Preparation and application of improved recombinant type III collagen peptides

Abstract

This invention provides the preparation and application of an improved recombinant type III collagen peptide, wherein the collagen contains the amino acid sequence shown in SEQ ID NO.1, and a tag sequence is fused to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1. The recombinant collagen of this invention contains only a small affinity tag during expression, facilitating purification and eliminating the need for enzymatic digestion, greatly simplifying the process steps and reducing process costs while maintaining the excellent characteristics of the original recombinant collagen peptide Co8 (SEQ ID NO.1), still exhibiting high expression levels, high hydrophilicity, and high stability.

Description

Invention Field

[0001] This invention belongs to the field of genetic engineering technology. Specifically, this application provides the preparation and application of improved recombinant type III collagen peptides. Background Technology

[0002] Collagen is a fibrous protein composed of three intertwined peptide chains, accounting for 25%-33% of the body's protein content. It is mainly found in the skin, muscles, and bones, providing tensile strength and flexibility to tissues and organs. It also participates in various vital activities within the body, including tissue formation and maturation, intercellular communication, movement, immunity, joint lubrication, blood pressure control, wound healing, tissue repair, and organ aging. Due to its superior efficacy and characteristics, collagen is widely used in food, medicine, biomaterials, beauty and skincare, and animal feed. With the expanding demand for skincare, beauty, and nutritional supplements in China, the collagen market is poised for continued growth.

[0003] Currently, collagen is mainly extracted from animal sources, primarily pigs, cattle, and fish, and mainly includes type I, II, and III collagen. The processing technology is relatively mature and the cost is low, but there are risks of immune rejection and allergies. Recombinant collagen peptides, with their composition and structure, are more easily absorbed by the human body. Although the cost is higher, they possess good biocompatibility, low immunogenicity, and the ability to promote tissue regeneration, and have gained widespread market and consumer acceptance.

[0004] Due to their large molecular weight and poor water solubility, full-length collagen sequences typically cannot be directly expressed recombinantly. Currently available or under development recombinant collagen peptides are mainly peptide sequences designed by truncation and splicing based on natural sequences. These peptides often need to possess good hydrophilicity, stability, and biological activity, as well as high expression levels suitable for industrial production. Therefore, the sequence screening and design of recombinant collagen peptides, and the acquisition of superior industrial strains, are technical challenges in this field, often representing a significant technological hurdle for related companies.

[0005] Currently, recombinant collagen peptides contain long fusion peptides (tens or hundreds of amino acids) during expression. Direct application in products may trigger an immune response. Therefore, the fusion tag needs to be removed during the preparation process, and a second affinity chromatography is required to remove the TEV protease and the removed fusion peptide. This undoubtedly increases the complexity of the process, production costs, and process difficulty.

[0006] Therefore, there is an urgent need in this field to develop an improved recombinant type III collagen peptide that contains only a small affinity tag during expression, which is easy to purify and does not require enzymatic digestion. This would greatly simplify the process steps, reduce the process cost, and maintain the excellent properties of the original recombinant collagen peptide, while still having high expression levels, high hydrophilicity, and high stability. Summary of the Invention

[0007] The purpose of this invention is to provide an improved recombinant type III collagen peptide that contains only a small affinity tag during expression, which is easy to purify and does not require enzymatic digestion. This greatly simplifies the process steps, reduces the process cost, and maintains the excellent characteristics of the original recombinant collagen peptide, while still having high expression level, high hydrophilicity, and high stability.

[0008] In a first aspect, the present invention provides a recombinant collagen protein, characterized in that the collagen protein comprises the amino acid sequence shown in SEQ ID NO.1, and a tag sequence is fused to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0009] In another preferred embodiment, the tag sequence is selected from the group consisting of: Strep-Tag II tags, 6×His tags, 8×His tags, FLAG-tags, Poly-Arg tags, Twin strep tags, or combinations thereof.

[0010] In another preferred embodiment, the collagen has the structure shown in Formula I from the N-terminus to the C-terminus:

[0011] Z0-L-Z1-L-Z2(I)

[0012] In the formula,

[0013] Z0 is either an unlabeled or first-labeled sequence;

[0014] L represents an empty or linked peptide;

[0015] Z1 is the amino acid sequence shown in SEQ ID NO.1;

[0016] Z2 is a sequence without a second tag or a second tag sequence;

[0017] "-" is the key;

[0018] Among them, Z0 exists and Z2 can be non-existent, or Z2 exists and Z0 is non-existent, or Z0 and Z2 exist simultaneously.

[0019] In another preferred embodiment, the first tag sequence and the second tag sequence are each independently selected from the group consisting of: Strep-Tag II tags, 6×His tags, 8×His tags, FLAG-tags, Poly-Arg tags, Twin strep tags, or combinations thereof.

[0020] In another preferred embodiment, Z0 is a Strep-Tag II tag and Z2 is a 6×His tag.

[0021] In another preferred embodiment, Z0 is none, and Z2 is a 6×His label.

[0022] In another preferred embodiment, the collagen may be modified or unmodified.

[0023] A second aspect of the present invention provides an isolated polynucleotide encoding a recombinant collagen as described in the first aspect of the present invention.

[0024] In another preferred embodiment, the polynucleotide is a codon-optimized polynucleotide.

[0025] In another preferred embodiment, the nucleotide sequence is selected from the group consisting of:

[0026] (a) The nucleotide sequence is as shown in either SEQ ID NO.3 or 5;

[0027] (b) The nucleotide sequence has ≥95% identity with the sequence shown in either SEQ ID NO. 3 or 5, preferably ≥98%, more preferably ≥99%; and

[0028] (c) A nucleotide sequence complementary to the nucleotide sequence described in (a) or (b).

[0029] In another preferred embodiment, the nucleotide sequence includes a DNA sequence, a cDNA sequence, or an mRNA sequence.

[0030] In another preferred embodiment, the nucleotide sequence includes single-stranded and double-stranded sequences.

[0031] A third aspect of the present invention provides a carrier comprising the polynucleotides described in the second aspect of the present invention, and the carrier does not contain fusion peptide elements and linker peptides for fusion expression with a target protein.

[0032] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, viral vectors, transposons, or combinations thereof.

[0033] In another preferred embodiment, the vector comprises one or more promoters operatively linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectivity marker, nucleic acid restriction site, and / or homologous recombination site.

[0034] In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, SV40, poxvirus, or combinations thereof.

[0035] In another preferred embodiment, the carrier includes an expression carrier, a shuttle carrier, and an integration carrier.

[0036] In another preferred embodiment, the fusion peptide element includes, but is not limited to, TrxA, S-Tag, T7-Tag, enterokinase recognition site, and thrombin recognition site.

[0037] A fourth aspect of the present invention provides a host cell containing a vector as described in the third aspect of the present invention.

[0038] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell.

[0039] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0040] The fifth aspect of the present invention provides a method for preparing recombinant collagen as described in the first aspect of the present invention, comprising the steps of:

[0041] (i) Under suitable conditions, host cells as described in the fourth aspect of the present invention are cultured to obtain the recombinant collagen as described in the first aspect of the present invention; and

[0042] (ii) Optionally, the recombinant collagen obtained in step (i) may be enzymatically digested, purified, and / or isolated.

[0043] A sixth aspect of the present invention provides a composition comprising:

[0044] (I) The recombinant collagen according to the first aspect of the present invention; and

[0045] (II) Pharmaceutically acceptable carriers or cosmetically acceptable excipients.

[0046] In another preferred embodiment, the composition is used for one or more purposes selected from the group consisting of:

[0047] (a) Promotes cell proliferation;

[0048] (b) Promotes cell migration;

[0049] (c) Promotes cell adhesion;

[0050] (d) Promotes cell differentiation.

[0051] In another preferred embodiment, the cells include fibroblasts, epidermal cells, mucosal epithelial cells, dermal cells, and skin mesenchymal cells.

[0052] In another preferred embodiment, the composition further contains other substances that can (a) promote cell proliferation; and / or (b) promote cell migration; and / or (c) promote cell adhesion; and / or (d) promote cell differentiation.

[0053] In another preferred embodiment, the excipients acceptable for use in cosmetics are selected from the group consisting of: moisturizers, skin conditioning agents, thickeners, emollients, emulsifiers, antioxidants, preservatives, UV protectants, film-forming agents, oil-soluble gelling agents, organically modified clay minerals, resins, fragrances, salts, pH adjusters, conditioning agents, chelating agents, cooling agents, anti-inflammatory agents, skin-beautifying ingredients, vitamins, amino acids, nucleic acids, inclusion compounds, solvents (such as water), or combinations thereof.

[0054] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of liquid dosage forms, semi-solid dosage forms or solid dosage forms, more preferably ointments, creams, emulsions, oils, powders, solutions, gels, sprays, foams, suspensions, lotions or sticks.

[0055] In another preferred embodiment, the composition is a face cream, lotion, serum, cleanser, or essence.

[0056] In another preferred embodiment, the composition is a pharmaceutical composition.

[0057] In another preferred embodiment, the composition further includes excipients.

[0058] The seventh aspect of the present invention provides the use of the recombinant collagen described in the first aspect of the present invention for preparing a composition or product for (a) promoting cell proliferation; and / or (b) promoting cell migration; and / or (c) promoting cell adhesion; and / or (d) promoting cell differentiation.

[0059] In another preferred embodiment, the composition or product includes tissue-engineered products, cosmetics, health products, or pharmaceuticals.

[0060] An eighth aspect of the present invention provides a method for (a) promoting cell proliferation; and / or (b) promoting cell migration; and / or (c) promoting cell adhesion; and / or (d) promoting cell differentiation, comprising the steps of:

[0061] Apply an effective amount of the recombinant collagen of the first aspect of the present invention or the composition of the sixth aspect of the present invention to the desired object.

[0062] In another preferred embodiment, the application includes topical application to the skin, topical application to mucous membranes, and wound dressing.

[0063] In another preferred embodiment, the application helps to improve the skin barrier, smooth fine lines, moisturize, whiten and lighten spots, shrink pores, and reduce dark circles; repair the mucosal barriers of the nasal cavity, oral cavity, vagina, and anus; stop bleeding and promote wound healing, and prevent scar formation.

[0064] In another preferred embodiment, the object is a human or a non-human mammal.

[0065] In another preferred embodiment, the non-human mammal includes pet dogs, cats, monkeys, rabbits, rats, or mice.

[0066] A ninth aspect of the present invention provides a method for (a) promoting cell proliferation; and / or (b) promoting cell migration; and / or (c) promoting cell adhesion; and / or (d) promoting cell differentiation, comprising the steps of:

[0067] Cells are cultured in the presence of the recombinant collagen described in the first aspect of the present invention or the composition described in the sixth aspect of the present invention, thereby (a) promoting cell proliferation; and / or (b) promoting cell migration; and / or (c) promoting cell adhesion; and / or (d) promoting cell differentiation.

[0068] In another preferred embodiment, the cells include fibroblasts, epidermal cells, mucosal epithelial cells, dermal cells, and skin mesenchymal cells.

[0069] In another preferred embodiment, the cells are cells cultured in vitro.

[0070] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0071] On the other hand, this application provides the use of the above-mentioned recombinant collagen in the preparation of tissue-engineered products, cosmetics, health products, food or pharmaceutical compositions.

[0072] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0073] Figure 1 The assembly of the modified recombinant type III collagen peptide and its tag on the expression plasmid is shown.

[0074] Figure 2 The expression solubility of Co8-2, Co8-3 and Co8 is shown.

[0075] Figure 3 The expression levels of Co8, Co8-2, and Co8-3 in shake flasks were shown.

[0076] Figure 4 The expression of the comparative sequences Co11, F3, F4, F7, F8, F9, F48, and F51 on the pET-32a vector before and after the removal of the fusion peptide is shown.

[0077] Figure 5 The changes in purity of Co8-2 protein before and after purification are shown.

[0078] Figure 6 The stability of the Co8-2 protein was demonstrated.

[0079] Figure 7 The results of the recombinant collagen cell proliferation activity assay are shown in a micrograph.

[0080] Figure 8 The results of the cell proliferation activity test of recombinant collagen are shown – proliferation rate.

[0081] Figure 9 The results of the cell migration-promoting activity assay for recombinant collagen are shown.

[0082] Figure 10 The results of the cell adhesion-promoting activity test of recombinant collagen are shown.

[0083] Figure 11 The results of the DPPH radical scavenging rate test for Co8-2 are shown.

[0084] Figure 12 The results of the Co8-2 hyaluronidase inhibition rate test are shown. Detailed Implementation

[0085] Through extensive and in-depth research and screening, the inventors have developed a recombinant collagen for the first time. The recombinant collagen contains the amino acid sequence shown in SEQ ID NO.1, and a tag sequence is fused to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0086] Specifically, this invention designs a modified recombinant type III collagen peptide containing only a small affinity tag during expression, facilitating purification and eliminating the need for enzymatic digestion. This significantly simplifies the process, reduces costs, and maintains the excellent characteristics of the original recombinant collagen peptide Co8 (SEQ ID NO.1), retaining high expression levels, high hydrophilicity, and high stability. When expressing the modified protein, the production strain does not need to express long and useless fusion peptides, reducing ineffective conversion of bioenergy and thus improving bioconversion efficiency and increasing protein yield and output ratio through process optimization. Furthermore, this invention surprisingly discovers that the modified recombinant collagen peptide Co8-2 exhibits superior biological activity compared to Co8, demonstrating stronger cell proliferation and migration activity. Therefore, the modified Co8-2 has greater advantages in industrial scale-up and has better development value for applications in functional skincare products, health foods, and non-implantable medical devices. This invention was completed based on these findings.

[0087] In this application, sequence identity refers to identity calculated using algorithms known in the art, including but not limited to blast and GenPast.

[0088] In this application, 95% or more of the sameness includes, but is not limited to, integer percentages such as 95%, 96%, 97%, 98%, and 99%, as well as the sameness with decimal places that may be obtained based on the number of variation points or the algorithm, including but not limited to 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, and 99.1%.

[0089] The coding sequence of recombinant collagen can be designed and obtained by those skilled in the art using existing knowledge of amino acid coding rules and design tools, based on factors such as host requirements.

[0090] The conventional strategies and tools used in the transformation, culture, and expression steps of the expression strains in the preparation method of this application can be routinely selected by those skilled in the art, and suitable parameters can be determined through literature review and experimental verification. For example, transformation methods such as calcium chloride, protoplasts, and electroporation can be used, as well as identification methods such as resistance marker screening and direct identification of recombinants; the expression strains can be Escherichia coli, Bacillus subtilis, yeast, insect cells, mammalian cells, plant cells, etc.; and induction systems such as IPTG, sugar induction, nisin, and pH induction can be used during the expression process.

[0091] The expression vectors used in this application may be various commercially available / documented / researched vectors, including but not limited to Pllp, pMBP, pET, pBAD, pCAI, and pPOW series vectors.

[0092] The excipients in the compositions of this application include, but are not limited to, pharmaceutical, cosmetic, health product, and food-grade excipients, including but not limited to fillers, solvents, cosolvents, dispersants, viscosity modifiers, antioxidants, sweeteners, binders, pH adjusters, etc.; those skilled in the art can select these excipients according to conventional practices in the field. The compositions of this application may also contain other functional ingredients, such as moisturizing, whitening, and tissue repair components from traditional Chinese and Western medicines, chemical components, and cytokines.

[0093] The health products and pharmaceutical compositions in this application can be: injectable preparations, such as injection solutions, powder injections, etc.; oral preparations, such as tablets, capsules, oral liquids, etc.; and topical preparations, such as ointments, sprays, patches, etc. Tissue-engineered products can be cosmetic or tissue repair products. Food and cosmetic products can be those in forms known to be available in the food and cosmetic fields.

[0094] The recombinant collagen of the present invention

[0095] As used herein, the terms "recombinant collagen of the present invention" and "recombinant type III collagen peptide of the present invention" are used interchangeably and refer to recombinant collagen comprising the amino acid sequence shown in SEQ ID NO.1 and having a tag sequence fused to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0096] In a preferred embodiment, the recombinant collagen of the present invention has the structure shown in Formula I from the N-terminus to the C-terminus:

[0097] Z0-L-Z1-L-Z2(I)

[0098] In the formula,

[0099] Z0 is either an unlabeled or first-labeled sequence;

[0100] L represents an empty or linked peptide;

[0101] Z1 is the amino acid sequence shown in SEQ ID NO.1;

[0102] Z2 is a sequence without a second tag or a second tag sequence;

[0103] "-" is the key;

[0104] Among them, Z0 exists and Z2 can be non-existent, or Z2 exists and Z0 is non-existent, or Z0 and Z2 exist simultaneously.

[0105] As used herein, the term "connector peptide" refers to a connector peptide that has sufficient length and flexibility to ensure that the two connected proteins have sufficient spatial freedom to perform their functions, preferably a flexible connector (or flexible linker).

[0106] In a preferred embodiment, the modified recombinant type III collagen peptide of the present invention removes all the original fusion sequences (including fusion peptide elements and linker peptides) on the plasmid backbone when constructing the expression plasmid. The coding sequence of the recombinant collagen peptide containing a small affinity tag is directly and tightly linked after the start codon (ATG) in the expression cassette, followed by the stop codon (TAA, TGA, TAG, preferably TAA).

[0107] This invention involves the packaging and removal of fusion peptide elements and linker peptides designed in a recombinant expression vector, thereby achieving pure expression of the target protein.

[0108] The small affinity tag is preferably a 6×His tag, an 8×His tag, a Strep-Tag II tag, a FLAG tag, a Poly-Arg tag (composed of five or six consecutive arginine residues), or a Twin strep tag (composed of two Strep-tag II residues linked together), with a 6×His tag being more preferred. It can also contain two tags simultaneously, facilitating the use of two different affinity chromatography methods to obtain recombinant protein of higher purity. The tag can be located at the N-terminus or C-terminus of the recombinant collagen peptide, preferably at the C-terminus, or two tags can be selected located at the N-terminus and C-terminus respectively (see details for specific assembly methods). Figure 1 ).

[0109] Fusion peptide elements designed on the backbone of E. coli expression plasmids, such as TrxA on the pET-32a plasmid, typically promote the expression, solubility, or stability of exogenous proteins. Removing these elements significantly reduces the expression performance of exogenous proteins. This invention investigated several coding sequences for recombinant collagen and attempted to construct them onto vector plasmids with the N-terminal fusion peptide removed. The results showed that most sequences were not expressed or weakly expressed. The Co8 sequence, due to its superior expression performance, remained highly expressed and completely soluble even on vector plasmids with the fusion peptide element completely removed, without any change in stability. The inherent molecular characteristics of Co8 enabled these improvements, which are unparalleled by other molecules.

[0110] As used herein, the term "recombinant collagen" also includes variants having the aforementioned activities. These variants include (but are not limited to): deletions, insertions, and / or substitutions of 1-3 amino acids (typically 1-2, more preferably 1), and additions or deletions of one or more amino acids (typically up to 3, preferably up to 2, more preferably up to 1) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the function of the protein. Similarly, adding or deleting one or more amino acids at the C-terminus and / or N-terminus typically does not alter the structure and function of the protein. Furthermore, the term also includes the polypeptides of the invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0111] This invention also includes active fragments, derivatives, and analogs of the aforementioned collagen peptides. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the function or activity of the fusion protein of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusing a polypeptide with another compound (e.g., a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6×His). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0112] A preferred class of active derivatives refers to polypeptides formed by replacing up to three, more preferably up to two, and even more preferably up to one amino acid with an amino acid of similar or analogous properties compared to the amino acid sequence of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0113] Table A

[0114]

[0115]

[0116] This invention also provides analogs of the recombinant collagen of this invention. These analogs may differ from the polypeptides of this invention in terms of amino acid sequence, or in the form of modifications that do not affect the sequence, or both. Analogs also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the polypeptides of this invention are not limited to the representative polypeptides exemplified above.

[0117] Furthermore, the recombinant collagen of this invention can be modified. Modifications (typically without altering the primary structure) include chemically derived forms of the peptide, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the peptide to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include peptides modified to improve their resistance to proteolysis or optimize their solubility.

[0118] The term "polynucleotide of the present invention" may include polynucleotides encoding the recombinant collagen of the present invention, or may include polynucleotides with additional coding and / or non-coding sequences.

[0119] This invention also relates to variants of the aforementioned polynucleotides that encode fragments, analogs, and derivatives of polypeptides or recombinant collagen having the same amino acid sequence as those of this invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the recombinant collagen it encodes.

[0120] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%.

[0121] The recombinant collagen and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, purified to homogenization.

[0122] The full-length polynucleotide sequences of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0123] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0124] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0125] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0126] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE (RACE-cDNA end amplification) method is preferred. Primers used for PCR can be appropriately selected based on the sequence information disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0127] Modified expression carrier

[0128] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the modified vectors of the present invention or the recombinant collagen coding sequences of the present invention, and methods for generating the polypeptides of the present invention via recombinant technology.

[0129] Using conventional recombinant DNA technology, the polynucleotide sequence of this invention can be used to express or produce recombinant collagen. Generally, the following steps are involved:

[0130] (1). Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the recombinant collagen of the present invention, or with a modified recombinant expression vector containing the polynucleotide (including the polynucleotide described in the present invention, and the vector does not contain a fusion peptide element fused to the target protein).

[0131] (2) Host cells cultured in a suitable culture medium;

[0132] (3) Isolate and purify proteins from culture media or cells.

[0133] In this invention, the polynucleotide sequence encoding recombinant collagen can be inserted into the recombinant expression vector modified by this invention. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0134] The modified vector of the present invention does not contain fusion peptide elements that are fused to express the target protein, such as TrxA, S-Tag, T7-Tag, enterokinase recognition site, and thrombin recognition site on the pET-32a plasmid.

[0135] As used herein, the term "fusion peptide element" refers to a fixed polypeptide or oligopeptide sequence, such as an affinity tag, protease recognition site, or signal peptide, designed into a recombinant expression vector. The polynucleotide sequence encoding this fusion peptide element is located in the same expression cassette as the inserted polynucleotide sequence encoding the target protein. It can be expressed upstream or downstream of the target protein, forming a complete polypeptide containing the fusion peptide element, the target protein, and / or a linker peptide. Fusion peptide elements often have specific functions, such as increasing the expression level of the target protein, promoting the solubility of the target protein, guiding the secretion of the target protein into the extracellular space, or translocating it to specific subcellular structures. Some elements are also designed to facilitate subsequent purification, labeling, and recognition by enzymes or antibodies.

[0136] Furthermore, in this invention, the modified vector does not contain any linking peptides that fuse with the target protein.

[0137] Methods well known to those skilled in the art can be used to construct expression vectors containing the recombinant collagen-encoding DNA sequence of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0138] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0139] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0140] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells, such as yeast; and plant cells (such as ginseng cells).

[0141] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0142] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0143] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0144] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0145] The recombinant collagen in the above methods can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified using various separation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0146] The main advantages of this invention include:

[0147] (1) This invention has developed a modified recombinant type III collagen peptide for the first time. When expressed, it contains only a small affinity tag, which is easy to purify and does not require enzymatic digestion. This greatly simplifies the process steps and reduces the process cost while maintaining the excellent characteristics of the original recombinant collagen peptide Co8 (SEQ ID NO.1). It still has high expression level, high hydrophilicity and high stability. When the production strain expresses the modified protein, it does not need to express a long and useless fusion peptide, which reduces the ineffective conversion of bioenergy. This is conducive to improving the bioconversion efficiency and improving the protein yield and output ratio through process optimization. It is more advantageous when scaled up for industrialization.

[0148] (2) This invention has surprisingly discovered that the improved recombinant collagen peptide Co8-2 exhibits superior biological activity compared to Co8, demonstrating stronger cell proliferation, cell migration, and adhesion activities. In vitro experiments show that Co8-2 has significant anti-wrinkle, soothing, and firming effects. Therefore, the improved version of Co8-2 has greater development value when applied to functional skincare products, health foods, and non-implantable medical devices.

[0149] (3) This invention examined the coding sequences of multiple recombinant collagens and attempted to construct them onto vector plasmids with the N-terminal fusion peptide removed. As a result, most sequences were not expressed or were weakly expressed. Due to its superior expression performance, the Co8 sequence was highly expressed and completely soluble even on vector plasmids with the fusion peptide element completely removed, and its stability remained unchanged. The molecular characteristics of the Co8 sequence itself enabled the above improvements, which is unparalleled by other molecules.

[0150] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the reagents and materials used in the embodiments of the present invention are commercially available products.

[0151] Example 1. Molecular design and expression of Co8-2

[0152] Co8-2 is a polypeptide sequence of 434 amino acids (SEQ ID NO. 2) formed by adding an 8-amino acid Strep tag (Strep-Tag II tag) to the N-terminus and a 6×His tag to the C-terminus of the Co8 core sequence (ZL202211556168.1, SEQ ID NO. 4) (SEQ ID NO. 1 in this invention, totaling 420 amino acids) (obtained from Wuhan Jiaweida Biotechnology Co., Ltd.). The nucleic acid sequence encoding SEQ ID NO. 2 was calculated according to the central dogma, and after codon optimization tailored to the preferences of *E. coli*, it was inserted into the exogenous sequence expression frame of the pET-28a vector (Novagen, catalog number: 70777), which has all fusion peptides removed (fusion peptides are peptide segments fused with the target protein, including fusion peptide elements and linker peptides). Specifically, the sequence between the start codon ATG and the stop codon TAA was removed, and the optimized exogenous sequence was directly inserted between ATG and TAA. The optimized Co8-2 coding sequence is shown in SEQ ID NO. 3.

[0153] Co8 core sequence:

[0154] GRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGR NGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRP(SEQ ID NO.1)

[0155] Co8-2 sequence:

[0156] WSHPQFEKGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPHHHHHH(SEQ ID NO.2)

[0157] Optimized Co8-2 coding sequence:

[0158]

[0159] Molecular construction: The modification of the vector plasmid, the synthesis and insertion of the exogenous sequence were all entrusted to Genscript Biotech Inc. After receiving the constructed pET28a-Co8-2 plasmid, the researchers transformed it into BL21(DE3) (Jinsha Biotech, catalog number: SEC14) host bacteria. The specific operation method is as follows: ① Add 1 μL (approximately 10 ng) of plasmid to 100 μL of E. coli competent cells BL21(DE3), gently mix, and incubate on ice for 30 min; ② Heat shock the mixture in a 42℃ water bath for 45 s, then quickly incubate on ice for 2 min; ③ Add 5... 1) Incubate 0.00 μL of antibiotic-free LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) at 37°C and 220 rpm for 40 min; 2) Spread 200 μL of the bacterial culture evenly on an LB agar plate containing kanamycin (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 50 μg / mL kanamycin); 3) Invert the plate and incubate overnight at 37°C until clearly visible colonies appear.

[0160] Culture and Expression: Single colonies were picked from the LB agar plates and cultured overnight at 37°C and 220 rpm in 2 mL LB liquid medium (containing 50 μg / mL kanamycin) (BioSharp, catalog number BS152). The colonies were then transferred to 200 mL LB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37°C and 220 rpm until the OD600 reached 0.6-1.0. IPTG (BioSharp, catalog number BS119) at a final concentration of 0.2 mM was added to induce expression at 18°C ​​and 220 rpm for 16-20 h. The cells were collected by centrifugation and stored at -20°C or immediately subjected to lysis and purification.

[0161] Cell lysis: The bacterial precipitate was resuspended in lysis buffer (20 mM PPB buffer, 500 mM sodium chloride, pH 7.4) (relevant reagents purchased from Sinopharm Group). The resuspending ratio was 1 g of bacterial sludge to 20 mL of lysis buffer. After thorough resuspending, the precipitate was aliquoted into 30 mL centrifuge tubes and the cells were disrupted using an ultrasonic cell disruptor (Ningbo Xinzhi, SCIENTZ-IID). The samples were placed in an ice-water bath, and the ultrasonic disruption was performed using a Φ6 amplitude transformer at 250 W, with a 3-second on-time and 3-second off-time, for a total duration of 25 min. The resulting lysate was incubated at 33°C for 30 min and then centrifuged at 12000 rpm for 20 min to ensure complete separation of soluble proteins from inclusion bodies or bacterial fragments.

[0162] Analysis and Detection: SDS-PAGE was used to monitor and analyze the expression of the target protein. The specific procedure was as follows: 40 μL of the bacterial cell lysate before centrifugation and the supernatant after centrifugation were sampled and mixed with 10 μL of 5× reducing protein loading buffer (Biosharp, catalog number: BL502A). The mixture was then placed in a metal bath at 95℃ for 5 min for heat denaturation. After centrifugation at 12000 rpm for 10 min, 5 μL of the supernatant was added to the wells of an SDS-PAGE protein gel (GenScript Biotech, catalog number M00930). Electrophoresis was performed at 160V for 1 h. The gel was then stained and destained using an automated staining and destaining system (GenScript Biotech, catalog number M00930), and photographed on a white light plate. The theoretical molecular weight of the recombinant collagen peptide Co8-2 was 42.2 kDa. The detection results are as follows: Figure 2 As shown in Figure A, the target band is located between 40kD and 55kD, indicating a high expression level, and the target protein is mostly present in the supernatant.

[0163] Example 2. Molecular design and expression of Co8-3

[0164] Co8-3 is a polypeptide sequence (SEQ ID NO. 4) of 426 amino acids formed by adding a 6×His tag only to the C-terminus of the Co8 core sequence (SEQ ID NO. 1, 420 amino acids). The nucleic acid sequence encoding SEQ ID NO. 4 was calculated according to the central dogma, and after codon optimization tailored to the preferences of *E. coli*, it was inserted into the foreign sequence expression frame of the pET-32a vector (Novagen, catalog number 69015) after removing all fusion peptides (fusion peptides are peptide segments fused to the target protein, including fusion peptide elements and linker peptides). Specifically, the optimized foreign sequence was directly inserted between the start codon ATG and the stop codon TAA. The optimized Co8-3 coding sequence is shown in SEQ ID NO. 5.

[0165] Co8-3 sequence:

[0166] GRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPHHHHHH(SEQ ID NO.4)

[0167] Optimized Co8-3 coding sequence:

[0168]

[0169] Co8-3 uses the pET-32a vector and requires transformation and screening with 100 μg / mL ampicillin (BioSharp, catalog number BS923). Ampicillin is also added during cell culture and fermentation expression. The molecular construction, culture expression, cell lysis, and analysis of Co8-3 are identical to those in Example 1, except for the vector plasmid and antibiotic.

[0170] The theoretical molecular weight of recombinant collagen peptide Co8-3 is 41.2 kDa, and the test results are as follows: Figure 2 As shown in Figure B, the target band is located between 40kD and 55kD, indicating a high expression level. Furthermore, the target protein is present in the supernatant, demonstrating soluble expression.

[0171] Example 3. Comparison of expression yields of Co8-2, Co8-3, and Co8

[0172] Co8 was cultured, expressed, lysed, and detected by electrophoresis using the same method as in Example 1. The results are as follows: Figure 2 As shown in Figure C, the electrophoresis results show that Co8, Co8-2, and Co8-3 are all soluble and expressed at high levels. The expression levels of Co8-2 and Co8-3 after removing the vector fusion peptide appear to be higher than or similar to Co8.

[0173] For accurate quantitative analysis, 2L shake flasks containing 600mL LB medium were used for bacterial culture and expression induction. Co8, Co8-2, and Co8-3 glycerol bacteria, frozen at -80℃, were inoculated at a ratio of 1:1000 (v / v) into 200mL LB liquid medium (containing the corresponding antibiotics) and cultured overnight at 37℃ and 220rpm. The next day, the cells were transferred at a ratio of 1:50 (v / v) to 600mL LB liquid medium (containing antibiotics) and cultured at 37℃ and 220rpm for 2.5h. IPTG was then added to a final concentration of 0.2mM, and the culture was continued at 18℃ and 220rpm for 17-21h. The bacterial cells were collected by centrifugation, washed once with pure water, and weighed wet. The lysis supernatant was prepared according to the bacterial lysis method in Example 1.

[0174] The supernatant of the lysis buffer was diluted 2-, 4-, 6-, 8-, and 10-fold with lysis buffer. BSA standard solution (Sangon Biotech, catalog number C500642-0001) was successively diluted with lysis buffer to 1.25 mg / mL, 1.0 mg / mL, 0.75 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. 40 μL of each of the serially diluted lysis buffer supernatant or BSA solution was added to 10 μL of 5× reducing protein loading buffer and mixed thoroughly. The mixture was then heat-denatured at 95°C for 5 min in a metal bath. After centrifugation at 12000 rpm for 10 min, 10 μL of the supernatant was added to 11-well SDS-PAGE gels. Electrophoresis was performed at 160 V for 1 h. The gels were then stained and destained using an automated staining and destaining system, and photographed on a white light plate.

[0175] The gel images were analyzed using GelAnalyzer software built into the Mona Quick Gel 6200 gel imaging system. Lane and band analysis were performed to obtain the grayscale values ​​of the BSA bands. A scatter plot was plotted based on the correlation between grayscale values ​​and BSA protein concentration, and a standard curve was obtained using linear fitting. Similarly, the grayscale values ​​of the recombinant collagen bands in each sample were analyzed. Grayscale values ​​falling within the standard curve range were substituted into a function to calculate the content of recombinant collagen in each sample. Furthermore, based on parameters such as sample dilution factor, culture volume, bacterial wet weight, and bacterial resuspension ratio, the amount of collagen expressed per gram of bacterial cells and per liter of bacterial culture were calculated. The expression levels of Co8, Co8-2, and Co8-3 cells per gram were 52.6 mg / g, 67.5 mg / g, and 54.9 mg / g, respectively, and the expression levels of Co8, Co8-2, and Co8-3 bacterial cultures per liter were 108.2 mg / L, 146.1 mg / L, and 168.4 mg / L, respectively (see...). Figure 3 (AB). This indicates that removing the fusion peptide from the Co8 plasmid backbone did not decrease its expression level; in fact, the expression level of Co8-2 cells increased to some extent. Co8-3, due to the use of the pET-32a vector, had a significantly increased bacterial density, which in turn significantly increased the expression level per unit volume of bacterial solution.

[0176] Example 4. Expression of other recombinant collagen peptides after defusion (comparative example)

[0177] This invention also attempts to design fusion peptides (including fusion peptide elements and linker peptides) for several other recombinant collagen peptides with good expression performance, including Co11, F3, F4, F7, F8, F9, F48, and F51. The coding sequences of these peptides were inserted into the multiple cloning site of the unmodified pET-32a vector via BamHI and XhoI restriction sites, and fused with TrxA, His-Tag, and S-Tag tags for expression. Tests showed high expression levels and excellent water solubility. Subsequently, the expression plasmids were modified to remove the longer N-terminal fusion peptides, retaining only the six-amino acid His tags at the N-terminus (Co11) or C-terminus (F3, F4, F7, F8, F9, F48, F51). Expression tests were performed again, and the expression levels of the above collagen peptides decreased significantly, with some even showing no significant expression. In contrast, the Co8 sequence exhibits exceptionally superior expression performance, maintaining high expression levels and good water solubility even after complete removal of the fusion peptide, a conclusion fully demonstrated in Example 3. The expression levels of the coding sequences for Co11, F3, F4, F7, F8, F9, F48, and F51 on the pET-32a vector before and after removal of the fusion peptide are compared in Table 1 and [Table data missing]. Figure 4 , AH.

[0178] Table 1. Comparative sequence expression before and after removal of the fusion peptide on the pET-32a vector.

[0179]

[0180]

[0181] The recombinant collagen peptides F3, F4, F7, F8, F9, F48, and F51 mentioned above have a His tag with 6 amino acids added to the C-terminus, but no tag at the N-terminus.

[0182] The aforementioned recombinant collagen peptide Co11 has a His tag with 6 amino acids added to the N-terminus, but no tag at the C-terminus.

[0183] Note: "+" indicates that the target protein is expressed, and the more "+" signs, the higher the expression level; "-" indicates that the target protein is not expressed or has no obvious expression.

[0184] Example 5. Fermentation of Co8-2

[0185] Glycerin-containing bacteria pET28a-Co8-2 / BL21(DE3) (the strain constructed in Example 1) stored at -80℃ were inoculated into LB medium containing kanamycin sulfate and cultured overnight at 37℃ and 220 rpm. The next day, the culture was transferred to fresh LB medium and cultured for another 4-6 hours at 37℃ and 220 rpm. OD was then measured. 600The culture medium is between 2.0 and 2.5, and can be inoculated into the fermentation tank. A 7L glass fermentation tank (Baoxing, BIOTECH-7L) contains 3L of basal culture medium (citric acid monohydrate 1.7g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 9.2g / L, ferrous sulfate heptahydrate 0.105g / L, anhydrous glucose 10g / L, 1M magnesium sulfate solution 2mL / L, 0.1M calcium chloride solution 0.2mL / L, M7 trace elements 1mL / L; glucose and magnesium sulfate are sterilized separately, cooled, and then combined into the tank; calcium chloride solution and trace elements are filtered and sterilized, and added before inoculation) (all reagents were purchased from Sinopharm Group). The tank is connected via silicone tubing to a feeding bottle (50% glucose, magnesium sulfate heptahydrate 4.8g / L, M7 trace elements 1mL / L, autoclaved), an ammonia bottle, and an antifoaming agent bottle. Seed culture was inoculated in shake flasks at a 5% (v / v) ratio, with kanamycin sulfate (final concentration 50 mg / L) added simultaneously. Initial stirring was set at 200 rpm, aeration at 1 vvm, flask temperature automatically controlled at 37°C, pH 7.0 with automatic alkali pump control, and dissolved oxygen at 40%, linked to stirring (200-900 rpm) and aeration (1-3 vvm). Once the carbon source in the basal medium was depleted and dissolved oxygen rapidly recovered, feeding was initiated at an initial rate of 5 g / L·h. The feeding rate was then adjusted based on dissolved oxygen levels to maintain dissolved oxygen at 30% ± 5%, with additional oxygen supplementation as needed. OD 600 After reaching 50°C, the temperature was gradually lowered until it reached 20°C. Once stable, IPTG at a final concentration of 0.2 mM was added for induction. Dissolved oxygen was maintained at 30% ± 5% by adjusting the feeding rate, and supplemental oxygen was added as needed. After 18 hours of induction culture, the fermentation broth was harvested, and the cells were collected by centrifugation at 8000 rpm for 15 minutes. Under these fermentation conditions, the OD of the pET28a-Co8-2 / BL21(DE3) strain was... 600 The yield can reach over 180, and the wet weight of the bacteria can be harvested from each liter of fermentation broth, which is 220-280g.

[0186] Five grams of bacterial sludge were weighed and washed once with pure water. A lysate was prepared according to the cell lysis method in Example 1, and the expression level of Co8-2 was quantified according to the analytical method in Example 3. The results showed that the expression level of the pET28a-Co8-2 / BL21(DE3) strain under the above fermentation conditions reached (9.5±1.2) g / L, with a maximum of 11.8 g / L. This expression level has reached the industrial production level and can be further scaled up for industrial development.

[0187] Example 6. Purification and preparation of Co8-2

[0188] Take the bacterial sludge from Example 5, wash it once with pure water, and then resuspend it in lysis buffer (20 mM PB buffer, 500 mM sodium chloride, 20 mM imidazole, pH 7.4), with a ratio of 1 g of bacterial sludge to 20 mL of lysis buffer. Filter the resuspended solution through a 100-mesh sieve, pump it into a high-pressure homogenizer (Ningbo Xinzhi, SCIENTZ-150) for disruption, and cycle it 3 times at 700 bar under cold trap protection. Incubate the bacterial lysate in a 33°C water bath for 30-50 min, transfer it to a large centrifuge cup, centrifuge at 8000 rpm for 30 min at room temperature, and collect the supernatant, which is the crude Co8-2 sample.

[0189] Co8-2 crude sample was clarified and filtered using a depth filter (Cobate, catalog number CDFCDCSD0140PCP), with the pressure difference controlled to be less than 0.4 bar, and the turbidity of the filtered mixture < 20 NTU. Chromatographic purification was performed using an XK50 / 20 column (Borglon) packed with Ni-FF packing material (Borglon). The column was equilibrated to 5 bed volumes with equilibration buffer (20 mM PB buffer, 500 mM sodium chloride, 20 mM imidazole, pH 7.4) (reagents purchased from Sinopharm) at a flow rate of 90 cm / h, and the UV index was zeroed. The clarified crude sample was loaded onto the column at a flow rate of 45 cm / h until the loading reached 80%. The column was then rinsed with equilibration buffer until the conductivity and UV line levels were reached. Co8-2 protein was eluted with elution buffer (20 mM PB buffer, 150 mM sodium chloride, 500 mM imidazole, pH 7.4) (reagents purchased from Sinopharm) at a flow rate of 60 cm / h, and the eluent with UV280 > 100 mAu was collected. The column was then rinsed with elution buffer until the conductivity and UV line levels were reached, and 0.5 M... Wash the column with NaOH for 2 column volumes, and wash with pure water for at least 10 column volumes until the effluent is neutral; store the nickel-packed chromatography column with 20% ethanol.

[0190] The collected eluted samples were transferred to pretreated 15kD-retaining dialysis bags and placed in 1×PBS (100 times the sample volume) for dialysis. Dialysis was performed twice, each time for at least 8 hours. Finally, the dialyzed protein solution was sterilized by filtration using a 0.22μm syringe filter (Millipore, catalog number SLGV033RS) in a clean bench to obtain the final Co8-2 recombinant collagen product.

[0191] Figure 5 The changes in the purity of Co8-2 protein before and after purification are shown. After the above steps, the purity of Co8-2 protein reached over 95%.

[0192] Example 7. Stability test of Co8-2

[0193] Take the purified sample from Example 6, aliquot 200 μL / vial into several centrifuge tubes, label them, and store them at 2-8℃ and room temperature (18-25℃) respectively, with at least 30 tubes per group. Sample and test every 1-2 weeks for the first 3 months, and monthly thereafter. Take 40 μL of sample, add 10 μL of 5× reduced protein loading buffer, mix well, heat denature, and then perform SDS-PAGE analysis. See Example 1 (Analysis and Detection) for specific procedures.

[0194] The results showed that the purified Co8-2 protein did not degrade after one year of storage at 2-8℃. At room temperature (18-25℃), the target band shifted slightly upwards and underwent minor degradation, but the degradation rate was very slow, and even after one year, no significant degradation occurred. Co8-2 exhibits excellent stability among large protein molecules. The stability test results for Co8-2 are shown below. Figure 6 , AB.

[0195] Example 8. Test of Co8-2 cell proliferation activity

[0196] Samples of Co8-2 and Co8-3 were purified and prepared according to the method in Example 6. The preparation of Co8 samples requires additional steps of changing the medium, TEV enzyme digestion and secondary nickel column loading to remove the label. For specific operations, please refer to patent ZL201811438582.6.

[0197] NIH / 3T3 cells (fibroblasts) with a confluence of 70%-90% (Pronosai, catalog number CL-0171) were digested with TrypLE Select CTS (Gibco, catalog number A1285901) and resuspended in DMEM (Gibco, catalog number C11995500BT) containing 10% fetal bovine serum (Corning, catalog number 35-081-CV). The resuspending solution was then added at a concentration of 3 × 10⁻⁶. 4 cells / cm 2 The cells were seeded at a density of 15 mL / flask into T75 cell culture flasks and cultured adherently at 37°C and 6% CO2 for 12-20 h. The original culture medium was then aspirated, and the cells were rinsed once with 8-10 mL of PBS buffer. 15 mL of PBS solution containing 0.5 mg / mL of the recombinant collagen was added, and the cells were cultured for another 20-28 h at 37°C and 6% CO2. For the negative control, 15 mL of PBS solution without recombinant collagen was added, and the cells were cultured under the same conditions. Cell confluence was observed under a microscope and photographed. The culture supernatant was aspirated, and the cells were rinsed once with 8-10 mL of PBS buffer, digested with TrypLE Select CTS, and then samples were taken for cell counting to calculate the fold increase.

[0198] The results are as follows Figure 7 and Figure 8 As shown in the figure, after treatment with recombinant collagen solution, the adherent NIH / 3T3 cells exhibited significantly higher cell confluence than the negative control, with the Co8-2 experimental group showing the highest cell confluence, even exhibiting longitudinal overlap. Based on cell counting results after digestion, NIH / 3T3 cells cultured in 0.5 mg / mL Co8-2 solution for approximately 24 hours showed a 92.4% increase in cell number. Cultures in Co8-3 and Co8 solutions also showed increases of 75.5% and 74.0%, respectively, significantly higher than the 11.4% proliferation rate of the negative control group. This indicates that all three recombinant collagen proteins effectively promote cell proliferation, with Co8-2 exhibiting the best proliferative activity, which may be related to the short tags at both ends of its sequence.

[0199] Example 9. Assay of Co8-2's cell migration-promoting activity

[0200] Samples of Co8-2 and Co8-3 were purified and prepared according to the method in Example 6. The preparation of Co8 samples requires additional steps of changing the medium, TEV enzyme digestion and secondary nickel column loading to remove the label. For specific operations, please refer to patent ZL201811438582.6.

[0201] Cell migration assays were performed using the scratch assay, with cells cultured in 6-well plates at a concentration of 6 × 10⁶ cells / well. 4 cells / cm 2 NIH / 3T3 cells were seeded and seeded in DMEM complete medium containing 10% fetal bovine serum. 2 mL of the medium was evenly spread in each well of the plate and cultured at 37°C and 6% CO2 for (16±3) h until the cell confluence reached 70%-90%. A 10 μL sterile pipette was used to streak a line from top to bottom in the center of the bottom of the well containing the cells. The original medium and detached cells were removed, and the plate was washed once with 1 mL of DMEM medium. 2 mL of DMEM medium containing 0.5 mg / mL of the above-mentioned recombinant collagen was added along the wall. The negative control group was cultured in DMEM medium without collagen. The plates were cultured at 37°C and 6% CO2 for (24±2) h. The healing status of the scratches was observed under an inverted microscope and photographed for recording.

[0202] The results are as follows Figure 9As shown in AD, after scratch treatment, NIH / 3T3 adherent cells cultured in DMEM medium containing recombinant collagen for 24 h showed a significant increase in cell density and migrated towards the scratch site, which was almost completely covered. In contrast, the negative control group without recombinant collagen showed no significant cell proliferation, the scratch remained, and there was no obvious cell migration or coverage. This indicates that all three recombinant collagens tested have good cell migration-promoting activity; compared with Co8-3 and Co8, the Co8-2 experimental group had higher cell confluence and more thorough scratch coverage, indicating that the tag-modified Co8-2 has superior cell migration-promoting ability.

[0203] Example 10. Assay of Co8-2's cell adhesion-promoting activity

[0204] Samples of Co8-2 and Co8-3 were purified and prepared according to the method in Example 6, and Co8 and three other recombinant collagen samples were prepared according to the method in patent ZL201811438582.6.

[0205] Recombinant collagen samples were diluted to 0.5 mg / mL using DMEM medium. 0.1 mL of collagen solution was added to each well (double replicate) and coated into 96-well ultra-low adsorption plates. The plates were incubated at 37°C and 6% CO2 for 1 h. After coating, the plates were washed twice with 0.2 mL PBS buffer, then incubated for 1 h with 0.1 mL 1% BSA-PBS solution for blocking. After blocking, the plates were washed twice again with 0.2 mL PBS buffer. NIH / 3T3 cells in logarithmic growth phase with confluence greater than 80% were collected, digested with trypsin for 2 min to prepare a single-cell suspension, and then digested with DMEM complete medium to terminate the digestion. The cell density was adjusted to 1 × 10⁶ cells based on the cell count results. 6 Cells / mL were added to each well of a sealed 96-well plate at a rate of 0.1 mL / mL. The plates were incubated at 37°C and 6% CO2 for 1 h for adherent culture. After incubation, each well was washed twice with 0.2 mL of PBS buffer. The adherence of the cells was then observed under an inverted microscope.

[0206] The results are as follows Figure 10 As shown, NIH / 3T3 cell suspensions treated with digestion exhibited excellent adhesion and retention after 1 hour of incubation in wells coated with recombinant collagen Co8-2, Co8-3, and Co8, with cells almost completely covering the entire well. In contrast, NIH / 3T3 cells showed almost no adhesion and growth in wells coated with recombinant collagen F3, F4, and F8, or in uncoated wells. This indicates that Co8-2, Co8-3, and Co8 proteins all possess excellent cell adhesion-promoting activity, and the activities of Co8-2 and Co8-3 were not affected after fusion peptide removal and tag modification.

[0207] Example 11. Test of the anti-wrinkle, soothing, and firming effects of Co8-2

[0208] The Co8-2 sample was purified and prepared according to the method in Example 6, and sent to Guangdong Jianxun Testing Technology Co., Ltd. for efficacy testing. The test included three effects: anti-wrinkle, soothing, and firming.

[0209] Anti-wrinkle: Excessive production of free radicals is a major cause of natural skin aging and photoaging, leading to wrinkles. Therefore, the ability to scavenge free radicals is one of the important indicators for evaluating anti-aging and anti-wrinkle cosmetics (raw materials). This experiment tested the DPPH free radical scavenging rate of the samples and compared it with the results of the negative control. If the scavenging rate of the test sample was higher than that of the negative control and the difference was statistically significant (P < 0.05), the test sample could be considered to have anti-wrinkle efficacy.

[0210] Test results are as follows Figure 11 As shown in the figure. Under laboratory conditions, the average DPPH free radical scavenging rate of the test sample Co8-2 was 16.08% ± 1.23%, which was significantly different from the negative control (P < 0.05). Moreover, the DPPH free radical scavenging rate of the positive control was > 25%, indicating that the reaction system was effective and that the sample Co8-2 had anti-wrinkle effects.

[0211] Soothing: Hyaluronidase exists in the extracellular matrix of connective tissues and is associated with most IgE-mediated type I and T-cell-mediated type IV hypersensitivity reactions. Therefore, in vitro hyaluronidase inhibition assays can, to some extent, reflect the soothing, anti-allergic, and anti-inflammatory effects of cosmetics (raw materials) on the skin. This experiment uses the Elson-Morgan colorimetric reaction to detect the consumption rate of hyaluronic acid substrate, thereby determining the hyaluronidase inhibition rate of the sample. Comparison with a negative control is used to determine whether the test sample has a soothing effect.

[0212] Test results are as follows Figure 12 As shown in the figure. Under the experimental conditions, the hyaluronidase inhibition rate of the test sample Co8-2 was 14.72±1.13%, which was significantly higher than that of the negative control (1.43±0.02%). SPSS software analysis and a two-tailed test were performed, and the significance factor P<0.05, indicating that the sample Co8-2 has a significant soothing effect.

[0213] Firming: Elastase can specifically break down elastin in connective tissue, causing elastin loss, skin laxity, and decreased elasticity. External stimuli such as ultraviolet radiation can promote the increase of elastase in dermal cells. Substances that can inhibit elastase activity can effectively slow down the degradation rate of elastin, thereby helping to maintain skin elasticity and achieve a firming effect. This experiment detected elastase activity in the test sample group and the negative control group. If the test sample group showed an inhibitory effect on enzyme activity compared with the negative control group, and the difference was statistically significant (P < 0.05), the test sample was considered to have a firming effect.

[0214] The test results are shown in Table 2. Under the experimental conditions, the elastase inhibition rate of the test sample group was 18.47%. SPSS software analysis and a two-tailed test showed a significance factor P < 0.05, indicating that the test sample had a significant inhibitory effect on enzyme activity, thus demonstrating that sample Co8-2 has a significant firming effect.

[0215] Table 2 Elastase Inhibition Rate Test

[0216]

[0217]

[0218] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A recombinant collagen, characterized in that, The collagen is fused with a tag sequence at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1, wherein the tag sequence is selected from Strep-Tag II tags, 6×His tags, or combinations thereof.

2. The recombinant collagen as described in claim 1, characterized in that, The collagen has the structure shown in Formula I from the N-terminus to the C-terminus: Z0-L-Z1-L-Z2 (I) In the formula, Z0 is either an unlabeled or first-labeled sequence; L represents an empty or linked peptide; Z1 is the amino acid sequence shown in SEQ ID NO.1; Z2 is a sequence without a second tag or a second tag sequence; "-" is the key; Among them, Z0 exists and Z2 is not present, or Z2 exists and Z0 is not present, or both Z0 and Z2 exist simultaneously.

3. The recombinant collagen as described in claim 2, characterized in that, The first tag sequence and the second tag sequence are each independently selected from Strep-Tag II tags or 6×His tags.

4. The recombinant collagen as described in claim 2, characterized in that, Z0 is a Strep-Tag II tag, and Z2 is a 6×His tag.

5. The recombinant collagen as described in claim 2, characterized in that, Z0 is none, and Z2 is a 6×His tag.

6. The recombinant collagen as described in claim 2, characterized in that, The amino acid sequence of the recombinant collagen is shown in SEQ ID NO.2 or 4.

7. An isolated polynucleotide, characterized in that, The polynucleotide encodes the recombinant collagen as described in any one of claims 1-6.

8. The polynucleotide of claim 7, characterized in that, The polynucleotide sequence is selected from the following group: (a) The polynucleotide sequence is as shown in either SEQ ID NO. 3 or 5; and (b) The polynucleotide sequence has ≥95% identity with the sequence shown in either SEQ ID NO.3 or 5.

9. A carrier, characterized in that, The vector comprises the polynucleotide as described in claim 7 or 8, and the vector does not contain the coding sequences for the fusion peptide element and the linker peptide fused to the target protein.

10. A host cell, characterized in that, The host cell contains the vector as described in claim 9.

11. A method for preparing the recombinant collagen as described in claim 1, characterized in that, Including the following steps: (i) Culturing the host cells as described in claim 10 under suitable conditions to obtain the recombinant collagen as described in claim 1; and (ii) Optionally, the recombinant collagen obtained in step (i) may be enzymatically digested, purified, and / or isolated.

12. A composition, characterized in that, The composition contains: (I) The recombinant collagen according to any one of claims 1-6; and (II) Pharmaceutically acceptable carriers or cosmetically acceptable excipients.

13. The composition according to claim 12, characterized in that, The composition is a face cream, lotion, serum, facial cleanser, or essence.

14. Use of the recombinant collagen according to any one of claims 1-6 in the preparation of cosmetics for improving the skin barrier, smoothing fine lines, moisturizing, whitening and fading spots, shrinking pores or lightening dark circles.

15. Use of the recombinant collagen of any one of claims 1-6 in the preparation of a medicament for repairing the mucosal barrier of the nasal cavity, oral cavity, vagina or anorectal cavity, or for promoting wound healing or preventing scar formation.

16. A non-therapeutic method for (a) promoting cell proliferation, (b) promoting cell migration and / or (c) promoting cell adhesion, characterized in that, Including the following steps: Cells are cultured in the presence of the recombinant collagen of claim 1 or the composition of claim 12, thereby (a) promoting cell proliferation, (b) promoting cell migration and / or (c) promoting cell adhesion. The cells in question are cells cultured in vitro. The cells are selected from epidermal cells, mucosal epithelial cells, or dermal cells.

17. The method as described in claim 16, characterized in that, The cells are selected from fibroblasts or skin mesenchymal cells.

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