High-purity recombinant collagen, preparation method therefor and use thereof

Abstract

Provided are a high-purity recombinant collagen, a preparation method therefor and a use thereof. The recombinant collagen consists of a recombinant collagen monomer, and the recombinant collagen monomer has an amino acid sequence as shown in any one of SEQ ID NOs: 1-22. The easy-break site and hydrophobicity of a collagen are optimized, the obtained recombinant collagen monomer and the recombinant collagen prepared therefrom have high cell activity (adhesion, migration and proliferation), have a triple helix structure, have high stability and high purity, and have no cytotoxicity, large-scale production can be achieved, and production costs are reduced.

Description

High-purity recombinant collagen, preparation methods and applications

[0001] This application claims priority to Chinese Patent Application No. 202510126121.9, filed on January 27, 2025, entitled "High-purity recombinant collagen, preparation method and application thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of biomedical materials, specifically to a high-purity recombinant collagen, its preparation method, and its applications. Background Technology

[0003] Collagen is an abundant high-molecular-weight protein in living organisms, accounting for 20% to 30% of total protein. Currently, about 28 types of natural collagen have been discovered, widely distributed in the connective tissues of skin, bones, tendons, and various organs, participating in physiological processes such as wound healing, skin aging, blood clotting, and joint lubrication. As a key biomaterial, collagen has significant applications in multiple fields, including medicine, cosmetics, and the food industry.

[0004] Natural type III and type IIIII collagen are fibrous proteins with complex multilayered structures. They have large molecular weights, and their helical regions are composed of repeating amino acid sequences of Gly-XY, where Y is usually hydroxyproline. Three homologous or heterologous protein chains form a triple helix structure through intermolecular forces such as hydrogen bonds and electrostatic forces, which further stack to form a more advanced fibrous structure.

[0005] Collagen on the market is mainly derived from animals such as pigs, cattle, and fish, and is obtained through extraction processes such as acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. However, animal-derived collagen has some drawbacks, including immunogenicity and allergy risks due to differences in amino acid sequences compared to human collagen, the potential presence of viruses, possible damage to the collagen structure during extraction, and religious and ethical issues.

[0006] With advancements in synthetic biology, scientists are now utilizing gene recombination technology to express recombinant human collagen in microorganisms such as *E. coli* and yeast, enabling large-scale production. Compared to traditional animal-derived extraction methods, recombinant collagen production offers several advantages: its amino acid sequence is identical to that of natural human collagen, eliminating the immunogenicity issues associated with animal sources; it avoids the risk of transmission from animal-derived viruses; and microbial fermentation is inexpensive, has a short cycle, and is suitable for large-scale production. The *Pichia pastoris* expression system, in particular, combines the advantages of prokaryotic and eukaryotic expression systems, offering ease of operation, high expression levels, low cost, suitability for industrial-scale production, and a certain capacity for post-translational modification of exogenous proteins. Through genetic engineering, recombinant collagen expressed in *Pichia pastoris* can form a triple-helix conformation, exhibiting biological activity closer to natural collagen, making it a more advanced biomaterial. Furthermore, *Pichia pastoris* has high protein secretion efficiency and low self-protein secretion, facilitating high-volume secretion and purification of exogenous proteins while avoiding toxic side effects during fermentation.

[0007] Currently, recombinant collagen expression mostly uses human collagen sequences. However, recombinant collagen produced by existing technologies often exhibits a random coiled structure, lacking regular secondary structures, and even less so tertiary or quaternary structures. Because water-soluble proteins are easy to process and use, amino acid sequences are often chosen for their high water solubility. This leads to linear recombinant collagen being easily degraded by proteases in the host. During purification and processing, due to the low content of hydrophobic regions, it is difficult to fold into stable higher-order structures. Unstable side chains and residues are prone to deamidation, oxidation, and hydrolysis, potentially generating process-related impurities and degradation products, increasing immunogenicity.

[0008] Therefore, developing recombinant collagen with high purity and high stability is an important need in this field and has significant value for industrial applications. Summary of the Invention

[0009] This application aims to address at least one of the technical problems existing in the prior art, to at least some extent.

[0010] This application utilizes mass spectrometry to analyze fermentation products of highly active recombinant collagen, identifying easily cleaved amino acid sites and modifying their hydrophobicity. For example, experiments revealed that collagen cleavage is concentrated in certain amino acids, such as the GXY amino acid combination containing I, R, D, V, G, N, P, and Q, which is highly prone to cleavage. The GIK amino acid sequence exhibits a cleavage rate >50%. Furthermore, the first GXY at the N-terminus is easily lost. Further, this application modifies the hydrophobicity of recombinant collagen, enabling hydrophobic chromatography to play a role in purification, minimizing impurities and degradation products, and reducing immunogenicity. For example, this application found that fragments containing GPL, GIA, or GIT amino acids are more hydrophobic than other sequences, and the position and number of hydrophobic sequences are strongly correlated with the stability and solubility of collagen. Therefore, this application adds different numbers of hydrophobic fragments at different positions in the N-terminus, C-terminus, and middle of the sequence of recombinant collagen. While ensuring that the sequence is fully humanized and the solubility is maintained, the hydrophobic strength is adjusted to obtain a series of recombinant collagen molecules with significantly improved stability and high safety.

[0011] In recent years, with the development of synthetic biology, researchers have utilized gene recombination expression technology, using microorganisms (such as E. coli and yeast) as cell factories to mass-produce recombinant human collagen. Compared with traditional animal collagen extraction processes, recombinant collagen production has the following advantages: 1) The amino acid sequence of recombinant human collagen is identical to that of natural human collagen, thus eliminating the safety risks associated with animal immunogenicity; 2) It avoids the spread of animal-derived viruses; 3) Microbial fermentation processes are low-cost, have short production cycles, and are suitable for large-scale production. In recent years, the Pichia pastoris expression system has been widely used in the production of recombinant collagen. The inventors discovered that the Pichia pastoris expression system combines the advantages of both prokaryotic and eukaryotic expression systems, such as ease of operation, high expression levels, and low cost, facilitating large-scale industrial production, while also allowing for a certain degree of post-translational modification of exogenous proteins. Through genetic engineering, the inventors designed and modified the Pichia pastoris expression system, discovering that yeast-expressed humanized collagen has the ability to form a triple helix conformation. Humanized collagen with a triple helix conformation and biological activity is a high-grade biomaterial closely resembling natural collagen. In addition, the inventors also discovered that Pichia pastoris has a high protein secretion efficiency and a low self-protein secretion, thus enabling high-level secretion of exogenous proteins that are easy to purify and avoid the toxic side effects caused by the accumulation of fermentation products.

[0012] Therefore, in a first aspect of this application, a recombinant collagen monomer is provided. According to embodiments of this application, the recombinant collagen monomer has the amino acid sequence shown in any one of SEQ ID NO: 1 to 22. This application optimizes easily broken sites and adjusts the hydrophobicity of the protein by performing amino acid sequence mass spectrometry analysis on recombinant collagen, thereby obtaining a recombinant collagen monomer sequence with improved stability. This simplifies the purification process, improves protein recovery rate and storage stability, reduces production costs, and enhances safety.

[0013] In a second aspect, this application provides a recombinant collagen. According to embodiments of this application, the recombinant collagen comprises three of the recombinant collagen monomers described in the first aspect. Therefore, the recombinant collagen exhibits high stability, cell adhesion activity, and good hydrophilicity, and can be widely used in various fields such as beauty, medicine, and tissue engineering.

[0014] According to embodiments of the present invention, the recombinant collagen further includes at least one of the following additional technical features:

[0015] According to embodiments of the present invention, the recombinant collagen has a triple helix conformation. Therefore, it possesses biological activity and can be widely applied in various fields such as cosmetics, medicine, and tissue engineering.

[0016] According to embodiments of the present invention, the recombinant collagen monomers are homologous or heterologous.

[0017] According to an embodiment of the present invention, the recombinant collagen monomers are interconnected by hydrogen bonds or electrostatic forces.

[0018] In a third aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect.

[0019] In a fourth aspect, the present invention provides a carrier. According to embodiments of the present invention, the carrier comprises the nucleic acid molecule described in the third aspect.

[0020] In a fifth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell comprises the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, or expresses the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect.

[0021] According to embodiments of the present invention, the recombinant cells further include at least one of the following additional technical features:

[0022] According to an embodiment of the present invention, the recombinant cells are eukaryotic cells or prokaryotic cells.

[0023] According to an embodiment of the present invention, the eukaryotic cell is Pichia pastoris, Saccharomyces cerevisiae, animal cell or plant cell.

[0024] According to an embodiment of the present invention, the prokaryotic cells are Escherichia coli, Bacillus subtilis, or Bacillus licheniformis.

[0025] In a sixth aspect, the present invention provides a method for preparing recombinant collagen. According to an embodiment of the present invention, the method comprises: culturing the recombinant cells described in the fifth aspect under conditions suitable for protein expression to obtain the recombinant collagen.

[0026] In a seventh aspect of the invention, the invention provides the use of the recombinant collagen monomer of the first aspect or the recombinant collagen of the second aspect in the preparation of a medicament for use in cosmetic, wound repair, joint lubrication, and gynecological applications.

[0027] In an eighth aspect of the invention, the invention proposes the use of the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect in the preparation of food.

[0028] In a ninth aspect, the present invention provides a medicament. According to embodiments of the invention, the medicament comprises the recombinant collagen monomer described in the first aspect or the recombinant collagen described in the second aspect.

[0029] According to embodiments of the present invention, the drug further includes at least one of the following additional technical features:

[0030] According to an embodiment of the present invention, the drug further includes excipients.

[0031] According to an embodiment of the present invention, the excipient is selected from recombinant collagen hydrogel, recombinant collagen repair solution, recombinant collagen solution or recombinant collagen dressing.

[0032] In a tenth aspect of the invention, a composition is provided. According to embodiments of the invention, the composition comprises at least one of the following: the recombinant collagen monomer of the first aspect, the recombinant collagen of the second aspect, the nucleic acid molecule of the third aspect, the carrier of the fourth aspect, the recombinant cell of the fifth aspect, the recombinant collagen prepared by the method of the seventh aspect, and the medicament of the tenth aspect.

[0033] The beneficial effects of this application include at least the following:

[0034] 1. This application identifies the easily broken sites of recombinant collagen through mass spectrometry analysis, and obtains recombinant collagen with good hydrophilicity, easy water solubility, high concentration, and sufficient hydrophobicity, which is easy to purify, by adjusting the hydrophilicity and hydrophobicity of collagen.

[0035] 2. The recombinant collagen prepared in this application has high cell activity (adhesion, migration, proliferation), has a triple helix structure, high stability and purity, and is non-cytotoxic.

[0036] 3. The preparation method of recombinant collagen in this application can be mass-produced with high purity, while greatly reducing production costs.

[0037] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0038] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0039] Figure 1 shows an agarose gel electrophoresis diagram of plasmid linearization in an embodiment of this application, where Y326pink represents a circular plasmid and Y326pink SacI represents a linearized plasmid.

[0040] Figure 2 shows the SDS-PAGE results of the monoclonal strains in the embodiments of this application;

[0041] Figure 3 shows the SDS-PAGE results of the MMC fluid exchange sample in the embodiments of this application;

[0042] Figure 4 shows the mass spectrometry (TIC) results of the recombinant collagen prepared in the embodiments of this application;

[0043] Figure 5 shows the results of circular dichroism (CD) detection of the triple helix of the recombinant collagen prepared in the embodiments of this application;

[0044] Figure 6 shows the Tm values ​​of the recombinant collagen prepared in the embodiments of this application at different temperatures;

[0045] Figure 7 shows the detection results of the anti-Trypsin enzyme digestion experiment of the recombinant collagen prepared in the embodiments of this application;

[0046] Figure 8 shows the cytotoxicity test results of the recombinant collagen prepared in the embodiments of this application against NIH3T3 or HSF cells;

[0047] Figure 9 shows the detection results of the cell adhesion-promoting activity of the recombinant collagen prepared in the embodiments of this application on NIH3T3 or HSF cells;

[0048] Figure 10 shows the detection results of the cell migration-promoting activity of the recombinant collagen prepared in the embodiments of this application on NIH3T3 or HSF cells;

[0049] Figure 11 shows the cell morphology detection results of NIH3T3 or HSF cells prepared in the embodiments of this application;

[0050] Figure 12 shows the detection results of the cell proliferation-promoting activity of the recombinant collagen prepared in the embodiments of this application on NIH3T3 or HSF cells;

[0051] Figure 13 shows the detection flowchart of type I collagen secretion by ELISA of recombinant collagen prepared in the embodiments of this application.

[0052] Figure 14 shows the detection results of the collagen regeneration activity of the recombinant collagen prepared in the embodiments of this application on NIH3T3 or HSF cells.

[0053] Figure 15 shows the SDS-PAGE results of the supernatant from the fermentation tank in an embodiment of this application;

[0054] Figure 16 shows the SEC purity test results of the recombinant collagen obtained by fermentation and purification in the production tank in the embodiments of this application;

[0055] Figure 17 shows the SDS-PAGE stability test results of the recombinant collagen obtained by fermentation and purification in the production tank in the embodiments of this application. Detailed Implementation

[0056] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0057] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0058] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0059] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0060] In this document, the term "conserved modified amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding properties of a protein containing that amino acid sequence. This modification includes amino acid substitutions, additions, and deletions. Modifications can be introduced into the proteins of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0061] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0062] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0063] This application discloses a high-purity recombinant collagen, its preparation method, and its applications, which will be described in detail below.

[0064] Recombinant collagen monomers, recombinant collagen

[0065] In one aspect, this application provides a recombinant collagen monomer having at least one of the amino acid sequences shown in any one of SEQ ID NO: 1 to 22 and their conserved modified forms. This application optimizes easily broken sites and adjusts the hydrophobicity of the protein by performing amino acid sequence mass spectrometry analysis on recombinant collagen, thereby obtaining a recombinant collagen monomer sequence with improved stability. This simplifies the purification process, improves protein recovery and storage stability, reduces production costs, and enhances safety.

[0066] In another aspect of this application, a recombinant collagen is provided, comprising three of the recombinant collagen monomers described in the first aspect. Therefore, the recombinant collagen exhibits high stability, cell adhesion activity, and good hydrophilicity, and can be widely used in various fields such as beauty, medicine, and tissue engineering.

[0067] It should be noted that the "recombinant collagen" described in this invention is recombinant human type III collagen.

[0068] According to embodiments of the present invention, the recombinant collagen further includes at least one of the following additional technical features:

[0069] According to embodiments of the present invention, the recombinant collagen has a triple helix conformation. Therefore, it possesses biological activity and can be widely applied in various fields such as cosmetics, medicine, and tissue engineering.

[0070] According to embodiments of the present invention, the recombinant collagen monomers are homologous or heterologous.

[0071] According to an embodiment of the present invention, the recombinant collagen monomers are interconnected by hydrogen bonds or electrostatic forces.

[0072] Nucleic acid molecules, vectors, recombinant cells

[0073] In another aspect of this application, the present invention provides a nucleic acid molecule that encodes the aforementioned recombinant collagen monomer or recombinant collagen.

[0074] In one aspect of the invention, a carrier is provided, the carrier comprising the nucleic acid molecules described above.

[0075] Those skilled in the art can clone DNA molecules encoding the recombinant collagen monomers, recombinant collagen, or fragments thereof described in this invention into a vector (particularly an expression vector), and then transform host cells. The recombinant collagen monomers, recombinant collagen, or fragments thereof can be obtained through induced expression. Therefore, this invention also provides (isolated) nucleic acids encoding the aforementioned recombinant collagen monomers, recombinant collagen, or fragments thereof, and recombinant vectors containing such nucleic acids. The nucleic acid is preferably an expression cassette obtained by genetic engineering methods.

[0076] In another aspect of the invention, the invention provides a recombinant cell comprising the aforementioned nucleic acid molecules, vectors, or expressing the aforementioned recombinant collagen monomers or recombinant collagen.

[0077] According to embodiments of the present invention, the recombinant cells further include at least one of the following additional technical features:

[0078] According to an embodiment of the present invention, the recombinant cells are eukaryotic cells or prokaryotic cells.

[0079] According to an embodiment of the present invention, the eukaryotic cell is Pichia pastoris, Saccharomyces cerevisiae, animal cell or plant cell.

[0080] According to an embodiment of the present invention, the prokaryotic cells are Escherichia coli, Bacillus subtilis, or Bacillus licheniformis.

[0081] It should be noted that the recombinant cells described in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma fissicerum, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. The eukaryotic cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0082] Methods for preparing recombinant collagen

[0083] In one aspect of the present invention, a method for preparing recombinant collagen is provided, the method comprising: culturing the aforementioned recombinant cells under conditions suitable for protein expression in order to obtain the recombinant collagen.

[0084] It should be noted that the "conditions suitable for protein expression" mentioned in this application specification refer to conditions suitable for the expression of the recombinant collagen described in this application. Those skilled in the art will readily understand that suitable conditions for recombinant collagen expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The "conditions suitable for protein expression" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the recombinant collagen according to the specific environment of their laboratory.

[0085] use

[0086] In another aspect of the invention, the invention proposes the use of the aforementioned recombinant collagen monomers or recombinant collagen in the preparation of pharmaceuticals for cosmetic, wound repair, and joint lubrication purposes.

[0087] Regarding beauty, including but not limited to (1) promoting collagen production (recombinant collagen III can promote the production of type I and type III collagen in the human body), increasing skin elasticity, and replenishing lost collagen; (2) moisturizing: recombinant collagen III can increase the water content of the skin, restore the skin's water absorption and storage function, and keep the skin moisturized; (3) anti-aging: as we age, the content of type III collagen in the skin gradually decreases. By supplementing recombinant collagen III externally, we can help maintain skin elasticity and firmness, and reduce wrinkles and sagging; (4) whitening and brightening: recombinant collagen III can prevent melanin deposition, reduce the damage of ultraviolet rays to the skin, lighten existing dark spots, and repair acne marks and scars on the epidermis; (5) shrinking pores: enhancing the skin's water retention capacity, maintaining the skin's water-oil balance, enhancing skin firmness, fading fine lines and wrinkles, refining pores, and making the skin plump and moisturized.

[0088] In medical applications: Recombinant collagen III can repair epidermal cells, thus playing a role in repairing the skin barrier and can be used for wound repair in various parts of the body. Wound healing: During the human wound healing process, the expression of type III collagen increases significantly. It participates in the formation and remodeling of the new extracellular matrix, providing fundamental support for the development of new tissue. Recombinant collagen III applied to wound dressings can not only promote the migration and proliferation of fibroblasts but also absorb liquid to form a protective gel, accelerating the natural healing process. Tissue engineering: Recombinant collagen III is used as a scaffold material to support cell attachment, proliferation, and differentiation. As a major component of the extracellular matrix, it provides a good platform for mimicking the natural biological environment. For example, in the regeneration of skin, blood vessels, and other soft tissues, it acts as a "scaffold" for cell growth, promoting the formation of new tissue. Absorbable sutures: As a natural biomaterial, recombinant collagen III can be absorbed by the human body, reducing the long-term residue of foreign bodies in the patient's body. Compared to traditional synthetic absorbable materials, recombinant collagen III sutures may offer better biocompatibility and less inflammatory response, which is crucial for improving the quality of wound healing and reducing postoperative complications.

[0089] Other applications: Anti-tumor effects: Recent studies have shown that type III collagen, by participating in the tumor microenvironment, can maintain the dormant state of tumor cells, thereby inhibiting tumor proliferation and exhibiting anti-tumor effects. Vaccine design: Recombinant human type III collagen can serve as a matrix for trimeric antigens, used in the design and optimization of vaccines, such as COVID-19 and influenza vaccines.

[0090] In another aspect of the invention, the invention proposes the use of the aforementioned recombinant collagen monomers or recombinant collagen in the preparation of food.

[0091] Drugs, Compositions

[0092] In one aspect of the invention, a medicament is provided, the medicament comprising the recombinant collagen monomer or recombinant collagen described above.

[0093] According to embodiments of the present invention, the drug further includes at least one of the following additional technical features:

[0094] According to embodiments of the present invention, the drug further includes excipients. The excipients are any pharmaceutically acceptable excipients and are not particularly limited thereto.

[0095] According to an embodiment of the present invention, the excipient is selected from recombinant collagen hydrogel, recombinant collagen repair solution, recombinant collagen solution or recombinant collagen dressing.

[0096] The term "pharmaceutically acceptable" indicates that a drug can be administered to a subject without producing adverse physiological reactions that would impede the administration of the composition. For example, "pharmaceutically acceptable carrier" refers to a carrier useful in the preparation of a generally safe, non-toxic, and desirable pharmaceutical composition. Preferably, examples of such carriers or diluents include, but are not limited to: water, saline, Ringer's solution, glucose, mannitol, dextran, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, and liposomes and non-aqueous mediators, such as non-volatile oils, may also be used.

[0097] In this invention, "patient" or "subject" generally refers to mammals, such as primates and / or rodents, especially humans or rats.

[0098] In another aspect of the invention, the invention provides a composition comprising at least one of the following: the recombinant collagen monomer, recombinant collagen, nucleic acid molecule, carrier, recombinant cell, recombinant collagen prepared by the method described above, and the drug described above.

[0099] The sequences in this application are shown in Table 1.

[0100] Table 1

[0101] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0102] Example 1: Construction of Pichia pastoris strain

[0103] Based on the characteristics of collagen, and to facilitate subsequent purification, the Pichia pastoris secretion system was selected to express the candidate molecule, using the plasmid pPinkα-HC (purchased from Thermofisher / A11153) as the vector. First, the designed collagen sequences (SEQ ID No: 1-22) were codon-optimized for the Pichia pastoris expression system, followed by full-gene synthesis. Vector construction was commissioned to BGI Genomics. The target sequence for expressing the collagen was inserted into the pPinkα-HC vector via XhoI and KpnI restriction enzyme sites.

[0104] After linearizing and digesting the plasmid pPinkα-HC-Y109 (vector from BGI Genomics) with restriction endonucleases DraI or SalI, the plasmid was then transformed into Pichia pastoris Pink competent cells by electroporation to screen transformant plates. The specific method is a routine procedure in this field and will not be described in detail here.

[0105] Example 2: Construction and screening of stable Pichia pastoris cell lines

[0106] This embodiment utilizes the Pichia pastoris strain expressing the collagen constructed in Example 1 for research, and the specific details are as follows:

[0107] 1. Inoculation: The Pichia pastoris strain expressing collagen constructed in Example 1 was inoculated into LB medium containing 100 μg / mL ampicillin and cultured overnight at 37°C and 220 rpm with shaking.

[0108] 2. Plasmid extraction: Plasmids were extracted using a plasmid large-scale extraction kit (purchased from Tiangen Biotech, batch number DP117). The specific operation was performed according to the kit instructions, and the plasmid concentration and purity (A260 / A280) were tested.

[0109] 3. Linearization: 200 μg of the extracted plasmid was digested with enzymes at 37 °C for 8 h. The enzyme digestion reaction system is shown in Table 2.

[0110] The linearized plasmid was recovered using a DNA recovery kit (purchased from Tiangen Biotech, batch number DP214), and the DNA concentration and purity (A260 / A280) were detected. This method is a routine operation in the field and will not be described in detail here. The obtained agarose gel electrophoresis results are shown in Figure 1, in which the linearization of the plasmid can be seen.

[0111] Table 2: Linearized Enzyme Digestion Reaction System

[0112] 4. Construction of stable yeast cell lines: Prepare competent yeast cells by electroporation, electroporate linearized DNA molecules encoding collagen into competent cells, and spread the bacterial culture on MD solid plates. The specific operation steps will not be described in detail.

[0113] 5. Screening for low-level protein expression: Single clones of the strain from the above-mentioned Tubu plates were picked and inoculated into BMGY medium (prepared with 1% yeast extract, 2% peptone, 100mM potassium phosphate (pH 6.0), 1.34% YNB, 0.00004% Biotin, and 1% glycerol) and cultured overnight at 28°C with shaking at 240 rpm. The next day, the original medium was discarded and replaced with BMMY medium (prepared with 1% yeast extract, 2% peptone, 100mM potassium phosphate (pH 6.0), 1.34% YNB, 0.00004% Biotin, and 0.5% methanol), and expression was induced for 1 day with 1% methanol. The supernatant of the bacterial culture was collected, and the collagen secreted into the medium was detected by SDS-PAGE. Clones with high expression levels were screened, and the glycerol-containing bacteria were cryopreserved at -80°C. This method is a routine procedure in this field and will not be described in detail here. The SDS-PAGE results of the cloned expression are shown in Figure 2.

[0114] 6.500mL shake flask protein expression: The selected strains with high collagen expression levels (clones #5 and #14) were inoculated into 50mL BMGY medium and cultured for 1 day. The original medium was discarded and replaced with BMMY medium to start induction expression. The final methanol concentration was 1%. Induction expression was carried out for 2 days, with methanol added every 24 hours.

[0115] 7. Centrifuge to collect the supernatant of the culture medium of the strain to obtain the collagen proteins.

[0116] Example 3: Detection of Recombinant Collagen Breakdown

[0117] 3.1 Preparation of collagen samples for mass spectrometry

[0118] The supernatant of the yeast fermentation broth obtained in Example 2 was heated at 60°C for 10 min. The inactivated sample was filtered through a 0.45 μm filter into a clean container. The chromatography column was thoroughly rinsed with purified water, then rinsed with 0.5 M NaOH, and finally rinsed with equilibration buffer before sample loading. The filtered sample was loaded at low temperature, and the flow-through was collected. The chromatography column was rinsed with elution buffer, and 1 CV of elution buffer was added for the first time. The column was incubated for 1 min and collected, labeled as tube 1. A total of 5 tubes of elution were collected, numbered 1-5 (Figure 3). The eluted sample was quantified using a BCA kit (purchased from Thermo Scientific, lot number 23236). The sample from the tube with the highest concentration was sent to our mass spectrometry analysis department for mass spectrometry analysis to analyze the protein sequence fragmentation.

[0119] 3.2 Mass spectrometry detection

[0120] The specific operation of mass spectrometry detection is as follows:

[0121] 1. Dilute the recombinant collagen obtained above with ultrapure water to a final concentration of 1 mg / mL, and take 200 μL into a sample vial.

[0122] 2. The molecular weight of collagen was determined by liquid chromatography-mass spectrometry (C4-ESI-MS). The instrument used was a SCIEX ZenoTOF 7600 high-resolution mass spectrometer system, with an XBridge Protein BEH C4 column. The injection volume was 1 μL, and isocratic elution mode (35% acetonitrile aqueous solution containing 0.1% formic acid) was used. The positive ion mode was used for scanning, with the ion source temperature set at 450℃, the spray voltage set at 5500V, and the primary mass range set at 300–2000 Da.

[0123] 4. Data processing (deconvolution) analysis was performed using SCIEX Biologics Explorer software. Protein molecular weight identification was based on primary mass spectrometry of the intact protein. Search parameters were: no fixed modifications; methionine oxidation, glutamine, and asparagine deamination were set as variable modifications; the protein library sequence included the intact Y197 sequence and fragment sequences. Because Y197 lacks disulfide bonds, the molecular weight of each fragment could be directly determined using this method.

[0124] The molecular weight identification (TIC) by mass spectrometry is shown in Figure 4. Based on the theoretical sequence, the break site is assumed step by step from the N-terminus / C-terminus. The assumed fragment molecular weight is then compared with the detected value. If the detected molecular weight is consistent with the theoretical value, it is inferred that the site is the break site. The fragment molecular weight analysis of Y197 protein is shown in Table 3.

[0125] Table 3: Fragment Molecular Weight

[0126] Note: Range represents the region of peak elution time, ordered from smallest to largest. Y197-F1 (SEQ ID NO:23), Y197-F2 (SEQ ID NO:24), Y197-F3 (SEQ ID NO:25), Y197-F4 (SEQ ID NO:26), and Y197-F5 (SEQ ID NO:27) are all fragments of Y197, as shown in the sequence below. In the post-translational modifications, 4*Loss_one_repeat+N-term_loss_G+N-term_loss_PGSPGPAGQQ represents the loss of four complete repeat sequences in the Y197 sequence, along with the loss of PGSPGPAGQQ and G at the N-terminus. Other proteins follow the same pattern.

[0127] Table 4

[0128] Example 4: Detection of the triple helix structure of recombinant collagen

[0129] To confirm the tertiary structure of recombinant collagen, this embodiment utilizes circular dichroism spectroscopy for collagen detection. First, the sample was diluted to 200 μg / mL with 50 mM NaAc, pH 5.0 solvent and stored at -80°C for later use. After complete freezing, it was placed on ice and rapidly transferred to 4°C, then allowed to stand overnight.

[0130] The specific procedures for variable-temperature circular dichroism (CD) detection are as follows:

[0131] The wavelength was set to 190–260 nm, the temperature to 4–40 °C, and the heating rate to 2 °C / min. After zeroing with 50 mM NaAc and pH 5.0 buffer, the CD value of the sample was detected at specific temperatures. The CD spectrum is shown in Figure 5, where Y326-ON-20C and WYM-ON-20C represent Y326-4 °C over night-20 °C detection, respectively. At low temperature (10 °C), the curve shows a negative peak near 195 nm and a positive peak near 221 nm. As the temperature increases, the positive peak near 221 nm disappears, indicating that collagen has a triple helix structure at low temperatures, and begins to unwind as the temperature gradually increases. Furthermore, GraphPad was used to plot the ellipticity at 221 nm at different temperatures, and a fitted curve was drawn to calculate the Tm value of collagen, as shown in Figure 6 (detection results of Y326) and Table 5. Different sequence designs affect the Tm value of recombinant collagen.

[0132] Table 5: Tm values ​​of different collagens

[0133] Example 5: Detection of anti-Trypsin enzyme digestion of recombinant collagen

[0134] This embodiment uses an anti-trypsin enzyme digestion experiment to detect the triple helix structure of collagen. Under specific conditions, a certain concentration of trypsin enzyme can degrade collagen single-chain molecules, while collagen molecules can resist trypsin digestion after forming a triple helix structure. Therefore, the anti-trypsin enzyme digestion experiment can analyze the triple helix structure of collagen.

[0135] 1. Transfer the collagen samples (Y197, Y326, and Y306) to be tested from -80℃ ice to 4℃ and let them stand overnight;

[0136] 2. Set up a group without trypsin and a group with trypsin. In the group with trypsin, 1 ng of trypsin was added for every 2 μg of collagen (Table 6).

[0137] Table 6: Trypin digestion conditions

[0138] 3. Set the above temperature gradient in the PCR amplification instrument; incubation time: 1 hour.

[0139] 4. After the reaction was complete, transfer the reaction sample to ice. Immediately add 5 μL of 5X Loading Buffer (non-reducing) to each tube and perform SDS-PAGE analysis. The results are shown in Figure 7.

[0140] Example 6: Effects of recombinant collagen on cell viability

[0141] 6.1 Cytotoxicity Detection

[0142] After digestion of NIH3T3 or HSF cells, centrifuge to remove the supernatant, resuspend in detection medium (DMEM + 10% FBS) for viability testing, and adjust the NIH3T3 or HSF cell concentration to 4*10⁻⁶. 5 Cells / mL were seeded into sterile 96-well plates using a multipipe syringe, 50 μL per well; incubated at 37℃ for 24 h; the next day, the test samples were serially diluted according to Table 5, with a dilution factor of 2-fold, starting at 3 μg / mL, for a total of 9 concentration gradients, and the 10th group served as a blank control (PBS). The culture medium in the wells was removed; the prepared molecular and blank control solutions (PBS) were added. The plates were incubated at 37℃ for 24 h, and then 10 μL of CCK8 was added to each well, and the plates were incubated at 37℃ for 1 h; the absorbance of the 96-well plates at 450 nm was read using a microplate reader, the results were recorded and plotted for analysis, as shown in Figure 8. After the application of collagen, the activity of both cell types was 100% or higher, indicating good cell viability.

[0143] 6.2 Cell adhesion activity assay

[0144] Each sample was diluted to 200 μg / mL with PBS to prepare a total volume of 255 μL. After thorough mixing, 85 μL was added to 170 μL of PBS, and this serial dilution was repeated 7 times to obtain 8 concentrations (200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09 μg / mL). After dilution, the samples were added to 96-well plates at 50 μL / well, with 3 replicates. The samples were then added to untreated 96-well cell culture plates. After incubation at 4°C overnight, the cell culture plates were washed once with PBS. A 1% BSA solution was prepared, and 100 μL / well of 1% BSA was added to each well of the 96-well plates using a pipette, and the plates were incubated at room temperature for 1 h. After removing the BSA, 200 μL of PBS was added to each well, and the wells were washed once. The PBS was then removed. The NIH3T3 cell density was adjusted to 1*103. 6 cells / mL (HSF cell density is 5*10) 5 (cells / mL); seeded into 96-well plates using a multipipeline, 100 μL / well, and incubated at 37°C for 1 h; slowly removed the culture medium and washed four times with PBS using a plate washer; prepared by adding 10 μL of CCK-8 reagent to every 100 μL of culture medium, mixed thoroughly, and added to 96-well plates, 100 μL / well, incubated at 37°C in a 5% CO2 cell culture incubator for 1 h, and then removed; the absorbance of the 96-well plate was read at 450 nm using a microplate reader, and the results were recorded. The experimental results are shown in Figure 9, where, within a certain range, the activity of collagen molecules in promoting NIH3T3 and HSF cell adhesion increased with increasing concentration.

[0145] 6.3 Cell migration activity assay

[0146] Each sample was serially diluted to different concentrations (333.33 / 37.04 / 4.12 / 1.37 / 0.46 / 0.15 μg / ml groups), and 50 μl / well was added to four non-treated 96-well plates, with two replicates. The NC group was coated with 50 μl / well PBS (Fillderm, purchased from Changchun Botai, batch number 20221008) at 4℃. After overnight incubation, the cell culture plates were washed once with 200 μl / well PBS. Single-cell suspensions were prepared, 2 x 10 cells per well. 3 Cells were cultured at 37°C in a 5% CO2 incubator. After 4 hours, the movement of fibroblasts in the cell culture plate was tracked using a high-content screening workstation. Six fields of view were captured per well, for a total of nine imaging sessions, with a total imaging time window of 2 hours and 6 minutes. The tracking results were analyzed using the high-content screening workstation's analysis module, and the cell movement speed was statistically analyzed. The results are shown in Figure 10, indicating that recombinant collagen molecules effectively promoted fibroblast migration.

[0147] 6.4 Cell morphology detection

[0148] After culturing NIH3T3 and HSF cells at 37°C and 5% CO2 for 24 h, the supernatant in the cell culture wells was removed, Calcein-AM staining solution was added, and the cells were incubated at 37°C for 30 min. Cell morphology was then observed and photographed using a high-content screening workstation. Six fields of view were used per well for cell morphology observation and statistical analysis. The results are shown in Figure 11, indicating that recombinant collagen molecules can effectively help maintain the morphology of fibroblasts.

[0149] 6.5 Cell proliferation activity assay

[0150] After culturing NIH3T3 and HSF cells at 37°C and 5% CO2 for 72 h, the supernatant was removed, and PBS diluted with water was added. The Luminescent Cell Viability Assay assay solution was prepared in batches of 100 μL per well. After shaking and incubation at room temperature for 10 min, 90 μL was transferred to a white, opaque 96-well plate. Chemiluminescence was detected using a multi-functional microplate reader, and the results were statistically analyzed. The results are shown in Figure 12. The application of recombinant collagen molecules effectively promoted the proliferation of fibroblasts.

[0151] 6.6 Activity Assay for Collagen Regeneration

[0152] Supernatant samples (333.33 / 37.04 / 4.12 μg / ml groups and NC group) were collected after 72 h of NIH3T3 and HSF culture for analysis. The NIH3T3 sample was diluted 10 times, and the HSF sample was diluted 2 times. The detection procedure is shown in Figure 13. The results are shown in Figure 14. The addition of recombinant collagen can promote the secretion of more type I collagen by fibroblasts.

[0153] Example 7: Induced Expression of Recombinant Collagen

[0154] This embodiment investigated the conditions for inducing collagen Y326 expression. Fermentation was conducted in a 5L fermenter with an initial fermentation volume of 2.5–3L. Aeration was achieved with compressed air at a rate of ≥2L / min. Stirring was performed at ≥200rpm. The initial culture temperature was 28℃–30℃, and the induction temperature was set at 30℃–32℃. Sugar replenishment was initiated 3–4 hours after sugar consumption, at a rate of 10–20mL / h. After sugar replenishment ceased and dissolved oxygen recovered for 0.5–1 hour, the inducing agent was added at a rate of 3–10mL / L·h. Nitrogen replenishment was performed once daily after induction, adding 5–15g / L of the initial nitrogen source. Fermentation lasted 48–96 hours. The fermentation broth was centrifuged at 10000–12000g, and the supernatant was collected. The purity and concentration of collagen in the fermentation broth supernatant were detected by SDS-PAGE and SEC methods. Table 7 shows that 30℃ and 32℃ had no significant effect on purity and expression level. Figure 15 shows that the purity of the fermentation supernatant was >90% in the SDS-PAGE test.

[0155] Table 7: Effect of temperature on expression levels and purity (fermentation conditions for two batches of Y326)

[0156] Example 8: Purification of recombinant collagen

[0157] The supernatant collected in Example 7 was inactivated at 60°C for 10 minutes. The inactivated fermentation supernatant was collected after deep filtration and 0.22μm sterilization filtration and stored at 2°C to 8°C.

[0158] The chromatography column was cleaned with cleaning buffer, and rinsed with equilibration buffer until acidic. Samples were loaded, and the column was rinsed with equilibration buffer for 3 column volumes, followed by elution buffer for 5 column volumes. The eluted sample was collected. The eluted sample buffer was replaced using an ultrafiltration membrane. The sample was adjusted to a 1 mol / L ammonium sulfate solution using ammonium sulfate stock solution, and hydrophobic chromatography was performed. The equilibration column was rinsed with equilibration buffer for 3 column volumes, and then linearly eluted with 1 mol / L ammonium sulfate and 50 mmol / L acetate buffer for 10 column volumes. Samples were collected in fractions, and their purity was determined. Samples with a purity >95% were combined and ultrafiltered into an acetate buffer system. The purity of the collected samples was determined by SDS-PAGE and SEC assays. The results are shown in Figures 16 and 17. The sample purity was consistently higher than 99%, and the protein exhibited high stability in the stock solution, maintaining a purity above 99% even after 7 days at 4°C or 25°C.

[0159] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0160] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A recombinant collagen monomer, characterized in that, The recombinant collagen monomer has an amino acid sequence shown in any one of SEQ ID NO:1 to 22.

2. A recombinant collagen, characterized in that, Includes the recombinant collagen monomers described in claim 1.

3. The recombinant collagen according to claim 2, characterized in that, The recombinant collagen has a triple helix conformation; Optionally, the recombinant collagen monomers are homologous or heterologous; Optionally, the recombinant collagen monomers are interconnected by hydrogen bonds or electrostatic forces.

4. A nucleic acid molecule, characterized in that, The recombinant collagen monomer of claim 1 or the recombinant collagen of any one of claims 2 to 3 is encoded.

5. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 11.

6. A recombinant cell, characterized in that, The product comprises the nucleic acid molecule of claim 4, the vector of claim 5, or expresses the recombinant collagen monomer of claim 1 or any one of claims 2 to 3; Optionally, the recombinant cells are eukaryotic cells or prokaryotic cells; Optionally, the eukaryotic cells are Pichia pastoris, Saccharomyces cerevisiae, animal cells, or plant cells; Optionally, the prokaryotic cells are Escherichia coli, Bacillus subtilis, or Bacillus licheniformis; Preferably, the recombinant cells are Pichia pastoris.

7. Use of the recombinant collagen monomer according to claim 1 in the preparation of recombinant collagen.

8. A method for preparing recombinant collagen, characterized in that, include: The recombinant cells of claim 6 are cultured under conditions suitable for protein expression in order to obtain the recombinant collagen.

9. Use of the recombinant collagen monomer of claim 1 or the recombinant collagen of any one of claims 2 to 3 in the preparation of a medicament, wherein the medicament is used for cosmetic purposes, wound repair, and joint lubrication.

10. Use of the recombinant collagen monomer of claim 1 or the recombinant collagen of any one of claims 2 to 3 in the preparation of food.

11. A drug, characterized in that, The drug comprises the recombinant collagen monomer of claim 1 or the recombinant collagen of any one of claims 2 to 3.

12. The medicament according to claim 11, characterized in that, The drug further includes excipients; Optionally, the excipients are selected from recombinant collagen hydrogel, recombinant collagen repair solution, recombinant collagen solution, or recombinant collagen dressing.

13. A composition, characterized in that, It includes at least one of the following: the recombinant collagen monomer of claim 1, the recombinant collagen of any one of claims 2-3, the nucleic acid molecule of claim 4, the carrier of claim 5, the recombinant cell of claim 6, the recombinant collagen prepared by the method of claim 8, and the drug of any one of claims 11-12.

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