Preparation method and application of humanized type III collagen

Abstract

The present invention provides a preparation method and application of humanized type III collagen. The amino acid sequence of natural type III collagen is modified to facilitate fermentation preparation and separation and purification, and the collagen can effectively adapt to the in vivo environment. The fermentation preparation method is optimized, and a yeast expression system is utilized, using methanol and sorbitol dual induction to significantly improve expression efficiency. The collagen can promote skin repair after damage, especially has a good protective effect against ultraviolet damage, can resist oxidative stress, and regulate the secretion of inflammatory factors.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a preparation method and application of humanized type III collagen. Background Art

[0002] Collagen is the main dermal component of human skin and has advantages such as biocompatibility and biodegradability. Its material-cell interaction is conducive to extracellular matrix (ECM) remodeling and tissue regeneration (see Arai Y, Choi B, Kim BJ, et al. Cryptic ligand on collagen matrix unveiled by MMP13 accelerates bone tissue regeneration via MMP13 / Integrinα3 / RUNX2 feedback loop. Acta Biomater. 2021; 125: 219–230.). The molecular composition of type III collagen is a homotrimer, in which the (Gly-XY)n tripeptide repeat sequence is the main feature of the helical region. Type III collagen is rich in blood vessels, intestines and skin, and is upregulated during the growth and development of organisms and wound healing (see Walimbe T, Calve S, Panitch A, et al. Incorporation of types I and III collagen in tunablehyaluronan hydrogels for vocal fold tissue engineering. Acta Biomater. 2019; 87: 97–107). During wound healing, type III collagen can induce inflammatory cells and fibroblasts to migrate to the wound site, promoting the formation and repair of connective tissue (see Makuszewska M, Bonda T, M, et al. Expression of collagen type III in healing tympanic membrane. Int J Pediatr Otorhi. 2020; 136: 110-196). Type III collagen is also a natural hemostatic substance that can induce platelet adhesion and aggregation to the bleeding site and activate some coagulation factors to initiate the coagulation cascade reaction (see Kuivaniemi H, Tromp G. Type III collagen (COL3A1): gene and protein structure, tissue distribution, and associated diseases. Gene. 2019; 707: 151-171). Studies have found that recombinant human type III collagen has a practical effect in promoting extracellular matrix remodeling, upregulating the synthesis of type I and type III collagen in the body, and alleviating skin photoaging caused by ultraviolet rays (see Wang J, Qiu H, Xu Y, et al. The biological effect of recombinant humanized collagen on damaged skin induced by UV-photoaging: an invivo study. Bioact Mater. 2022; 11: 154–165). Type III collagen has unique physiological functions and is widely used in food, beauty, medicine and other fields, and the market demand is increasing. At present, a variety of collagen-based medical devices have been approved for marketing, such as Matrix Wound Healing and Collagen Implant I.

[0003] Currently, almost all collagen used in biomedicine, pharmaceutical applications and tissue engineering medical products is of animal origin, among which bovine collagen dominates in both quality and quantity (see Fertala A. Three decades of research on recombinant collagens: reinventing the wheel or developing new biomedical products? Bioengineering. 2020; 7(4): 155). Most collagen products on the market are extracted from mammalian tissues and organs, requiring a large amount of raw materials. Safety aspects should be considered when using animal-derived collagen, including the risks of infectious viruses, pathogenicity and allergic reactions (see Fushimi H, Hiratsuka T, Okamura A, et al. Nishimura I. Recombinant collagen polypeptide as aversatile bone graft biomaterial. Communications Materials. 2020; 1(1): 871–8713), such as prions, HIV, foot-and-mouth disease virus, etc. (see Kotler ED, Marshall WS, Gareta EG. Sources of collagen for biomaterials in skin wound healing. Bioengineering. 2019; 6: 56). Meanwhile, religious sensitivity, purification, and batch-to-batch variation are other limiting factors for native collagen (see Liu D, Zhang X, Li T, et al. Extraction and characterization of acid- and pepsin-soluble collagens from the scales, skins and swim-bladders of grass carp (Ctenopharyngodon idella) Food Biosci. 2015; 9: 68–74).

[0004] The biosynthesis of collagen has made great progress in recent years. The expression of collagen in plant and animal cells, yeast, and Escherichia coli systems and the large-scale production through high-density fermentation technology are promising options (see Xiang ZX, Gong JS, Li H, et al. Heterologous expression, fermentation strategies and molecular modification of collagen for versatile applications. Crit Rev Food Sci Nutr. 2023; 63(21): 5268-5289). Tang et al. used Escherichia coli as a host and carried out small-scale production of human-like collagen in a 10L bioreactor at 28°C under the induction of 0.1mM isopropyl-β-d-thiogalactopyranoside (IPTG), ultimately achieving a yield of 0.26g L-1. IPTG is toxic to the strain, so a strategy using lactose, a lower-cost inducer, was used instead. Ultimately, approximately 0.7 g L⁻¹ of the collagen binding domain fusion protein was obtained in a 3-L fermentor (see Fruchtl M, Sakon J, Beitle R. Alternate carbohydrate and nontraditional inducer leads to increased productivity of a collagen binding domain fusion protein via fed-batch fermentation. J Biotechnol. 2016;226:65–73). Escherichia coli is the most common prokaryotic host, but its poor post-translational modification capacity, proneness to inclusion body formation, and endogenous pyrogens have long plagued researchers. Furthermore, plant and animal cell systems are cumbersome to manipulate and require stringent culture conditions, leaving them a long way from industrial production.

[0005] Yeast is a safe and popular eukaryotic host for expressing pharmaceutical proteins. It not only has complete post-translational modification functions, but also avoids the formation of inclusion bodies and the production of endotoxins. Researchers have attempted to use Pichia pastoris to ferment and produce modified type III collagen (see Xiang ZX, Gong JS, Shi JH, et al. High-efficiency secretory expression and characterization of the recombinant type III human-like collagen in Pichia pastoris. Bioresour Bioprocess. 2022, 9(1): 117). Therefore, the present invention is to develop a novel method for preparing type III collagen, modify the amino acid sequence of natural type III collagen, replace some amino acids, and then use a yeast system for biosynthesis, and optimize the fermentation conditions. The obtained type III collagen has good biological activity, which provides an effective way for the preparation and application of collagen. Summary of the Invention

[0006] A first aspect of the present invention provides a type III collagen, characterized in that the amino acid sequence of the type III collagen is shown in SEQ ID NO: 2.

[0007] The present invention modifies the partial sequence of the α1 chain of human wild-type type III collagen, replaces some hydrophobic residues with hydrophilic residues, and replaces some acidic amino acid residues with basic amino acid residues. This can improve the water solubility of collagen and also increase its bioavailability, so that it can adapt to the weakly alkaline environment in the body.

[0008] The second aspect of the present invention provides a method for preparing the type III collagen, characterized in that the method comprises: introducing a nucleotide sequence encoding the type III collagen into a yeast expression vector, fermenting and culturing the yeast, inducing it with methanol and sorbitol, and harvesting the type III collagen.

[0009] Furthermore, the nucleotide sequence encoding the type III collagen is shown in SEQ ID NO: 3.

[0010] Furthermore, the yeast is Pichia pastoris. Preferably, the Pichia pastoris is GS115.

[0011] Furthermore, the culture medium used in the culturing process includes: 15 g / L glycerol, 5 g / L mannitol, 20 g / L peptone, 8 g / L yeast extract, 0.5 g / L manganese sulfate, 0.5 g / L sodium molybdate dihydrate, 0.3 g / L magnesium sulfate, 0.2 g / L cobalt chloride, 0.06 g / L zinc sulfate, 0.05 g / L copper sulfate and 0.02 g / L chromium chloride.

[0012] Furthermore, the culturing step includes: using YPD medium to culture the yeast in a shaking incubator at 30°C and 220 r / min; inoculating the yeast into the culture medium at an inoculum size of 5%, and culturing in a shaking incubator at 30°C and 260 r / min; when the OD600 reaches 4-6, adding 1.2% methanol and 0.5% sorbitol by volume to induce expression for 72 hours; after the induction is completed, centrifuging at 5000 r / min for 10 minutes to collect the fermentation supernatant, and separating and purifying to obtain type III collagen.

[0013] The third aspect of the present invention provides a use of the type III collagen in the preparation of a product for protecting skin damage.

[0014] Furthermore, the damage is ultraviolet damage.

[0015] Furthermore, the product is a cosmetic.

[0016] Beneficial effects

[0017] The present invention provides a preparation method and application of humanized type III collagen, which has the following beneficial effects:

[0018] (1) The sequence structure of natural type III collagen was modified to improve the hydrophilicity of the protein, facilitate fermentation preparation and separation and purification, and effectively adapt to the in vivo environment;

[0019] (2) The fermentation preparation method was optimized, and the yeast expression system was used, using methanol and sorbitol dual induction to significantly improve the expression efficiency;

[0020] (3) The collagen can promote skin repair after damage, especially has a good protective effect against ultraviolet damage, can resist oxidative stress, and regulate the secretion of inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Relative cell viability determination;

[0022] Figure 2 :Histological observation of animal skin;

[0023] Figure 3 : SOD level detection;

[0024] Figure 4 : MDA level detection;

[0025] Figure 5 :IL-1β level detection;

[0026] Figure 6 : IL-6 level detection. DETAILED DESCRIPTION

[0027] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0028] Example 1 Collagen Molecular Design

[0029] Natural collagen is a high-molecular-weight, insoluble fibrin protein with numerous Gly-XY repeats in its sequence. Furthermore, during synthesis, it is subject to strict post-modification regulation and self-assembly, making collagen expression difficult. Studies have demonstrated molecular modification of type III collagen, replacing hydrophobic amino acids other than Pro with hydrophilic amino acids, and expressing it in the Pichia pastoris system, achieving high collagen production.

[0030] This study was based on a partial sequence of the α1 chain of wild-type human type III collagen, the amino acid sequence of which is shown in SEQ ID NO:1 and GenBank accession number KAI4037253.1. While retaining the Gly-XY basic backbone, some hydrophobic amino acids (such as Tyr, Leu, Ile, Val, and His, except Pro) were replaced with hydrophilic residues (such as Asn, Gln, Lys, and Thr). Furthermore, some acidic amino acids (such as Asp and Glu) were replaced with basic amino acids (such as Lys and Arg), and the amino acid sequence was partially truncated at the termini. This improved the collagen's water solubility and bioavailability, enhancing its adaptation to the weakly alkaline environment in the body. The amino acid sequence of the modified type III collagen is shown in SEQ ID NO:2. After codon optimization, the nucleotide sequence is shown in SEQ ID NO:3. EcoRI and NotI restriction sites were added at both ends, and the target sequence was obtained by gene synthesis.

[0031] Example 2 Preparation of collagen

[0032] In this study, Pichia pastoris was selected to express the target protein. Pichia pastoris is a methylotrophic yeast with low cost, relatively short expression time, and co-translational and post-translational processing capabilities. Furthermore, Pichia pastoris is amenable to post-translational modifications, such as O- and N-linked glycosylation and disulfide bond formation, enabling proper folding, solubility, stability, and appropriate biological activity. Therefore, this expression system can effectively express exogenous proteins.

[0033] In this study, the target gene was amplified using PCR and restriction sites were introduced. After double digestion with EcoRI and NotI, the target gene was ligated into the expression vector pPIC9K, designated pPIC9K-muCL. This vector was transformed into Escherichia coli DH5α and selected for kanamycin resistance. Single colonies were cultured, and the recombinant plasmid pPIC9K-muCL was extracted using a plasmid extraction kit. The recombinant plasmid pPIC9K-muCL was linearized with SacI endonuclease and electroporated into Pichia pastoris GS115. Positive clones were screened, and the recombinant strains were designated GS115-muCL.

[0034] Yeast culture medium is a key factor in improving target protein expression. This invention improves upon the conventional yeast BMGY medium. In terms of carbon sources, mannitol was added to the glycerol already present in BMGY medium, enriching the carbon source variety. As for nitrogen sources, peptone and yeast extract remained the primary nitrogen sources, but the ratio was adjusted. Trace elements were also added to promote yeast growth. After optimization, the culture medium comprised: 15g / L glycerol, 5g / L mannitol, 20g / L peptone, 8g / L yeast extract, 0.5g / L manganese sulfate, 0.5g / L sodium molybdate dihydrate, 0.3g / L magnesium sulfate, 0.2g / L cobalt chloride, 0.06g / L zinc sulfate, 0.05g / L copper sulfate, and 0.02g / L chromium chloride, with the balance being water.

[0035] Induced expression is also a key factor affecting the expression of the target protein. The most common way to induce expression in Pichia pastoris is to use methanol induction, but methanol has a certain toxic effect on cells. If the dose is too large, it may kill the cells, and if the dose is too small, it is difficult to induce expression. Therefore, using methanol induction alone is often difficult to achieve the purpose of efficient expression of exogenous proteins. In the Pichia pastoris expression system, one of the most famous carbon sources that can be used with methanol is sorbitol. Sorbitol does not induce or inhibit the AOX promoter, so using sorbitol instead of glycerol in the mixed substrate method can reduce cell growth rate and increase the formation rate of specific products. In the present invention, methanol and sorbitol are used for mixed induction to increase the expression of the target protein. Preliminary experiments have shown that compared with the use of methanol alone, the protein expression amount can be increased by about 20-35%.

[0036] The GS115–muCL strain was cultured in YPD medium at 30°C and 220 rpm in a shaker. The modified medium was then inoculated with GS115–muCL at a 5% inoculum, with the volume not exceeding 20% ​​of the shake flask volume. The culture was shaken at 30°C and 260 rpm. When the OD600 reached 4–6, expression was induced for 72 hours by adding 1.2% methanol and 0.5% sorbitol. After induction, the supernatant was centrifuged at 5000 rpm for 10 minutes and the fermentation supernatant was collected. The target protein, designated muCL, was isolated and purified by Ni2+ affinity chromatography.

[0037] Example 3 Collagen promotes cell proliferation

[0038] In this section, we used the human epidermal cell line HaCaT and the fibroblast cell line BJ-5ta to study the physiological activity of collagen. The experiments were conducted using commercially available type III recombinant collagen (purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd., trade name Yuelixin) as a control sample. The type III recombinant collagen was designated as CL protein. The specific steps are as follows:

[0039] Take the above cells in the exponential growth phase and transfer 1×10 5 Cells were seeded in 96-well plates and randomly divided into three groups, with three replicates per group: the muCL group, with 50 ng / mL of muCL protein added to each well; the CL group, with 50 ng / mL of CL protein added to each well; and the control group, with an equal volume of culture medium added to each well. After 48 hours of culture, cell proliferation activity was determined using the MTT assay. Cells were incubated with 10 μL of 12 mM MTT solution at 37°C in the dark for 4 hours. The culture medium was decanted, and the cells were washed three times with PBS. The resulting formazan salt was dissolved in DMSO, and its absorbance was measured at 490 nm using a spectrophotometer.

[0040] Relative cell viability = (OD value of experimental well - OD value of blank control well) / (OD value of control well - OD value of blank control well), with the well containing only culture medium as the blank control well.

[0041] The results are as follows Figure 1 As shown, collagen can promote the proliferation of skin epidermal cells and fibroblasts, and the modified muCL has a stronger ability to promote cell proliferation, which is significantly improved compared with the control group, indicating that it has good application prospects in treating skin damage, promoting skin repair, and maintaining the normal physiological function of skin tissue.

[0042] Example 4 Collagen against skin oxidative damage

[0043] 4.1 UV skin damage modeling and drug administration

[0044] Mice were irradiated with UVB using an ultraviolet phototherapy device. Forty Balb / c mice were randomly divided into four groups, with 10 mice in each group. The normal group (NC group) was shaved without irradiation. The other groups were irradiated with UVB every day, with an initial dose of 100 mJ / cm 2 , increase by 500mJ / cm per day 2 , reaching 300mJ / cm 2 Then keep it unchanged for a total of 2 weeks.

[0045] After irradiation, the model groups were given medications: muCL group: 100 mg / kg of recombinant muCL protein was applied to the back every day; CL group: 100 mg / kg of recombinant CL protein was applied to the back every day; control group: 100 mg / kg of normal saline was applied to the back every day. Each group was given medication for a total of 6 weeks.

[0046] 4.2 Pathological observation

[0047] After the experiment, the mice in each group were killed, and 1 cm × 1 cm skin of the irradiated area on the back was taken and fixed with 4% paraformaldehyde. Pathological sections were prepared after dehydration, embedding, and sectioning. The sections were stained with HE and Masson, and the histopathological changes were observed under an optical microscope.

[0048] The results are as follows Figure 2 As shown, the normal group showed neatly arranged collagen fibers in the dermis, with a well-organized skin structure. The control group showed significantly thinner dermis, with fewer collagen fibers, disordered arrangement, and uneven density. Treatment with muCL and CL resulted in thicker, more uniform dermis, a more intact structure, and clearer layers. Furthermore, the muCL group showed better recovery, with neatly arranged dermal fibers and a more complete structure. These results demonstrate that the recombinant collagen provided by this invention can alleviate UV-induced skin damage in mice and promote the recovery of skin physiological functions.

[0049] 4.3 Oxidative stress detection

[0050] UVB can indirectly damage skin cell DNA by stimulating oxidative stress, causing skin cell necrosis. Reducing skin oxidative stress is an effective way to prevent skin photoaging. Therefore, this section measured oxidative stress-related indicators in skin tissue to examine the protective and repair effects of collagen on the skin. Appropriate amounts of dorsal skin from each group of mice were mixed with physiological saline, ground into a homogenate, and centrifuged to obtain the supernatant. Skin SOD and MDA levels were measured according to the instructions of the kit (purchased from Beijing Solebold Technology Co., Ltd.).

[0051] like Figure 3 、 Figure 4As shown, compared with the NC group, the SOD level in the skin of the control mice was significantly reduced, while the MDA level was significantly increased, indicating that oxidative stress caused severe damage to the skin. After treatment, SOD levels recovered and MDA levels decreased significantly, with the therapeutic effect being more pronounced in the muCL group. This demonstrates that the collagen provided in this application can effectively combat oxidative stress in skin tissue.

[0052] 4.4 Detection of inflammatory factor secretion

[0053] The skin tissue fluid of each group of mice was collected (prepared as in Section 4.3) and the IL-1β and IL-6 levels in the tissue fluid were detected using ELISA kits (purchased from Nanjing Jiancheng Technology Co., Ltd.). Figure 5 、 Figure 6 As shown, compared with the normal group, the levels of IL-1β and IL-6 in the skin of the control mice were significantly increased. After treatment, the levels of inflammatory factors in the treated groups were significantly reduced, and the IL-1β and IL-6 levels in the muCL group were closer to normal physiological levels, indicating that the collagen can inhibit the secretion of inflammatory factors and protect against UV-induced skin damage.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type III collagen, characterized in that: The amino acid sequence of the type III collagen is shown in SEQ ID NO:

2.

2. A method for preparing type III collagen according to claim 1, characterized in that: The method comprises: introducing a nucleotide sequence encoding the type III collagen into a yeast expression vector, fermenting and culturing yeast containing the yeast expression vector, inducing with methanol and sorbitol, and harvesting the type III collagen.

3. The method according to claim 2, characterized in that The nucleotide sequence encoding the type III collagen is shown in SEQ ID NO:

3.

4. The method according to claim 2, characterized in that The yeast is Pichia pastoris.

5. The method according to claim 2, characterized in that The culture medium used in the culture process included: 15 g / L glycerol, 5 g / L mannitol, 20 g / L peptone, 8 g / L yeast extract, 0.5 g / L manganese sulfate, 0.5 g / L sodium molybdate dihydrate, 0.3 g / L magnesium sulfate, 0.2 g / L cobalt chloride, 0.06 g / L zinc sulfate, 0.05 g / L copper sulfate and 0.02 g / L chromium chloride.

6. The method according to any one of claim 5, characterized in that The culturing step comprises: using YPD medium to culture the yeast in a shaking platform at 30°C and 220 r / min; inoculating the yeast into the culture medium according to claim 5 at an inoculum amount of 5%, culturing in a shaking platform at 30°C and 260 r / min, and when the OD600 reaches 4-6, adding 1.2% methanol and 0.5% sorbitol with a volume fraction to induce expression for 72 hours; after the induction is completed, centrifuging at 5000 r / min for 10 minutes to collect the fermentation supernatant, and separating and purifying to obtain type III collagen.

7. Use of the type III collagen according to any one of claims 1 to 6 in the preparation of a product for protecting against skin damage.

8. The use according to claim 7, characterized in that The damage is ultraviolet damage.

9. The use according to claim 7, characterized in that The product is a cosmetic.

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