Recombinant I-type collagen with triple helix structure and application of recombinant I-type collagen

Abstract

The invention provides a recombinant I-type collagen with a triple helix structure and application thereof, and belongs to the technical field of protein engineering, the amino acid sequence of the recombinant I-type collagen comprises n repetitive units, n is an integer of 1 or greater than 1, and the amino acid sequences of the repetitive units are as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7; the invention also provides a construction method of the expression system and a preparation method of the recombinant I-type collagen. The invention also provides practical industrial application according to the characteristics of the protein. The recombinant I-type collagen provided by the invention is high in purity and has a stable triple-helix structure.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering technology, and particularly relates to a recombinant type I collagen with a triple helix structure and its applications. Background Technology

[0002] Collagen has a triple helix structure, in which three polypeptide chains coil together in a spiral shape. Each polypeptide chain consists of a repeating triplet amino acid sequence of Gly-XY, where X and Y can be any amino acid. The most common triplet is proline-hydroxyproline-glycine (Gly-Pro-Hyp), accounting for approximately 10.5% of collagen triplets. Collagen is widely found in tissues such as skin, bone, and muscle, and is a major structural protein in the extracellular matrix of mammalian cells, playing various physiological functions in regulating cell proliferation, differentiation, and migration. Due to its excellent biological properties, collagen is widely used in tissue engineering, clinical medicine, the food industry, packaging materials, cosmetics, and medical aesthetics.

[0003] In recent years, collagen has been widely used in the medical field, such as as a component of biocompatible materials for artificial organ grafts, assisting in sustained drug release, artificial skin, and wound treatment matrices, as well as tissue fillers.

[0004] Many collagens currently used in medical devices are derived from non-human mammal species such as cattle or pigs. Reports indicate that patients may experience allergic reactions when these collagens are transplanted into humans. Furthermore, the risk of prion or pathogen contamination with collagen from non-human mammal species has become a major concern.

[0005] With the rise of the synthetic biology industry, researchers have turned their attention to systems for producing safe human collagen. For example, using mammalian cells, yeast, or E. coli to produce recombinant collagen. However, currently available recombinant collagen does not perform well due to two main reasons: firstly, it lacks a sophisticated triple helix structure; secondly, it is easily degraded, has poor function, and low yield, making it unsuitable for industrialization. Summary of the Invention

[0006] In view of this, the first objective of the present invention is to provide a recombinant type I collagen with a triple helix structure. The recombinant collagen provided by the present invention has an advanced structure, high purity, and good thermal stability.

[0007] Specifically, the recombinant type I collagen of the present invention is obtained by connecting and repeatedly tandemly linking multiple truncated regions. For example, the recombinant collagen of the present invention is obtained by truncating human type I collagen between 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or 1-2 amino acids.

[0008] The present invention discloses a recombinant type I collagen whose amino acid sequence contains n repeating units, where n is an integer of 1 or greater than 1, and the repeating units contain the fragment shown in GPPGPPGPAGKE (SEQ ID NO: 1).

[0009] Preferably, the repeating unit is formed by linking multiple amino acid residues to the N-terminus and / or C-terminus of GPPGPPGPAGKE (SEQ ID NO: 1).

[0010] The value of n is preferably selected from any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and more preferably from any one of 8, 9, 10, 11, 12, 13, 14, 15, 16.

[0011] The n repeating units are repeated in series, either by connecting the repeating units head to tail or by connecting the repeating units in series by a linker. The linker preferably contains glycine (Gly) and / or serine (Ser) residues, and preferably contains 1-5 amino acid residues.

[0012] Preferably, the repeating unit is formed by connecting multiple amino acid residues to the N-terminus and / or C-terminus of GPPGPPGPAGKE. Further, the amino acid sequence formed by the tandem repeating units of the present invention may also have multiple amino acid residues spliced ​​at its head (N-terminus) and tail (C-terminus), preferably 1-6 amino acids.

[0013] More preferably, the repeating unit is

[0014] X1X2X3GPPGPPGPAGKE、

[0015] X1X2X3X4X5X6GPPGPPGPAGKE、

[0016] GPPGPPGPAGKEX7X8X9,

[0017] GPPGPPGPAGKEX7X8X9X 10 X 11 X 12 ,

[0018] X1X2X3GPPGPPGPAGKEX7X8X9、

[0019] X1X2X3X4X5X6GPPGPPGPAGKEX7X8X9,

[0020] X1X2X3GPPGPPGPAGKEX7X8X9X 10 X 11 X 12 ,

[0021] Or X1X2X3X4X5X6GPPGPPGPAGKEX7X8X9X 10 X 11 X 12 Among them, X1, X4, X7, X 11 For glycine residues, X2, X3, X5, X6, X8, X9, X 11 X 12 It can be any amino acid residue.

[0022] Preferably, the amino acid sequence of the repeating unit is as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

[0023] In one embodiment, the recombinant type I collagen is named LY101 when the amino acid sequence shown in SEQ ID NO.2 is repeated 8 times and LY101-1 when repeated 16 times.

[0024] In one embodiment, the recombinant type I collagen is named LY102 when the amino acid sequence shown in SEQ ID NO.3 is repeated 8 times and LY102-1 when repeated 16 times.

[0025] In one embodiment, the recombinant type I collagen is named LY103 when the amino acid sequence shown in SEQ ID NO.4 is repeated 8 times and LY103-1 when repeated 16 times.

[0026] In one embodiment, the recombinant type I collagen is named LY111 when the amino acid sequence shown in SEQ ID NO.5 is repeated 8 times.

[0027] In one embodiment, the recombinant type I collagen is named LY131 when the amino acid sequence shown in SEQ ID NO.6 is repeated 8 times.

[0028] In one embodiment, the recombinant type I collagen is named LY137 when the amino acid sequence shown in SEQ ID NO.7 is repeated 8 times.

[0029] It should be understood that this invention essentially protects the amino acid sequence of recombinant type I collagen. Therefore, the mass production of proteins containing repeating unit sequences in the form of single-chain structures still falls within the protection scope of this invention.

[0030] In this invention, the recombinant type I collagen can be used as a domain to connect with domains from other sources to form a fusion protein, thereby constructing and expressing a novel target protein with multiple functions; it can also be linked with tags that help with protein expression, purification or detection.

[0031] In this invention, the introduction of N-terminal amino acids into the protein can be carried out in accordance with the method described in CN117511978A, which enhances the stability of the RNA secondary structure, slows down the protein translation rate, and allows the peptide chain to better form the target protein, thereby increasing the yield of exogenous protein.

[0032] In this invention, the amino acid introduced at the C-terminus of the protein can be an amino acid derived from human type I collagen.

[0033] Collagen, as a major component of the natural extracellular matrix, provides excellent support for cells through its unique triple helix structure and collagen fibers, creating a suitable microenvironment for cell proliferation and migration. However, current animal-derived collagen often suffers from varying degrees of damage to its natural cross-linked structure due to complex extraction methods or the introduction of excessive chemical reagents. Furthermore, many commercially available recombinant collagens fail to form higher-order structures and lack sufficient mechanical strength, making them easily hydrolyzed by proteases in vivo. Therefore, this invention addresses this issue by optimizing the structure and properties of collagen, such as improving thermal stability to ensure the raw material possesses sufficient mechanical strength and remains "inert" to proteases.

[0034] This invention characterizes the structure of collagen using circular dichroism (CD), a commonly used spectroscopic method in the field. CD is used to determine the structure of compounds with chiral structures that produce differential absorption between left and right rotations, and is mainly used to determine the asymmetry of molecular structures. Most biological macromolecules contain chiral groups and structures; therefore, CD is often used to measure and observe changes in the structure and conformation of biological macromolecules. The CD characteristic of the triple helix structure of collagen generally shows a positive absorption peak near 221 nm and a negative absorption peak near 195 nm (industry standard YY / T1849—2022). The positions of the absorption peaks shift with changes in the amino acid sequence and length. The thermal stability of collagen includes the thermal shrinkage temperature (Ts) of collagen fibers and the thermal denaturation temperature (Td) of collagen. The thermal shrinkage temperature of collagen fibers refers to the temperature at which collagen fibers undergo axial contraction when heated. The thermal denaturation temperature of collagen refers to the temperature at which the triple helix structure of collagen unwinds and forms individual single chains when heated in a medium to a certain temperature, reaching 50% unwinding. Therefore, CD spectroscopy can be used to study the helical structure of collagen and its thermal denaturation process.

[0035] This invention provides a gene encoding the recombinant type I collagen described above.

[0036] The encoding includes proteins produced by transcription of DNA molecules to form an RNA product, followed by translation; or proteins produced by transcription of DNA molecules to provide an RNA product, processing to provide a processed RNA product, followed by translation.

[0037] The present invention provides a recombinant vector expressing the recombinant type I collagen, comprising the aforementioned gene.

[0038] Vectors include any nucleic acid molecule derived from any source and capable of genome integration or autonomous replication (e.g., plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules), comprising nucleic acid molecules operatively linked with one or more nucleic acid molecules. Vectors may include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the host cell genome. Preferably, the vectors include, but are not limited to, pPICZαA and pPIC9k.

[0039] In this invention, the vector can be cloned with the recombinant human type I collagen gene alone, or it can be cloned with the proline hydroxylase gene and / or the lysine hydroxylase gene and the recombinant human type I collagen gene on the same vector.

[0040] The present invention provides recombinant cells expressing the recombinant type I collagen, wherein the recombinant cells are transfected with the recombinant vector.

[0041] The recombinant cells include, but are not limited to, Escherichia coli, yeast, mammalian cells, and plant cells.

[0042] The *E. coli* strain is BL21(DE3). The recombinant type I collagen encoding gene can be expressed alone or co-expressed in *E. coli* with the viral lysyl hydroxylase L230 gene, proline hydroxylase L593 gene, and / or *Bacillus anthracis* P4H gene. The yeast strain is *Pichia pastoris* X-33 or GS115. The mammalian cells can be Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK293) cells, or the fibrosarcoma cell line HT1080.

[0043] Methods for obtaining transduced cells expressing protein molecules by introducing a vector into other host cells are known. Similar methods can be used in this invention.

[0044] A second objective of the present invention is to provide a method for expressing the recombinant type I collagen.

[0045] In a preferred embodiment, the recombinant type I collagen is expressed in Pichia pastoris GS115, comprising the following steps:

[0046] 1) The gene was ligated into the vector pPICZαA to obtain a recombinant vector;

[0047] 2) The recombinant vector was transfected into Pichia pastoris GS115 to obtain recombinant cells;

[0048] 3) After culturing the recombinant cells, centrifuge and collect the supernatant;

[0049] 4) The recombinant type I collagen was obtained by separating it from the supernatant.

[0050] A third objective of this invention is to provide the application of the recombinant type I collagen and the recombinant type I collagen prepared by the aforementioned method in the preparation of medical devices or daily chemical products.

[0051] Currently, approximately 29 types of collagen are reported to exist in vertebrates. Based on primary structure, length of triple helix domains, molecular weight, size and shape of triple helix interruptions and terminal domains, collagen can be divided into four major categories: Category 1, fibrous collagen, includes types I, II, III, V, XI, XXIV, and XXVII. Types I, II, and III are more abundant in vertebrates, while types V and VI are less abundant but play an auxiliary role in the assembly of types I, II, and III. Category 2, triple helix fibrous collagen (FACIT), includes types IX, XII, XIV, XVI, XIX, and XXII. FACIT collagen does not form collagen, but it can interact with fibrillary collagen to regulate the formation and size of collagen fibers and control collagen synthesis in the extracellular matrix; the third type is reticular collagen, including types IV, VII, and XXVIII. Type IV collagen forms a fibrous network structure, while type VII collagen assembles into anchoring fibers that connect the epidermis to the dermis; the fourth type is discontinuous helical collagen (MACITs), including types XIII, XXIII, and XXV (Ye et al., 2023).

[0052] Based on this, the recombinant type I collagen of the present invention can be used in combination with one or more of recombinant or natural type I fibrous collagen, type II triple-helix fibrous collagen, type III reticular collagen and type IV discontinuous helix collagen. More preferably, it can be prepared and used by fusing expression with bioactive components such as type III collagen, type IV collagen, type VII collagen, type XVII collagen, fibronectin, and human epidermal growth factor. It can also be used by mixing with the above-mentioned bioactive components after separate preparation.

[0053] Subcutaneous injection filler

[0054] Subcutaneous filler injection is a technique that involves injecting filler material percutaneously into appropriate locations to achieve cosmetic results. Ideal soft tissue fillers should possess good safety, biocompatibility, and effectiveness. Currently, facial fillers primarily utilize non-autologous tissue injectables, such as single-component fillers like hyaluronic acid (HA) and collagen. Collagen is widely used in aesthetic medicine to repair skin defects and subcutaneous diseases. Localized facial collagen injections can achieve effects such as facial contour correction, wrinkle reduction, and scar repair.

[0055] Medical repair materials

[0056] Collagen materials possess a natural porous network structure, which allows them to rapidly absorb blood upon contact with the wound, promoting platelet aggregation. Simultaneously, they absorb tissue exudate, creating a favorable wound microenvironment and accelerating wound healing. Currently, there are numerous collagen-based soft tissue repair products available clinically, including collagen dressings, dura mater patches, artificial corneas, oral patches, hernia patches, and breast augmentation patches. Their applications include, but are not limited to, wound healing, skin repair, dermatitis, eczema, hemorrhoids, oral mucositis, oral ulcers, scars, and allergic rhinitis.

[0057] Based on this, the present invention provides a method for promoting wound repair. The method includes identifying tissue requiring repair, such as collagenous tissue (e.g., cornea, skin, bone), connective tissue (e.g., cartilage, ligaments, tendons, or basement membrane); and contacting the tissue with an agent that increases type I collagen. Wounds can be caused by trauma (e.g., connective tissue or muscle tears), incisions, or surgeries (e.g., transplants), such as heart transplants, lens transplants, joint replacements, hair follicle replacements, and skin grafts. In one scenario, the wound is used to repair wounds present in the eye, such as surgical incisions, corneal transplants, LASIK flap reconnection, cataract surgery, laser surgery, corneal transplants, penetrating keratoplasty, posterior lamellar keratoplasty, refractive surgery, corneal remodeling, or treatment of corneal lacerations. For example, the wound may be present in heart tissue, a heart valve, or further, as a result of heart valve replacement or transplantation. For example, the wound may be present in cartilage, tendons, or ligaments. For example, the wound may be present in epithelial tissue, such as in the dermis.

[0058] In one specific case, collagen sponge artificial dura mater was applied to patients with post-traumatic epilepsy. It was found that the collagen sponge artificial dura mater could effectively prevent cerebrospinal fluid leakage, had a good hemostatic effect, and did not increase the incidence of epilepsy. No infection or rejection reaction occurred in the patients after the operation, indicating that it has good tissue compatibility and can be used for intracranial tumors (including gliomas, meningiomas, pituitary adenomas, schwannomas and other congenital tumors, metastatic cancers, etc., intracranial aneurysms and vascular malformations, spinal tumors, and other lesions, germ cell tumors, epileptic foci, colloid cysts, inflammatory granulomas, etc. (Qin Guoqiang et al., 2014).

[0059] Medical transplant materials

[0060] Tissue engineering applies engineering and life science principles to the research of biological substitutes. It utilizes seed cells, scaffolds, and growth factors to construct tissues and organs similar to those of the human body, which are then transplanted back into the host's deficient or missing tissue or organ sites to form new tissues that closely resemble normal tissues. This restores the defective shape or partial function, achieving the goal of regenerating deficient tissues and organs. Currently, tissue engineering technology research has achieved certain results in the study of tissues and organs such as skin, liver, and kidneys. In another preferred embodiment, the transplantation device is an artificial device, such as a non-biological or semi-biological device (e.g., a device containing both biological and non-biological components). For example, the transplanted material can be an organ or part thereof, such as an artificial eye or lens, an artificial heart or heart valve, an artificial hair follicle, or artificial muscle; it can be artificial connective tissue, such as artificial cartilage, tendon, ligament, or basement membrane tissue; artificial bone tissue; an artificial tooth; or a prosthesis. In a preferred embodiment, the transplanted tissue is eye tissue, such as a lens; heart tissue, such as a heart valve; cartilage tissue, such as a tendon; muscle tissue; hair follicle; or epithelial tissue, such as a skin graft.

[0061] Type I fibrillary collagen is the main collagen in the heart muscle, accounting for 85% of total collagen, while type III accounts for 11%. Type I collagen or functional fragments thereof may help treat diseases of heart valves or other areas of the heart, or to create, treat, or maintain transplantable bioequivalents of parts of the heart. Type I collagen may help improve the success rate of such transplants, extend the lifespan of the heart and its valves, or improve the success rate of cardiac surgery.

[0062] Because type I collagen is expressed at the muscle-connective tissue boundary of the eye, type I collagen or functional fragments thereof may help treat eye diseases or create, treat, or maintain transplantable bioequivalents of parts of the eye. Type I collagen can be used to improve the success rate of such transplants, such as lens replacement surgery, or to extend the lifespan of the lens and its tissues, or to increase the success rate of ophthalmic surgeries, such as treating surgically induced endophthalmitis.

[0063] Type I collagen is the most common type of collagen and is essential for the structural integrity of various tissues. It is present in almost all connective tissues and is a major component of the interstitial matrix. It is currently used as a scaffold material in bone tissue engineering methods.

[0064] Mature dentin contains approximately 70% hydroxyapatite, 18% collagen, 2% non-collagenous proteins, and 10% water. COLⅠ (Collagen-Oxygen Composite) is the scaffold structure of the dentin matrix, combining collagen with hydroxyapatite to form a hydroxyapatite-mineralized collagen complex, which can be used as a osteoconductive coating and scaffold. For example, in dental restorations, collagen fills periodontal bone defects, accelerating periodontal bone hyperplasia and gingival regeneration. It is also the most abundant matrix protein in bone tissue.

[0065] Daily chemical products

[0066] Compositions containing recombinant collagen can be applied topically to the skin to reduce skin damage or promote the repair of damaged skin. For example, the compositions can protect the skin from UV damage, protect skin cells from the effects of exposure to oxidation, promote the repair of damaged skin by increasing cell vitality and / or increasing procollagen synthesis when applied to the skin, and / or promote the vitality of skin cells.

[0067] Based on this, the present invention can select a composition comprising at least one of the aforementioned proteins in a cosmetically or pharmaceutically effective amount, and at least one excipient or cosmetically or pharmaceutically acceptable adjuvant, according to practical applications. The dosage form of the composition includes, but is not limited to, creams, lotions, aqueous solutions, gels, oils, powders, muds, patches, films, or freeze-dried products; furthermore, to promote transdermal absorption of collagen molecules, patch products can be prepared using solid carriers such as non-woven fabrics and applied to the skin surface to prolong the action time of the collagen solution.

[0068] From the perspective of end products, the collagen of the present invention can be applied to daily chemical products. The composition containing recombinant collagen may also contain at least one additional ingredient, which includes other active ingredients, topical carriers and / or preservatives. Other active ingredients include sodium hyaluronate, tocopherol (vitamin E), tocopheryl acetate, citric acid, niacinamide panthenol, centella asiatica extract, squalane, jojoba seed oil, butyrospermum parkii fruit butter, glycyrrhiza glabra root extract, tetrahydromethylpyrimidine carboxylic acid, bifida ferment lysate, salicylic acid, hydroxypropyl tetrahydropyrantriol, retinol, purslane extract, tranexamic acid, meadowfoam seed oil, retinyl palmitate, trehalose, ceramide NP, 3-o-ethyl ascorbic acid, dipotassium glycyrrhizate, olive leaf extract, allantoin, bisabolol, and helianthus. Annuus seed oil, ascorbyl palmitate, acetyl hexapeptide-8, arginine, olive (olea europaea) fruit oil, ascorbic acid (vitamin C), sweet almond (prunus amygdalus dulcis) oil, ascorbyl glucoside, hydrogenated lecithin, glycyrrhiza inflata root extract, sour orange (citrus aurantium dulcis) peel oil, tea (camellia sinensis) leaf extract, rosadamascena flower water, phenylethyl resorcinol, carnosine, ascorbyl tetraisopalmitate, bifida ferment filtrate, grape (vitis vinifera) seed oil, rosa damascena flower oil, aloe barbadensis leaf water, camellia (camellia ajaponica) seed oil, galactosomalidase ferment filtrate, adenosine, grapefruit (citrus) Paradisi fruit peel oil, bergamot (Citrus aurantium bergamia) fruit oil, erythritol, tripeptide-1 copper, Melaleuca alternifolia leaf oil, licorice (Glycyrrhiza uralensis) root extract, dipeptide diaminobutyryl benzylamide diacetate, α-arbutin, asiaticoside, ergothionein.Topical carriers include those selected from liposomes, biodegradable microcapsules, emulsions, sprays, aerosols, powders, biodegradable polymers, mineral oils, triglyceride oils, silicone oils, glycerin, glyceryl monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, waxes, sorbitan monostearate, polysorbate, cetyl wax, cetearyl alcohol, 2-octyldodecanool, benzyl alcohol, cyclomethyl silicone oil, cyclopentadioxane, and water. Preservatives include those selected from tocopherol, diiodomethyl-p-tolyl sulfone, 2-bromo-2-nitropropane-1,3-diol, cis isomer 1-(3-chloroallyl)-3,5,7-triaza-1-azamonoadamantane chloride, glutaraldehyde, 4,4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, Germaben II, rosemary extract, and EDTA.

[0069] Specifically, the composition containing recombinant collagen is a personal care product that may contain other cosmetic ingredients suitable for human use. This personal care product can be used to prevent or treat damage to human skin or hair caused by external environmental factors. Applications can include skin cleansing and repair products, hair cleansing and repair products, such as face masks, serums, creams, soaps, cleansing pastes, cleansing milks, facial cleansers, shampoos, conditioners, lotions, and shower gels.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] 1) The recombinant type I collagen of the present invention is based on the original amino acid sequence of human type I collagen. Its molecular weight is significantly smaller than that of natural full-length collagen (300kDa). It has optimized functional active sites to fully exert biological functional activity.

[0072] 2) The recombinant type I collagen of this invention has a triple helix structure consistent with natural collagen and has thermal stability higher than human body temperature, which is beneficial to industrialization and downstream applications; it has good biocompatibility and is an excellent material for medical fillers. Attached Figure Description

[0073] Figure 1 This is an SDS-PAGE image of the purified recombinant type I collagen LY101, LY101-1, LY102, LY102-1, LY103, LY103-1, LY111, LY131, and LY137 of this invention.

[0074] Figure 2 The circular dichroism chromatograms of the recombinant type I collagen LY101, LY101-1, LY102, LY102-1, LY103, LY103-1, LY111, LY131, and LY137 of this invention at room temperature are shown.

[0075] Figure 3 The circular dichroism chromatograms of the recombinant type I collagen LY101, LY101-1, LY102, LY102-1, LY103, LY103-1, LY111, LY131, and LY137 of this invention at different temperatures are shown.

[0076] Figure 4 The results of in vitro enzymatic hydrolysis experiments of recombinant type I collagen LY111, LY131, and LY137 of this invention are shown. Detailed Implementation

[0077] In this invention, the gene can be synthesized by a biotechnology company. This invention does not impose any particular limitation on the preparation method of the recombinant vector and recombinant cells; conventional methods for preparing recombinant vectors and recombinant cells in the art can be used.

[0078] The present invention does not specifically limit the separation and purification method; conventional protein separation and purification methods in the art can be used. Preferred technical solutions are described in the embodiments.

[0079] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0080] Example 1: Expression, isolation, and purification of recombinant type I collagen of the present invention

[0081] 1. Sequence synthesis

[0082] GenScript Biotech Inc. was commissioned to synthesize gene fragments of the recombinant type I collagen (LY101, LY101-1, LY102, LY102-1, LY103, LY103-1, LY111, LY131, LY137) of this invention and construct them into the pPICZalphaA Pichia pastoris expression vector.

[0083] 2. Construction of Pichia pastoris expression system

[0084] 2.1 Electric Rotor

[0085] Plasmid pPICZalphaA was amplified by PCR and linearized at the SacI site, followed by PCR fragment recovery (ThermoGeneJET PCR Purification Kit). Competent GS115 cells were thawed on ice, and 5-10 μg of the linearized plasmid was added. The cells were incubated on ice for 15 min, and the plasmid and competent cells were added together to a pre-chilled electroporation cuvette. Immediately after electroporation, 1 ml of pre-chilled sorbitol was added, and the mixture was transferred to a sterile 1.5 ml centrifuge tube and activated at 30°C and 230 rpm for 2 h. The cells were centrifuged at 5000g for 1 min, 300 μl of supernatant was discarded, and the cells were resuspended and plated on YPDSZ agar plates (tryptone 20 g / L, yeast extract 10 g / L, D-sorbitol 182.1 g / L, 20% glucose, 100 μg / ml bleomycin) and incubated at 30°C and 230 rpm for 48 h.

[0086] 2.2 Remove the plate containing colonies, pick out single colonies, and copy them onto YPD solid medium with high concentration (400 μg / ml or 800 μg / ml) bleomycin for screening.

[0087] 2.3 Remove the plates containing colonies, pick single colonies, and inoculate them into 50 ml of YPD liquid medium (containing 100 μg / ml bleomycin) for overnight seed culture at 30°C and 230 rpm. Inoculate the overnight seed culture into 1 L of BMGY medium at a 1:50 ratio for scale-up. Incubate at 30°C and 230 rpm for 48 h. Centrifuge the BMGY seed culture at 3000 rpm for 5 min, discard the supernatant, resuspend in sterile water, centrifuge again at 3000 rpm for 5 min, discard the supernatant, resuspend in 1 L of BMMY medium (without methanol), and add 1% methanol for induction. After 72 h of induction, centrifuge and collect the supernatant for purification.

[0088] 2.4 Ion exchange purification

[0089] The target protein was dialyzed into buffer A (20 mM PB, pH 4.0). The protein sample was filtered through a 0.22 μm pore size and 13 mm diameter filter membrane, followed by cation exchange using an AKTApure column (5 ml SP column). Linear elution with buffer B (20 mM PB and 1 M NaCl, pH 7.4) yielded a highly pure target protein. SDS-PAGE was used to verify the protein's molecular weight and purity. Protein loading buffer (with added DTT) was added to the protein sample, mixed well, and placed in a metal bath. The mixture was heated at 95°C for 10 min, centrifuged at 1500 rpm for 1 min, and the supernatant was analyzed by SDS-PAGE. The results are shown below. Figure 1As shown. Protein concentration was determined by BCA method to estimate protein yield. Based on protein purity, concentration, liquid replacement, and lyophilization were performed for preservation.

[0090] Example 2: Circular dichroism (CD) detection of structural features of recombinant type I collagen

[0091] The recombinant type I collagen lyophilized powder prepared in Example 1 was dissolved in 20 mM PB buffer to a concentration of 0.5 mg / ml. The sample was further diluted to a concentration of 0.01 mg / ml and a volume of 3 ml. The sample was transferred to a 10 mm × 10 mm sample cell of a circular dichroism chromatograph. The scanning wavelength range was set to 190 nm–260 nm, the scanning speed to 100 nm / min, and the scanning temperature to room temperature. The CD spectra were the average of three scans. Collagen has three left-handed α-helical peptide chains, which are wound in parallel, right-handed helical configurations to form a "rope-like" triple helix structure. This structure exhibits a characteristic absorption spectrum in the far ultraviolet region (185–250 nm), with a negative peak around 195 nm and a positive peak around 221 nm. The results are as follows: Figure 2 As shown, the recombinant type I collagen of the present invention has a maximum characteristic positive peak at 221 nm and a negative peak at 195 nm. Based on the known circular dichroism chromatographic characteristics of the triple helix structure of collagen, the recombinant type I collagen samples LY101, LY101-1, LY102, LY102-1, LY103, LY103-1, LY111, LY131, and LY137 of the present invention all have a triple helix structure.

[0092] The Circular Dichroism (RPN) value is calculated by measuring the CD value (circular dichroism) of a collagen solution at a specific wavelength. Specifically, the RPN value is the ratio of the absolute values ​​of the positive peak intensity to the negative peak intensity, i.e., RPN = positive peak intensity / |negative peak intensity|. Changes in the RPN value can reflect changes in collagen structure to some extent. When the triple helix structure of collagen is significantly disrupted, the RPN value drops to 0 or even negative. For example, the RPN value of fully denatured collagen or gelatin is 0 or even negative. During heating, the triple helix structure of collagen is affected, leading to changes in the RPN value. The main reason is that increased temperature affects the hydrogen bonds within the collagen molecule, thus affecting its conformation. The RPN value can be used to assess the structural integrity and degree of denaturation of collagen. By monitoring changes in the RPN value, the structural stability of collagen under different conditions (such as heating, irradiation, etc.) can be understood.

[0093] The RPN values ​​of the recombinant type I collagen samples LY101, LY101-1, LY102, LY102-1, LY103, LY103-1, LY111, LY131, and LY137 of the present invention are shown in Table 1.

[0094] Table 1

[0095]

[0096]

[0097] Example 3: Real-time thermal stability of recombinant type I collagen determined by circular dichroism (CD) chromatography.

[0098] The collagen sample was diluted according to the method described in Example 2, and the thermal stability of the protein was tested by real-time temperature increase at a rate of 1℃ / min. The CD spectra were the average of three scans, and the data were analyzed and calculated using software. The results are as follows: Figure 3 As shown, based on the disappearance of the characteristic positive peak at 221 nm, the thermal stability of the recombinant type I collagen samples LY101 and LY101-1 of this invention is 33℃ and 35℃, respectively; the thermal stability of the recombinant type I collagen samples LY102 and LY102-1 of this invention is 41℃ and 42℃, respectively; the thermal stability of the recombinant type I collagen samples LY103 and LY103-1 of this invention is 44℃ and 32℃, respectively; and the thermal stability of the recombinant type I collagen samples LY111, LY131, and LY137 of this invention is 39℃, 40℃, and 34℃, respectively. Among these, the real-time thermal stability of LY102, LY102-1, LY103, LY111, and LY131 is higher than human body temperature, indicating that the triple helix structure formed by the protein is tightly wound, which is beneficial for use as implantation, filling, and other related materials.

[0099] Example 4: In vitro anti-degradation experiment to detect the stability of recombinant type I collagen

[0100] Recombinant type I collagen lyophilized powders (LY111, LY131, LY137) prepared in Example 1 and commercially available bovine type I collagen (animal-derived extract) were dissolved in PBS buffer to prepare solutions with a concentration of 0.5 mg / mL, and allowed to equilibrate to room temperature. Trypsin (purchased from Promega) was added to each collagen solution to make the enzyme-to-substrate mass ratio 1:200. The mixtures were incubated in constant temperature water baths at 30°C and 37°C, and samples were taken at time points of 0 min, 30 min, 60 min, 120 min, and 240 min. 50 μL of the reaction solution was taken, and protein loading buffer containing DTT was added. The mixture was immediately vortexed and mixed. The sample was heated in a metal bath at 95°C for 10 min, followed by centrifugation at 1500 rpm for 1 min. The supernatant was collected for later use. 10 μL of the supernatant was used for SDS-PAGE electrophoresis (conditions: constant voltage 160 V, electrophoresis time 40 min). Observe the band distribution after Coomassie brilliant blue staining.

[0101] like Figure 4 As shown, the recombinant type I collagen LY111 and LY131 of this invention still showed clear target protein bands after 240 minutes of trypsin digestion, while commercially available bovine type I collagen was completely degraded after 1 hour, leaving no residual bands. The results indicate that the recombinant collagen prepared by this invention has a certain resistance to trypsin degradation compared to animal-derived products, making it suitable for biomedical applications requiring long-term stability (such as tissue engineering scaffolds or sustained-release formulations).

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant type I collagen, characterized in that, Its amino acid sequence contains n repeating units, where n is an integer of 1 or greater than 1, and the repeating units contain the GPPGPPGPAGKE (SEQ ID NO: 1) fragment.

2. The recombinant type I collagen according to claim 1, characterized in that, The repeating unit is formed by linking multiple amino acid residues to the N-terminus and / or C-terminus of GPPGPPGPAGKE (SEQ ID NO: 1).

3. The recombinant type I collagen according to claim 1, characterized in that, The amino acid sequence of the repeating unit is shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:

7.

4. The recombinant type I collagen according to claim 1, characterized in that, The value n is selected from any one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

5. The recombinant type I collagen according to claim 1, characterized in that, The value of n is any one of 8, 9, 10, 11, 12, 13, 14, 15, and 16.

6. The gene encoding the recombinant type I collagen as described in claim 1.

7. A recombinant vector expressing the recombinant type I collagen according to claim 1, characterized in that, It contains the gene described in claim 5.

8. Recombinant cells expressing the recombinant type I collagen of claim 1, characterized in that, The recombinant cells contain the recombinant vector as described in claim 7.

9. The method for preparing recombinant type I collagen according to claim 1, characterized in that, Includes the following steps: 1) The gene described in claim 6 is recombined into the vector pPICZαA to obtain a recombinant vector; 2) The recombinant vector was transfected into Pichia pastoris (Komagataella phaffii) to obtain recombinant cells; 3) After culturing the recombinant cells, centrifuge and collect the supernatant; 4) The recombinant type I collagen was obtained by separating it from the supernatant.

10. The application of the recombinant type I collagen according to claim 1 and the recombinant type I collagen prepared by the preparation method according to claim 9 in the preparation of medical devices or daily chemical products.

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