERGAP and its 16 tandem repeats T16 possess cell adhesion activity, and it is believed that recombinant proteins containing multiple tandem repeats of triple-helical fragments can have more stable helical conformations or more favorable ligand binding configurations to facilitate cell membrane attachment and adhesion.

[0124] R. Strawn et al., Biopolymers 109 (2018), e23226 demonstrates that recombinant collagen-like proteins with multiple identical triple-helical peptide units arranged in tandem repeats can self-assemble into microfibers for use in the biopharmaceutical and industrial fields.

[0125] J. Yao et al., J. Biochem. 136 (2004) 643e649 reports that recombinant collagen-like proteins containing tandem repeats of collagen type I peptides show high cell adhesion activity.

[0126] A number of applications previously filed by the applicant also demonstrate that recombinant collagens composed of different numbers of repeating units of collagen can have excellent cell adhesion activity. Tandem repeats of repeating units in the number of 1-30 can have the same properties.

[0127] Therefore, when the present inventors demonstrated that the collagen of SEQ ID NO: 2 has cell adhesion activity, it will be understood by those skilled in the art that new collagens can be formed by performing other numbers of tandem repeats on SEQ ID NO: 1 or 8. Such formed collagens can have similar cell adhesion activity due to the retention of the adhesion fragments of SEQ ID NO: 1 or 8 such as GER or GEK and the peptide segments forming triple-helical structures. For example, the recombinant collagen of the present application can comprise 1 repeating unit of SEQ ID NO: 1 or 8, or can comprise 2 or more repeating units, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Each recombinant unit can be linked in tandem by a peptide bond, thereby forming a recombinant collagen.

[0128] Fusion protein

[0129] Also encompassed herein are fusion proteins comprising a collagen described herein and a protein for facilitating secretion, isolation, and / or purification of the collagen. It will be understood by one skilled in the art that the addition of these proteins is to facilitate secretion, isolation, and / or purification of the collagen without affecting the function of the collagen itself. In some embodiments, the protein for facilitating secretion, isolation, and / or purification of the collagen. In some embodiments, the protein is selected from the group consisting of a cleavage site sequence, a signal peptide, and a purification tag sequence. In some embodiments, the cleavage site sequence is a TEV protease cleavage site sequence. In some embodiments, the protein tag sequence is a His tag, a GST tag, a MBP tag, a SUMO tag, a Cytiva Protein Select tag, or a NusA tag. In some embodiments, the collagen is directly linked to the protein or linked through a linker, the linker being a flexible linker such as (G) a or (GGGGS) b wherein a and b are each independently an integer from 1-10 or 1-5 or 1-3.

[0130] Compositions

[0131] The recombinant humanized collagen type XX of the present application can be prepared as a composition. The composition can comprise the recombinant humanized collagen type XX described herein, a nucleic acid, a vector, and / or a host cell. The composition can further comprise a pharmaceutically and / or cosmetically acceptable carrier or solvent. The composition can be a pharmaceutical composition or a cosmetic composition for the purpose of a drug and / or a cosmetic. For example, the composition is one or more of a biological dressing (e.g., a collagen biological dressing), a human body bionics material, a plastic and aesthetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, a material for ophthalmology, a biomaterial for gynaecology, a material for neurorestoration and neuroregeneration, a material for liver tissue engineering, and a material for vascular repair and regeneration, a biomaterial for 3D printing of artificial organ, a cosmetic raw material, a pharmaceutical excipient, and a food additive.

[0132] The cosmetic composition can be a cosmetic composition for anti-wrinkle efficacy, skin oil control efficacy, skin repair efficacy, and / or skin soothing efficacy. The part to which the cosmetic composition is applied is not particularly limited, and can be the face, hands, legs, torso, and the like. For example, the skin oil control efficacy is a facial skin oil control efficacy.

[0133] The form of the composition is not particularly limited, as long as the intended function can be achieved. For example, the composition is a solid, liquid, or gel composition.

[0134] The composition can be applied in any suitable manner, for example, as a composition for oral and / or topical administration. The composition can also be prepared as a kit. The kit can contain additional ingredients, for example, auxiliary ingredients, such as a buffer, and contain instructions for use. In particular, the composition can be formulated into a suitable formulation, such as a liquid formulation. The formulation can contain a buffer, for example, a D-PBS buffer or a PBS buffer.

[0135] Methods

[0136] Provided herein is a method of adhering or anchoring cells, the method comprising contacting the cells with the recombinant humanized collagen type XX described herein, the composition, and / or the liquid formulation. The method of the present application can be performed in vitro to increase cell adhesion to a culture vessel. Alternatively, the method of the present application can also be performed in vivo.

[0137] Also provided herein is a cosmetic method comprising administering to a subject the recombinant humanized collagen type XX described herein, the composition, and / or the liquid formulation. The administration can be oral administration.

[0138] Screening methods

[0139] Provided herein are methods of screening for a collagen protein, comprising: mutating one or more amino acid residues of the amino acid sequence of SEQ ID NO: 9 to obtain a collagen protein library. Each collagen protein in the collagen protein library can be assayed for a biological activity, such as a cell adhesion activity. Collagen proteins with cell adhesion activity can be selected based on the activity of the collagen protein (e.g., compared to the parent collagen protein or bovine collagen type I). In one embodiment, the mutation is a substitution, an insertion, a deletion, and / or an addition. In one embodiment, the substitution is a conservative amino acid substitution. In one embodiment, the substitution, insertion, or deletion is a substitution, insertion, or deletion of 1-6 amino acid residues. In one embodiment, the addition is an addition of 1-15 amino acid residues. In one embodiment, the linker is a linker of 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In one embodiment, the addition is an addition of (Gly-X-Y)m at the N-terminus and / or C-terminus of the repeat unit, wherein X and Y are any amino acid residues, and m is 1-5. In one embodiment, Gly-X-Y is GER, GEK, GEP, or GTS. In one embodiment, the selected collagen protein comprises the amino acid sequence of SEQ ID NO: 1 or 8. In one embodiment, the method further comprises tandem repeating the selected collagen protein of SEQ ID NO: 9 or the repeat unit 2 or more times, such as 2-30 and any integer therebetween. In one embodiment, the method further comprises assaying a collagen protein comprising 2 or more repeat units for cell adhesion activity; and selecting a collagen protein comprising 2 or more repeat units with cell adhesion activity. In one embodiment, the number of repeat units of the selected collagen protein comprising 2 or more repeat units is 2-12 and any integer therebetween.

[0140] Examples

[0141] The following examples are provided to illustrate the present application. Those of ordinary skill in the art will understand that the examples are merely illustrative and are not limiting on the scope of the application, which is defined only by the appended claims.

[0142] Example 1-1: Construction, prokaryotic expression, and screening of humanized collagen XX fragments

[0143] 1. Large scale functional region screening was performed to obtain the following humanized collagen XX fragments.

[0144] Amino acid sequence of C20 collagen protein:

[0145] gtsgergppgtvgptglpgpkgergekgep

[0146] gtsgergppgtvgptglpgpkgergekgep

[0147] gtsgergppgtvgptglpgpkgergekgep

[0148] gtsgergppgtvgptglpgpkgergekgep (SEQ ID NO: 2).

[0149] The repeating unit of the amino acid sequence of C20 collagen protein is gtsgergppgtvgptglpgpkgergekgep (SEQ ID NO: 1).

[0150] The inventors designed the C20 collagen protein nucleotide sequence for the amino acid sequence of C20 collagen protein.

[0151] The nucleotide sequence of C20 collagen protein is:

[0152] GGATCCGAAAATCTGTATTTTCAGGGTACCAGCGGTGAACGTGGTCCGCCGGGTACAGTTGGTCCTACAGGTTTACCGGGTCCTAAAGGTGAACGTGGTGAAAAAGGTGAACCGGGTACCAGCGGTGAACGTGGTCCTCCTGGTACAGTTGGTCCGACCGGTTTACCGGGTCCGAAAGGTGAACGTGGTGAAAAAGGTGAACCGGGTACCAGCGGTGAACGTGGTCCTCCGGGTACAGTTGGTCCGACAGGTTTACCTGGTCCGAAAGGTGAACGTGGTGAAAAAGGTGAACCGGGTACCAGCGGTGAACGTGGTCCTCCTGGTACAGTTGGTCCGACAGGTTTACCGGGTCCGAAAGGTGAACGTGGTGAAAAAGGTGAACCGTAAAAGCTT (SEQ ID NO: 3).

[0153] 2. Construction of E. coli genetically engineered bacteria

[0154] The nucleotide sequence of C20 collagen protein was cloned into an expression vector, and then the expression vector was transformed into an E. coli expression strain to obtain the E. coli genetically engineered bacteria.

[0155] Specifically, according to the amino acid sequence of C20 collagen protein (SEQ ID NO: 2), the codon-optimized gene sequence of SEQ ID NO: 3 was optimized and selected. The nucleotide sequence of SEQ ID NO: 3 was synthesized. The nucleotide sequence of C20 collagen protein was inserted into the pET-32a expression vector (Beijing Lihe Huada Gene Technology Co., Ltd.) through the enzyme digestion sites of Kpn I (NEB, Cat No: R0136L) and Xho I (NEB, Cat No: R0146L), and the pET-32a-C20 expression vector was constructed. The expression vector was introduced into Escherichia coli BL21 (DE3), and the positive Escherichia coli genetically engineered bacteria were screened. The above operations were entrusted to Beijing Lihe Huada Gene Technology Co., Ltd.

[0156] 3. Fermentation culture of Escherichia coli genetically engineered bacteria

[0157] The successfully constructed pET-32a-C20 expression vector was transformed into Escherichia coli competent cells BL21 (DE3). The specific process is as follows:

[0158] (1). Take the Escherichia coli competent cells BL21 (DE3) out of the ultra-low temperature refrigerator and place it on ice. Add 2 μl of the expression plasmid to be transformed into the competent cells BL21 (DE3) and mix slightly for 2-3 times.

[0159] (2). Place the mixture on ice for 30 min, then heat shock at 42℃ for 45-90 s, and then place it on ice for 2 min.

[0160] (3). Transfer to a biological safety cabinet and add 700 μl of liquid LB medium, then incubate at 37℃ and 220 rpm for 60 min.

[0161] (4). Take 200 μl of the bacterial solution and evenly spread it on an LB plate containing ampicillin sodium.

[0162] (5). Incubate the plate in a 37℃ incubator for 15-17 h until uniform-sized colonies grow.

[0163] (6). Pick 5-6 single colonies from the transformed LB plate and place them in a shake flask containing an antibiotic stock solution. Incubate at 220 rpm and 37℃ in a constant temperature shaker for 7 h. Then cool the shake flask to 16℃, add IPTG to induce expression for a period of time, then divide the bacterial solution into centrifuge bottles, centrifuge at 8000 rpm and 4℃ for 10 min, collect the bacterial bodies, and record the weight of the bacterial bodies. Take a sample for electrophoresis detection.

[0164] (7). The collected bacteria were resuspended with the equilibration working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), and the bacterial solution was cooled to ≤15°C, homogenized, and high-pressure homogenized twice. After completion, the bacterial solution was collected. The homogenized bacterial solution was divided into centrifuge bottles and centrifuged at 17,000 rpm and 4°C for 30 min. The supernatant was collected, and the supernatant and precipitate were subjected to electrophoresis detection.

[0165] (8). The C20 collagen was purified and enzymatically cut, and the specific process was as follows: (1) crude purification: a. Water washing column material (Ni6FF, Cytiva), 5 CV. b. Equilibrium liquid (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) was used to equilibrate the column material, 5 CV. c. Sample loading: the supernatant after centrifugation was added to the column material, and after the liquid flow was completed, the flow-through liquid was taken for electrophoresis detection. d. Cleaning of impurities: 25 mL of impurity washing liquid (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) was added until the liquid flow was completed, and the impurity washing flow-through liquid was taken for electrophoresis detection. e. Collection of target protein: 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0) was added, and the flow-through liquid was collected for detection of protein concentration and calculation of protein amount, and electrophoresis detection. f. The column material was washed with 1 M imidazole working solution. g. The column material was washed with purified water. (2) Enzymatic cutting: the total amount of protein and the total amount of TEV protease were 50:1, TEV protease was added, and enzymatic cutting was performed at 16°C for 4 h, and sampling was performed for electrophoresis detection. The protein liquid after enzymatic cutting was placed in a dialysis bag and dialyzed at 4°C for 2 h, and then transferred to a new dialysis liquid and dialyzed at 4°C overnight. (3) Fine purification: a. Equilibrium column material: A liquid (20 mM Tris, 20 mM sodium chloride, pH 8.0) was used to equilibrate the column material at a flow rate of 10 ml / min. b. Sample loading: the flow rate was 5 ml / min, the sample was loaded and the flow-through liquid was collected, and electrophoresis detection was performed. c. Gradient elution: 0-15% B liquid (20 mM Tris, 1 M sodium chloride, pH 8.0) was set for 2 min, then maintained for 3 CV, 15-30% B liquid for 2 min, then maintained for 3 CV, 30-50% B liquid for 2 min, then maintained for 3 CV, 50-100% B liquid for 2 min, then maintained for 3 CV, and the peak was collected and subjected to electrophoresis detection (as fine protein). d. Cleaning of the column material. The protein was stored in a 4°C environment.

[0166] Concentration detection: an appropriate amount of sample was accurately taken, diluted 10-50 times with eluent, and thoroughly stirred with a glass rod. The absorbance was measured at 280 nm using a UV-visible spectrophotometer, and the protein concentration was calculated according to the formula C (mg / ml) = A280 x absorbance coefficient x dilution multiple (note: the absorbance value should be within 0.1-1).

[0167] The specific electrophoresis detection process is as follows: Take 40 μl of sample solution, add 10 μl of 5x protein loading buffer (250 mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), boil in 100℃ water for 10 min, then add 10 μl to each well of SDS-PAGE protein gel, run at 80V for 2 h, stain the protein with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20 min, and then destain with protein destaining solution (10% acetic acid, 5% ethanol).

[0168] Electrophoretic analysis results of C20 collagen purified protein showed that... Figure 1 .like Figure 1 As shown, the actual molecular weight of the C20 collagen of this invention is 11 kDa, indicating high purity. The actual molecular weight of the purified C20 collagen matches the expected molecular weight, demonstrating that the C20 collagen is correctly expressed.

[0169] Expected Examples 1-2: Prokaryotic Expression of Collagen 1-3

[0170] The following collagen proteins will be constructed using the same methods as C20 collagen construction, prokaryotic expression, and screening. The amino acid sequences of collagen proteins 1-3 will be submitted to a gene synthesis company for the synthesis of corresponding polynucleotide sequences. Collagen proteins 1-3 will be synthesized and expressed as described above. SDS-PAGE experiments will be used to verify the molecular weight of collagen proteins 1-3 and confirm that each protein is correctly expressed.

[0171] Collagen 1:

[0172] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0173] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0174] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0175] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0176] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0177] GTSGERGPPGTVGPTGLPGPKGERGEKGEP (SEQ ID NO: 5; number of repeats is 6), the TEV protease cleavage site ENLYFQ can be added to the front end of this sequence.

[0178] Collagen 2:

[0179] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0180] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0181] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0182] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0183] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0184] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0185] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0186] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0187] GTSGERGPPGTVGPTGLPGPKGERGEKGEP (SEQ ID NO: 6; number of repeats is 9), which sequence can be preceded by the TEV protease cleavage site ENLYFQ.

[0188] Collagen 3:

[0189] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0190] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0191] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0192] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0193] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0194] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0195] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0196] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0197] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0198] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0199] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0200] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0201] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0202] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0203] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0204] GTSGERGPPGTVGPTGLPGPKGERGEKGEP (SEQ ID NO: 7; number of repeats is 16), which can be preceded by the TEV protease cleavage site ENLYFQ.

[0205] Collagen 4:

[0206] GERGPPGTVGPTGLPGPKGERGEK (SEQ ID NO: 8; number of repeats is 16), which can be preceded by the TEV protease cleavage site ENLYFQ.

[0207] Example 2-1: Bioactivity detection of C20 collagen

[0208] The method for detecting the adhesion activity of C20 collagen can refer to the document 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:

[0209] (1) The concentration of the protein sample to be detected is detected by ultraviolet absorption method, including bovine type I collagen (China Food and Drug Inspection Research Institute, number: 380002) and C20 collagen provided by the application. Specifically, the ultraviolet light absorption of the sample at 215 nm and 225 nm is detected, and the protein concentration is calculated by using the empirical formula C (μg / mL) = 144 x (A215-A225), and it is noted that the detection should be performed under the condition of A215<1.5. The principle of this method is that the characteristic absorption of the peptide bond under far ultraviolet light is determined, which is not affected by the content of chromophore, has few interfering substances, is simple to operate, and is suitable for detecting human collagen and its analogues which are not colored by Coomassie brilliant blue. (The reference is Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45). After detecting the protein concentration, the concentration of all the proteins to be detected is adjusted to 0.5 mg / mL by PBS.

[0210] (2) 100 μL of various protein solutions (bovine type I collagen or C20 collagen) are added to a 96-well plate.

[0211] (3) 10 5 well-cultured 3T3 cells are added to each well, and incubated at 37°C for 60 min.

[0212] (4) Each well is washed with PBS for 4 times.

[0213] (5) The OD492nm absorbance is detected by using an LDH detection kit (Roche, 04744926001) according to the manufacturer's instructions. According to the value of the blank control, the adhesion rate of the cells can be calculated.

[0214] The adhesion rate calculation formula is as follows:

[0215]

[0216] The adhesion rate of the cells can reflect the adhesion activity of each protein. The higher the activity of the protein, the better external environment the protein can provide for the cells in a short time, and the better the cells can adhere.

[0217] The results are shown in Table 1. Figure 2 As can be seen from the comparison, the C20 collagen protein of the present application has a more excellent biological adhesion activity than the bovine type I collagen protein (PC group, 0.5 mg / ml). Figure 2 The relative cell adhesion activity of the C20 collagen protein relative to the bovine type I collagen protein is described, which indicates that the C20 collagen protein has a higher (more than 2 times) cell adhesion activity than the bovine type I collagen protein. It is unexpected that the C20 collagen protein has such a high cell adhesion activity.

[0218] Expected Example 2-2: Biological activity detection of collagen 1-3

[0219] The biological activity of collagen 1-3 will be detected by the same method as Example 2-1. According to the reports of prior art (for example, Chen Hua et al., Biochemical and Biophysical Research Communications 508 (2019) 1018e1023; Li Yang et al., Biomaterials 276 (2021) 121055), the recombinant collagen containing multiple tandem repeats of triple helix fragments can have a more stable helical conformation or a more favorable ligand binding configuration to promote cell membrane attachment and adhesion. The inventors expect that collagen 1-3 all have excellent cell adhesion activity. As described in subsequent Example 6, these collagens are indeed verified to have excellent cell adhesion activity.

[0220] Example 3: Circular dichroism spectrum of C20 collagen

[0221] 1) Sample preparation

[0222] Prepare phosphate buffer solution (PBS) 1X: dissolve 8 g NaCl, 0.2 g KCl, 3.62 g Na2HPO4.12H2O and 0.24 g KH2PO4 in 800 ml distilled water, adjust the pH value of the solution to 7.4 with HCl, and add water to 1 L. After high-pressure sterilization, store at room temperature. If the storage time is more than 1 week, filter through a 0.45 filter membrane before use.

[0223] Sample dissolution: dissolve the freeze-dried flocculent sample in PBS - each bottle contains 4 mg of C20 collagen protein, inject 2 mL of PBS into the Schlenk flask with a syringe (note that the Schlenk flask has negative pressure, and the syringe should be accurately injected with 2 mL of PBS) to a final concentration of 2 mg / mL, 4°C, overnight.

[0224] Note: Protein samples should be selected with UV absorption value A below 2, HT (instrument voltage) controlled between 170-700, > 700, the test result is not accurate enough, you can use the reduced sample concentration, use short wavelength cuvette, change the solvent, etc. The recommended concentration is 1-0.5 mg / mL as the highest concentration, and it is recommended to set the instrument sensitivity according to the instrument sensitivity.

[0225] Sample detection:

[0226] Gradient dilution sample configuration: 1. Take ice from the ice maker, and dilute the 4°C overnight sample by two times (note to change the gun head), take 500µl of the overnight sample, and dilute the PBS concentration to 1.0, 0.5, 0.25, and 0.125 mg / mL respectively.

[0227] 2) Instrument (JASCO Circular Dichroism Spectrometer, JASCO J-815) parameter setting

[0228] Band width: 1.0 nm

[0229] Step: 1.0 nm

[0230] Measurement range: 190-260 nm

[0231] Time-per-point: 1 s

[0232] Scanning speed: 50 nm / min

[0233] Repeats: 3 times

[0234] Measurement temperature: 4°C

[0235] 3) Standard CD scanning

[0236] Set the scanning wavelength to 190-260 nm for the blank buffer baseline test, and collect the circular dichroism absorption of the sample solution PBS in the range of 190-260 nm.

[0237] 4) Scan map processing.

[0238] Figure 3 The circular dichroism spectrum of C20 collagen is shown. C20 collagen has a negative peak near 195 nm and a positive peak near 221 nm, with a triple helix structure.

[0239] Expected Example 4: Yeast expression of collagen 1

[0240] The inventors have commissioned a gene synthesis company to design polynucleotide sequences suitable for expression in yeast (e.g. Pichia pastoris) from the amino acid sequences of collagen 1 and C20 collagen. The N-terminus of the above collagens can be added with the tag sequence of SEQ ID NO: 10.

[0241] Collagen 1:

[0242] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0243] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0244] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0245] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0246] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0247] GTSGERGPPGTVGPTGLPGPKGERGEKGEP (SEQ ID NO: 5)

[0248] C20 collagen

[0249] GTSGERGPPGTVGPTGLPGPKGERGEKGEP GTSGERGPPGTVGPTGLPGPKGERGEKGEP GTSGERGPPGTVGPTGLPGPKGERGEKGEP GTSGERGPPGTVGPTGLPGPKGERGEKGEP GTSGERGPPGTVGPTGLPGPKGERGEKGEP.

[0250] Cytiva Protein Select tag tag sequence: MVKIVSRKSLGVQNVYDIGVEKDHNFLLANGLIASN (SEQ ID NO: 10).

[0251] Cloning and expression in yeast host cells can be seen in Chinese patent application CN117126874A. The specific procedures are as follows: the commercially synthesized nucleotide sequence encoding is inserted into the pPiczal alpha A expression vector to obtain recombinant expression plasmid 1 (expressing collagen 1) and expression plasmid 2 (expressing C20 collagen).

[0252] 1. Plasmid digestion

[0253] Take the successfully constructed expression plasmids 1 and 2, use sac1 for enzyme digestion, according to the proportion of restriction enzyme enzyme digestion, 37℃ enzyme digestion for 2-3h, after enzyme digestion, detect the plasmid before and after enzyme digestion by nucleic acid gel electrophoresis, verify whether all enzyme digestion, and the band position after enzyme digestion is greater than before enzyme digestion.

[0254] 2. Plasmid purification

[0255] Use plasmid purification kit to purify the enzyme-digested plasmid to remove impurities such as ions, use 20-25ul sterilized purified water to dissolve, detect the plasmid concentration, and put the plasmid in 4℃ for standby.

[0256] 3. Yeast strain modification

[0257] Take the yeast competent cells (X33), and perform the following operations under the biosafety cabinet:

[0258] a. Take a 0.2cm electroporation cup, soak it in 75% alcohol for 10min for sterilization, rinse it repeatedly with sterile water for 3 times and dry it;

[0259] b. Place the electroporation cup in ice bath for 20min, and turn on the electroporator;

[0260] c. Add 80ul of competent cells and 5-15ug of enzyme-digested plasmid to a sterile 1.5ml EP tube, mix well with a pipette;

[0261] d. Transfer 100ul of the mixture to the electroporation cup, ice bath for 5min, wipe the outer surface of the electroporation cup, transfer the electroporation cup to the electroporator slot, click start, and perform electroporation;

[0262] e. After the electroporation is completed, add 1ml of 1M sterile sorbitol solution to the electroporation cup, seal it with a sealing film, and incubate at 30℃ for 2h;

[0263] f. Transfer the mixture in the electroporation cup to a sterile 10ml EP tube, then add 1ml of YPD liquid medium (yeast peptone 0.2g / L, yeast extract powder 0.1g / L) to the EP tube, and incubate at 30℃, 300rpm for 1h.

[0264] g. Plate: under the biosafety cabinet, take 200ul of the transformed and incubated bacterial solution and add it to the inverted 1-3 plates, and use a sterile spreader to evenly spread the plates, then incubate at 30℃ for 2-5 days.

[0265] h. Strain culture: plate culture to grow bacterial plaque, add 10 ml anhydrous glucose solution to 40 ml YPD liquid medium, when the medium temperature is reduced to room temperature; 50 ml medium is divided into 10 50 ml biological reaction tubes at 5 ml / tube, add bleomycin solution at a proportion of 2000 mg / L, pick 10 single clones on the plate and inoculate in 10 small tubes for 30°C, 300 rpm overnight culture; prepare 200 ml BMGY medium, after sterilization, add 20 ml 10*YNB stock solution and 200 ul 500*biotin stock solution in a shaking flask, mix evenly; divide 200 ml medium into 10 50 ml centrifuge tubes, 20 ml / tube; then take 4 ml of the cultured bacterial liquid and inoculate in 10 20 ml / tube centrifuge tubes, and culture overnight until the OD value of the bacterial liquid is between 3-10.

[0266] i. Inducing expression: prepare 500 ml BMMY medium, after sterilization, add 50 ml 10*YNB stock solution and 500 ul 500*biotin stock solution in a shaking flask, mix evenly; divide 500 ml medium into 10 250 ml shaking flasks, 50 ml / flask; centrifuge the cultured bacterial liquid at 1500 rpm for 10 min, and discard the supernatant; suspend the bacterial body precipitate in 10 centrifuge tubes with the medium in the 10 shaking flasks, and transfer to the respective shaking flasks, shake at 30°C, 300 rpm; every 24 h, take 1 ml to 1.5 ml of sample from the 10 shaking flasks into 1.5 ml EP tubes, and store at -20°C; add 1% methanol (filtered with a 0.22 um filter) to the shaking flasks for inducing expression, until the end of the 5th day; centrifuge the sample in the 1.5 ml EP tubes at 12000 rpm for 5 min, take the supernatant for electrophoresis detection, and observe the expression of the 10 clones respectively.

[0267] 4. Electrophoresis detection

[0268] The specific process is: take 40 ul of sample liquid, add 10 ul of 5* protein loading buffer (250 mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% beta-mercaptoethanol), boil in 100°C boiling water for 10 min, then add 10 ul per hole to the SDS-PAGE protein gel, run at 80V for 2h, then dye the protein with coomassie brilliant blue staining solution (0.1% coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20 min, and then decolorize with protein decolorizing solution (10% acetic acid, 5% ethanol).

[0269] The inventors will perform electrophoresis detection of the apparent molecular weight of the target protein. The inventors expect that collagen 1 and C20 collagen can be successfully expressed in the yeast host.

[0270] 5. The inventors will detect the bioactivity of collagen 1 and C20 collagen and circular dichroism detection. The inventors expect that the collagen 1 and C20 collagen expressed by yeast cells have cell adhesion activity and have a triple helix structure.

[0271] Example 5: Screening and activity determination of humanized collagen XX fragments

[0272] Large-scale functional region screening was performed on the amino acid sequence of collagen XX to obtain the following collagen XX target gene functional regions C1-C3, and the specific sequences are as follows. Polypeptide synthesis company was commissioned to synthesize C1-C3:

[0273] C1: GTSGERGPPGTVGPTGLPGPKGERGEKGEP (SEQ ID NO: 1);

[0274] C2: GERGPPGTVGPTGLPGPKGERGEK (SEQ ID NO: 8);

[0275] C3: GPPGTVGPTGLPGPK (SEQ ID NO: 9).

[0276] The adhesion activity of collagen C1-C3 was determined. The adhesion activity detection method can refer to 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:

[0277] (1) The concentration of the protein sample to be detected is detected by ultraviolet absorption method, including bovine collagen type I (China Food and Drug Inspection Research Institute, No. 380002) and collagen C1-C3 provided by the application. Specifically, the ultraviolet light absorption of the sample at 215 nm and 225 nm is detected respectively, and the protein concentration is calculated by using the empirical formula C (μg / mL) = 144 x (A215-A225), and it is noted that the detection should be carried out under the condition of A215<1.5. The principle of the method is that the characteristic absorption of the peptide bond under far ultraviolet light is determined, which is not affected by the content of chromophore, has few interfering substances, is simple to operate, and is suitable for detecting human collagen and its analogues which do not show color with coomassie brilliant blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45). After detecting the protein concentration, the concentration of all the proteins to be detected is adjusted to 0.5 mg / mL with PBS.

[0278] (2) 100 μL of various protein solutions (bovine collagen type I or collagen C1-C3) are added to a 96-well plate.

[0279] (3) 10 μL of well-cultured 3T3 cells are added to each well, and incubated at 37°C for 60 min. 5

[0280] (4) Each well is washed with PBS for 4 times.

[0281] (5) The OD492nm absorbance is detected by using an LDH detection kit (Roche, 04744926001) according to the manufacturer's instructions. According to the value of the blank control, the cell adhesion rate can be calculated.

[0282] The adhesion rate calculation formula is as follows:

[0283]

[0284] The adhesion rate of the cells can reflect the adhesion activity of each protein. The higher the activity of the protein, the better the external environment provided for the cells in a short time, and the better the help for cell adhesion. Figure 1 From the comparison, it can be known that compared with bovine collagen type I (PC group, 0.5 mg / ml), the collagen C1-C3 provided by the application has more excellent biological adhesion activity.

[0285] ​Chen Hua et al. Biochemical and Biophysical Research Communications 508 (2019) 1018e1023 disclose that both GERGAPGFRGPAGPNGIPGEKGPAGERGAP repeat unit and T16, a 16 tandem repeats of the repeat unit, possess cell adhesion activity, and it is believed that recombinant proteins containing multiple tandem repeats of triple-helical fragments can have more stable helical conformation or more favorable ligand binding configuration to facilitate cell membrane attachment and adhesion. Below, in view of the teachings of the prior art, the sequence of C1 collagen GTSGERGPPGTVGPTGLPGPKGERGEKGEP (SEQ ID NO: 1) is repeated multiple times to determine whether recombinant collagens having multiple repeats of SEQ ID NO: 1 also possess cell adhesion activity.

[0286] Example 6: Prokaryotic expression and activity determination of recombinant collagens

[0287] 1. Design of recombinant collagens

[0288] The C1 collagen GTSGERGPPGTVGPTGLPGPKGERGEKGEP (SEQ ID NO: 1) screened in Example 1 is repeated 6 times to obtain collagen T6 and 9 times to obtain collagen T9.

[0289] Collagen T6:

[0290] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0291] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0292] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0293] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0294] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0295] GTSGERGPPGTVGPTGLPGPKGERGEKGEP (SEQ ID NO: 5; number of repeats is 6).

[0296] Collagen T9:

[0297] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0298] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0299] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0300] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0301] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0302] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0303] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0304] GTSGERGPPGTVGPTGLPGPKGERGEKGEP

[0305] GTSGERGPPGTVGPTGLPGPKGERGEKGEP (SEQ ID NO: 6; number of repeats is 9).

[0306] 2. Construction of the genetically engineered E. coli bacteria

[0307] The nucleotide sequence of T6 or T9 collagen protein is cloned into an expression vector, and then the expression vector is transformed into an E. coli expression strain to obtain the genetically engineered E. coli bacteria.

[0308] Specifically, according to the amino acid sequence of T6 or T9 collagen protein (a TEV protease cleavage site ENLYFQ, SEQ ID NO: 4, can be added at the front end of the sequence), a codon-optimized gene sequence preferred by E. coli is selected and optimized. The polynucleotide sequence of T6 or T9 collagen protein is synthesized. The polynucleotide sequence of T6 or T9 collagen protein is inserted into a pET-32a expression vector (Beijing Lihe Huada Gene Technology Co., Ltd.) through Kpn I (NEB, Cat. No. R0136L) and Xho I (NEB, Cat. No. R0146L) enzyme cleavage sites to construct a pET-32a-T6 or pET-32a-T9 expression vector. The expression vector is introduced into E. coli BL21 (DE3) to obtain positive genetically engineered E. coli bacteria. The above operations are entrusted to Beijing Lihe Huada Gene Technology Co., Ltd.

[0309] 3. Fermentation culture of the genetically engineered E. coli bacteria

[0310] The successfully constructed pET-32a-T6 or pET-32a-T9 expression vectors were transformed into E. coli competent cells BL21 (DE3) respectively. The specific process is as follows:

[0311] (1). The E. coli competent cells BL21 (DE3) were taken out from the ultra-low temperature refrigerator and placed on ice. 2 μl of the expression plasmid to be transformed was added to the competent cells BL21 (DE3) and mixed slightly for 2-3 times.

[0312] (2). The mixture was placed in an ice bath for 30 min, then heat shocked at 42°C for 45-90 s, and then placed in an ice bath for 2 min after being taken out.

[0313] (3). Transferred to a biological safety cabinet, and then 700 μl of liquid LB medium was added, and then incubated at 37°C and 220 rpm for 60 min.

[0314] (4). 200 μl of the bacterial solution was uniformly coated on an LB plate containing ampicillin sodium.

[0315] (5). The plate was incubated in a 37°C incubator for 15-17 h until uniform-sized colonies were grown.

[0316] (6). Five to six single colonies were picked from the transformed LB plate and placed in a shake flask containing an antibiotic stock solution, and then incubated in a 37°C constant temperature incubator at 220 rpm for 7 h. After the culture, the shake flask was cooled to 16°C, and then IPTG was added to induce expression for a period of time. The bacterial solution was then divided into centrifuge bottles, centrifuged at 8000 rpm and 4°C for 10 min, the bacterial bodies were collected, and the weight of the bacterial bodies was recorded. The sample was taken for electrophoresis detection.

[0317] (7). The collected bacterial bodies were resuspended with a balanced working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0). The bacterial solution was cooled to ≤15°C, and then homogenized twice by high-pressure homogenization. After completion, the bacterial solution was divided into centrifuge bottles and centrifuged at 17000 rpm and 4°C for 30 min. The supernatant was collected, and the supernatant and precipitate were subjected to electrophoresis detection.

[0318] (8). The collagen is purified and enzyme cut, the specific process is: (1) crude purification: a. Water washing column material (Ni6FF, Cytiva), 5 column volumes (CV). b. Equilibrium liquid (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) equilibrates the column material, 5 CV. c. Sample loading: add the supernatant after centrifugation to the column material, until the liquid flow is complete, take the flow-through liquid for electrophoresis test. d. Remove impurities: add 25 mL of impurity washing liquid (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) to the liquid flow until the liquid flow is complete, and take the flow-through liquid for electrophoresis test. 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 liquid, detect the protein concentration to calculate the protein amount, and perform electrophoresis detection. f. Wash the column material with 1 M imidazole working solution. g. Purified water washes the column material. (2) Enzyme cutting: according to the ratio of total protein to total TEV protease of 50:1, add TEV protease, 16℃ enzyme cutting for 4h, take sample for electrophoresis detection. The enzyme cut protein liquid is placed in a dialysis bag and dialyzed at 4℃ for 2h, then transferred to a new dialysis liquid and dialyzed at 4℃ overnight. (3) Refining: a. Equilibrium column material: use A liquid (20 mM Tris, 20 mM sodium chloride, pH 8.0) to equilibrate the column material, at a flow rate of 10 ml / min. b. Sample loading: at a flow rate of 5 ml / min, sample loading and collection of flow-through liquid, and electrophoresis detection. c. Gradient elution: set 0-15% B liquid (20 mM Tris, 1 M sodium chloride, pH 8.0) for 2 min, then maintain 3 CV, 15-30% B liquid for 2 min, then maintain 3 CV, 30-50% B liquid for 2 min, then maintain 3 CV, 50-100% B liquid for 2 min, then maintain 3 CV, peak collection and electrophoresis detection (refined protein). d. Clean the column material. Store the protein in a 4℃ environment.

[0319] Concentration detection: accurately take an appropriate amount of sample, dilute 10-50 times with eluent, and mix well with a glass rod. Use a UV-visible spectrophotometer to measure the absorbance at 280 nm, and calculate the protein concentration according to the formula C (mg / ml) = A280 x absorbance coefficient x dilution factor (note: the absorbance value should be between 0.1-1).

[0320] The electrophoresis detection process is as follows: 40 μl of sample liquid is taken, 10 μl of 5x protein loading buffer (250 mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) is added, and the mixture is boiled in 100°C boiling water for 10 min, then 10 μl of SDS-PAGE protein gel is added to each well, and the voltage is 80V for 2h, then the protein is dyed with coomassie brilliant blue staining solution (0.1% coomassie brilliant blue R-250, 25% isopropyl alcohol, 10% glacial acetic acid) for 20 min, and then the protein is decolorized with a protein decolorizing solution (10% acetic acid, 5% ethanol).

[0321] The electrophoresis detection results of the T6 or T9 collagen protein purified protein are shown in Figure 5 As shown in Figure 5 , the actual molecular weight of T6 or T9 is consistent with the expected molecular weight, and both are correctly expressed.

[0322] Example 7: Biological activity detection of T6 or T9 collagen protein

[0323] The biological activity detection of the T6 or T9 collagen protein is carried out as described in Example 2-1. The results are shown in Figure 6 As can be seen from the comparison, the T6 or T9 collagen protein of the present application has a more excellent biological adhesion activity compared with bovine type I collagen (PC group, 0.5 mg / ml). Figure 6 The relative cell adhesion activity of the T6 or T9 collagen protein relative to bovine type I collagen is described, indicating that the T6 or T9 collagen protein has a higher cell adhesion activity than bovine type I collagen, which is unexpected.

[0324] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A collagen, wherein the collagen consists of 1-9 repeating units, wherein the amino acid sequence of the repeating unit is SEQ ID NO: 1, or the amino acid sequence of the collagen is SEQ ID NO: 8 or 9.

2. A fusion protein consisting of a collagen according to claim 1 and a protein for facilitating secretion, isolation and / or purification of the collagen; the protein is selected from the group consisting of an enzyme cleavage site sequence, a signal peptide and a purification tag sequence, the collagen is directly linked to the protein.

3. The fusion protein according to claim 2, wherein the enzyme cleavage site sequence is a TEV protease cleavage site sequence.

4. The fusion protein according to claim 2, wherein the purification tag sequence is a His tag, a GST tag, a MBP tag, a SUMO tag, a Cytiva Protein Select tag or a NusA tag.

5. A nucleic acid encoding the collagen according to claim 1 or the fusion protein of claim 3 or 4.

6. The nucleic acid according to claim 5, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO:

3.

7. A vector comprising the nucleic acid according to claim 5 or 6.

8. The vector according to claim 7, comprising a nucleotide sequence encoding a purification tag, a nucleotide sequence encoding a leader and / or a regulatory element.

9. The vector according to claim 8, wherein the purification tag is selected from the group consisting of a His tag, a GST tag, a MBP tag, a SUMO tag or a NusA tag, a Cytiva Protein Select tag.

10. The vector according to claim 8, wherein the regulatory element is selected from the group consisting of a promoter, a terminator and / or an enhancer.

11. A host cell comprising the nucleic acid according to claim 5 or 6 or the vector according to any one of claims 7-10.

12. The host cell according to claim 11, which is a eukaryotic cell or a prokaryotic cell.

13. The host cell according to claim 12, wherein the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell, and / or the prokaryotic cell is an E. coli cell.

14. The host cell according to claim 13, wherein the E. coli cell is E. coli BL21; the yeast cell is S. cerevisiae or P. pastoris.

15. The host cell according to claim 14, wherein the P. pastoris is P. pastoris.

16. A method of producing a collagen, comprising: (1) culturing the host cell according to any one of claims 11-15 under suitable culture conditions; (2) harvesting the host cell and / or the culture medium comprising the collagen; and (3) purifying the collagen or the fusion protein.

17. The method according to claim 16, wherein the purification step comprises (1) crude purification of the collagen on a Ni affinity column; (2) enzyme cleavage; and / or (3) ion exchange column fine purification of the collagen.

18. Collagen having a triple helix structure, comprising three identical single chain forms of the collagen according to claim 1.

19. Composition comprising the collagen according to claim 1, the fusion protein according to any one of claims 2-4, the nucleic acid according to claim 5 or 6, the vector according to any one of claims 7-10, the host cell according to any one of claims 11-15 and / or the collagen having a triple helix structure according to claim 18.

20. The composition according to claim 19, which is a pharmaceutical composition, a food composition or a cosmetic composition.

21. The composition according to claim 19 or 20, which is one or more of a biological dressing, a human bionics material, an orthopedic and cosmetic 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 blood vessel repair and regeneration material, a 3D printing artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient and a food additive.

22. The composition according to claim 21, wherein the biological dressing is a collagen biological dressing.

23. The composition according to claim 20, which comprises a pharmaceutically and / or cosmetically acceptable carrier.

24. The composition according to claim 20, which is a solid, liquid or gel composition.

25. The composition according to claim 20, which is an orally and / or topically administrable composition.

26. The composition according to claim 20, which is a kit.

27. The composition according to claim 20, which is a liquid formulation comprising the collagen according to claim 1 and a pharmaceutically and / or cosmetically acceptable carrier.

28. The composition according to claim 27, wherein the carrier is a buffer.

29. The composition according to claim 28, wherein the buffer is a D-PBS buffer or a PBS buffer.

30. An in vitro method of promoting cell adhesion or attachment, the method comprising contacting a cell with the collagen according to claim 1, the collagen having a triple helix structure according to claim 18 and / or the composition according to any one of claims 19-29.

31. Use of the collagen of claim 1, the fusion protein of any one of claims 2-4, the nucleic acid of claim 5 or 6, the vector of any one of claims 7-10, the host cell of any one of claims 11-15, the collagen having triple helix structure of claim 18, and / or the composition of any one of claims 19-29 in the preparation of one or more of a biological dressing, a human bionics material, a plastic and cosmetic 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, a blood vessel repair and regeneration material, and a 3D printed artificial organ.

32. Use of the collagen of claim 1, the fusion protein of any one of claims 2-4, the collagen having triple helix structure of claim 18, and / or the composition of any one of claims 19-29 in the preparation of a kit for promoting cell adhesion or attachment.

33. A method of forming a collagen in a triple helix structure form, comprising the step of dissolving the collagen of claim 1 in a single chain form in water or a buffer.

34. The method of claim 33, wherein the buffer is a D-PBS buffer or a PBS buffer.

35. A cosmetic method comprising administering to a subject the collagen of claim 1, the collagen having triple helix structure of claim 18, and / or the composition of any one of claims 19-29, the method being for non-therapeutic purposes.

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