Recombinant collagen having skin penetration effect, preparation method therefor, and use thereof

Abstract

Provided are a recombinant collagen having a skin penetration effect, a preparation method therefor, and the use thereof, belonging to the technical fields of protein engineering and genetic engineering. A novel recombinant collagen is designed and obtained by means of methods such as screening, replacement, splicing, etc.; the nucleotide sequence thereof is optimized for codon preference of Komagataella pastoris, and then a cell-penetrating peptide is introduced at the N terminus of the sequence; a corresponding genetically-engineered yeast strain is constructed and obtained, and fermentation is performed to successfully prepare the recombinant collagen. Compared with commercially available recombinant collagens, the prepared recombinant collagen has better free radical scavenging performance and skin penetration performance, thereby effectively solving the problem of poor skin penetration of existing collagens, and further improving the biological activity and efficacy of same.

Description

A recombinant collagen with transdermal effect, its preparation method and application

[0001] Cross-reference to related applications

[0002] This invention claims priority to Chinese Patent Application No. 202411466639.9, filed on October 21, 2024, entitled "A Recombinant Collagen with Transdermal Effect and its Preparation Method and Application", the entire contents of which are incorporated herein by reference and constitute a part of this invention for all purposes. Technical Field

[0003] This invention belongs to the fields of protein engineering and genetic engineering technology, specifically relating to a recombinant collagen with transdermal effect, its preparation method, and its application. Background Technology

[0004] Collagen is the most abundant and widely distributed functional protein in mammals, accounting for 25%–30% of total protein. It plays a crucial role in connecting and supporting bones, skin, and joints. Collagen can also interact with various macromolecules such as integrins, decorative proteins, fibronectin, heparin, and matrix metalloproteinases, playing an important role in regulating tissue regeneration. To date, 29 types of collagen have been discovered. Among them, type III collagen is mainly found in connective tissues such as skin, tendons, ligaments, and joints, forming the extracellular matrix network structure. It is the most abundant type of collagen in the dermis and is therefore widely used in cosmetic skincare, medical devices, and medical aesthetics products.

[0005] Type III collagen obtained from animal tissues suffers from low content, difficulty in purification, and contamination by animal-derived viruses, which limits its application and development to some extent. Recombinant collagen, synthesized using genetic engineering technology, possesses higher bioactivity and biocompatibility, lower immunogenicity, and a lower risk of missed pathogen detection, thus attracting significant attention. Furthermore, the stratum corneum, as the body's natural barrier, often restricts the entry of large molecules into the dermis. Currently, commercially available collagen molecules generally have large molecular weights, making it difficult for them to cross the epidermal barrier and reach the superficial dermis to exert their biological functions. Cell-penetrating peptides are short peptides that can carry large molecules into cells and the skin. Novel protein molecules formed by linking cell-penetrating peptides with collagen using molecular biology methods may possess excellent transdermal activity. Therefore, using genetic engineering and protein engineering techniques to artificially design and synthesize recombinant collagen with an amino acid sequence similar to type III collagen and good transdermal effects holds promise for solving the aforementioned problems. Based on this technology, the applicant has previously developed a recombinant transdermal collagen, SPACE-Col7 (CN118359703A). However, the antioxidant and transdermal properties of this recombinant transdermal collagen still need to be improved. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a recombinant collagen with transdermal effects, its preparation method, and its applications. Specifically, the recombinant collagen SPACE-Col4 produced by the present invention exhibits superior free radical scavenging and transdermal properties compared to commercially available recombinant collagen and the previously reported SPACE-Col7 (CN118359703A). Based on the above research findings, the present invention is thus completed.

[0007] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a recombinant collagen protein having:

[0009] (a1) The amino acid sequence as shown in SEQ ID NO.1;

[0010] (a2) A protein derived from the amino acid sequence shown in SEQ ID NO.1 by substitution and / or deletion and / or addition of one or more amino acid residues and having the same function;

[0011] (a3) Other genes that encode proteins that have more than 90% similarity to the amino acid sequence composition shown in SEQ ID NO.1 and have the protein activity shown in SEQ ID NO.1.

[0012] The recombinant collagen is further modified with a transdermal peptide, thereby effectively enhancing its transdermal properties. The transdermal peptide can be modified at the carboxyl terminus (C-terminus) or amino terminus (N-terminus) of the recombinant collagen, preferably at the amino terminus.

[0013] In a second aspect, the present invention provides a polynucleotide capable of encoding the aforementioned recombinant collagen.

[0014] A third aspect of the present invention provides a recombinant expression vector comprising the aforementioned polynucleotide.

[0015] In a fourth aspect, the present invention provides a host bacterium containing the above-described recombinant expression vector or chromosome integrating the above-described polynucleotides or capable of expressing the above-described recombinant collagen.

[0016] A fifth aspect of the present invention provides a method for preparing the recombinant collagen, the method comprising: culturing the host bacteria to express the recombinant collagen; and separating and purifying the recombinant collagen.

[0017] A sixth aspect of the present invention provides the use of the above-described recombinant collagen in the preparation of cosmetics, medical aesthetic products, or pharmaceuticals.

[0018] A seventh aspect of the present invention provides a cosmetic product comprising at least the aforementioned recombinant collagen.

[0019] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0020] The above technical solution designs a novel recombinant collagen protein through screening, replacement, splicing and other methods. Its nucleotide sequence is optimized by Pichia pastoris codon preference, and then a membrane-penetrating peptide is introduced at the amino terminus of the sequence. The corresponding genetically engineered yeast strain is constructed and successfully fermented to produce recombinant collagen protein.

[0021] Experiments have shown that the recombinant collagen prepared by the above technical solution has better free radical scavenging and transdermal properties compared with commercially available recombinant collagen, thus effectively solving the current problem of difficult transdermal collagen production and further improving its bioactivity and efficacy, thus having good practical application value. Attached Figure Description

[0022] Figure 1 shows the SDS-PAGE and Western Blot results of the fermentation supernatant of recombinant collagen SPACE-Col4 in Example 4 of the present invention; wherein, A is the SDS-PAGE protein electrophoresis diagram, in which lane M represents the standard protein with a molecular weight of 180kDa; lane 1 represents the fermentation supernatant of control strain P. pastoris GS115 / pPIC9K; lane 2 represents the fermentation supernatant of recombinant strain P. pastoris GS115 / pPIC9K-SPACE-Col4; and B is the Western Blot result diagram.

[0023] Figure 2 shows the antioxidant experiments of different collagens in Example 6 of the present invention; wherein, Comparative Example 1 is a commercially available recombinant collagen; Comparative Example 2 is a recombinant transdermal collagen SPACE-Col7; Col4 is a recombinant collagen Col4 prepared based on Example 5 of the present invention; SPACE-Col4 is a recombinant collagen SPACE-Col4 prepared based on Example 5 of the present invention.

[0024] Figure 3 is a comparison of the transdermal performance test results of different collagens in Example 7 of the present invention; wherein, the control group is commercially available recombinant collagen; experimental group 1 is recombinant transdermal collagen SPACE-Col7; experimental group 2 is recombinant collagen Col4 prepared based on Example 5 of the present invention; SPACE-Col4 is recombinant collagen SPACE-Col4 prepared based on Example 5 of the present invention.

[0025] Figure 4 shows fluorescence micrographs of pig skin in the fluorescence penetration test of different collagens in Example 7 of the present invention; the control group is the comparative experimental group of commercially available recombinant collagen; experimental group 1 is recombinant transdermal collagen SPACE-Col7; experimental group 2 is recombinant collagen Col4 prepared based on Example 5 of the present invention; SPACE-Col4 is recombinant collagen SPACE-Col4 prepared based on Example 5 of the present invention. Detailed Implementation

[0026] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] As mentioned earlier, the collagen molecules currently used in the market are generally large in size, making it difficult for them to cross the epidermal barrier and enter the superficial dermal tissue to exert their biological effects.

[0029] In view of this, in a typical embodiment of the present invention, a recombinant collagen is provided, the recombinant collagen having:

[0030] (a1) The amino acid sequence as shown in SEQ ID NO.1;

[0031] (a2) A protein derived from the amino acid sequence shown in SEQ ID NO.1 by substitution and / or deletion and / or addition of one or more amino acid residues and having the same function;

[0032] (a3) Other genes that encode proteins that have more than 90% similarity to the amino acid sequence composition shown in SEQ ID NO.1 and have the protein activity shown in SEQ ID NO.1.

[0033] In another specific embodiment of the present invention, the recombinant collagen is further modified with a transdermal peptide, thereby effectively enhancing its transdermal properties. The transdermal peptide may be modified at the carboxyl terminus (C-terminus) or amino terminus (N-terminus) of the recombinant collagen, preferably at the amino terminus.

[0034] The membrane-penetrating peptide may be the membrane-penetrating peptide SPACE (amino acid sequence as shown in SEQ ID NO.3), the membrane-penetrating peptide TAT (amino acid sequence as shown in SEQ ID NO.4), or the membrane-penetrating peptide ANTP (amino acid sequence as shown in SEQ ID NO.5); more preferably, the membrane-penetrating peptide is the membrane-penetrating peptide SPACE.

[0035] In another specific embodiment of the present invention, a polynucleotide is provided, the polynucleotide being capable of encoding the above-mentioned recombinant collagen;

[0036] The polynucleotides are selected from:

[0037] (b1) A nucleotide sequence as shown in either SEQ ID NO.2 or SEQ ID NO.6;

[0038] (b2) and (b1) have nucleotide sequences that encode proteins with the same amino acid sequence, but are different in sequence due to the degeneracy of the genetic code.

[0039] (b3) A nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (b1) or (b2) and encodes the same functional protein;

[0040] (b4) is a complementary nucleotide sequence to either (b1) or (b3).

[0041] In another specific embodiment of the present invention, a recombinant expression vector is provided, the recombinant expression vector comprising the above-mentioned polynucleotide.

[0042] In this invention, the recombinant expression vector is obtained by effectively linking the above-mentioned polynucleotide to the expression vector. The expression vector is a plasmid, specifically any plasmid vector in the pPIC series. Further, the expression vector is the pPIC9K plasmid.

[0043] In another specific embodiment of the present invention, a host bacterium is provided, wherein the host bacterium contains the above-mentioned recombinant expression vector or chromosome integrating the above-mentioned polynucleotide or is capable of expressing the above-mentioned recombinant collagen.

[0044] The host bacteria can be eukaryotic or prokaryotic bacteria, such as bacteria, fungi, actinomycetes, etc. Further, the host bacteria can be yeast, a type of fungus. The yeast can be Pichia pastoris (such as Pichia pastoris GS115, KM71, or SMD1168).

[0045] In another specific embodiment of the present invention, a method for preparing the recombinant collagen is provided, the method comprising: culturing the above-mentioned host bacteria to express the recombinant collagen; and separating and purifying the recombinant collagen.

[0046] Furthermore, the separation and purification includes steps such as hollow fiber clarification, ultrafiltration membrane concentration and replacement, ion exchange chromatography, ultrafiltration membrane desalting, and lyophilization, which are not specifically limited here.

[0047] In another specific embodiment of the present invention, the above-mentioned recombinant collagen is provided for use in the preparation of cosmetics, medical aesthetic products or pharmaceuticals.

[0048] The cosmetics described herein can be applied to the skin to achieve antioxidant and skin barrier repair effects. Therefore, the cosmetics described in this invention can be skin care products. The cosmetic dosage form can be an aqueous solution, emulsion, cream, etc. At the same time, based on the above-mentioned basic cosmetic categories, other cosmetic categories can be further derived and prepared, such as facial cleanser, sunscreen, toner, face cream, eye cream, essence water (lotion), hand cream (ointment), body lotion, and face mask, etc.

[0049] In this invention, the medical aesthetic products refer to products used in the field of medical aesthetics. Some commonly used medical aesthetic products include medical aesthetic masks, dressings, etc. The use of these products requires professional medical personnel or relevant guidance.

[0050] In this invention, the drug can be a drug-device combination product, which refers to a product composed of a drug and a medical device, and manufactured as a single entity. One specific embodiment of the drug-device combination product can be a topical dressing containing the aforementioned recombinant collagen.

[0051] In another specific embodiment of the present invention, a cosmetic product is provided, the cosmetic product comprising at least the above-mentioned recombinant collagen.

[0052] Furthermore, the cosmetics mentioned are specifically skincare products.

[0053] Of course, the cosmetics may also contain other ingredients permitted in any cosmetic field, including but not limited to suspending agents, emulsifiers, emollients, moisturizers, pH adjusters, thickeners, and fragrances. Those skilled in the art can choose to add these ingredients according to the actual situation, and no specific limitations are made here.

[0054] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The following examples illustrate test methods with specific conditions, which are generally performed under conventional conditions.

[0055] The components of the culture medium used in the examples are as follows:

[0056] YPD medium: yeast extract 10g / L, peptone 20g / L, glucose 20g / L.

[0057] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, K₂HPO₄ 3 g / L, KH₂PO₄ 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10⁻⁶ -4 g / L, glycerol 10g / L.

[0058] BMMY medium: yeast extract 10 g / L, peptone 20 g / L, K₂HPO₄ 3 g / L, KH₂PO₄ 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10⁻⁶ -4 g / L, methanol 10mL / L.

[0059] Example 1: Sequence Design of Recombinant Collagen

[0060] In this embodiment, the amino acid sequence of natural human type III collagen α1 chain protein (serial number NP_000081) was analyzed. Based on the characteristics of natural collagen amino acid sequences and reported active sites, an amino acid sequence was finally obtained through screening, replacement, and splicing. The amino acid sequence is shown in SEQ ID NO.1, which is the recombinant collagen Col4.

[0061] Example 2: Construction of a recombinant collagen gene expression system

[0062] Based on the codon bias of Pichia pastoris, the sequence of recombinant collagen Col4 was optimized, and the optimized nucleotide sequence is shown in SEQ ID NO.2. Subsequently, the codon-optimized nucleotide sequence of recombinant collagen Col4 was synthesized by GenScript Biotech Co., Ltd., and cloned into the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K to obtain the recombinant expression vector pPIC9K-Col4. DNA sequencing confirmed the correct recombinant sequence. After linearizing the recombinant expression plasmid pPIC9K-Col4 with SalI rapid digestion, it was electroporated into P. pastoris GS115 expression host cells. The obtained recombinant transformants were screened with genimycin G418 to obtain high-copy recombinant Pichia pastoris GS115 / pPIC9K-Col4.

[0063] Example 3: Shake-flask fermentation of recombinant collagen engineered yeast strain

[0064] The obtained recombinant engineered strain *P. pastoris* GS115 / pPIC9K-Col4 was cultured in shake flasks. The specific fermentation steps were as follows: Single colonies were picked and inoculated into 40 mL of YPD medium and cultured at 30℃ and 200 rpm for 24 h. A 10% inoculum was then transferred to 40 mL of initial expression medium (BMGY) and cultured at 30℃ and 200 rpm for 24 h. The cells were collected by centrifugation, washed with physiological saline, and then transferred to 40 mL of induction expression medium (BMMY) and cultured at 30℃ and 200 rpm. Pure methanol was added to the medium every 24 h until a final concentration of 1.0% (v / v) was reached, and expression was induced for 96 h.

[0065] Example 4: Construction and protein expression of a high-copy strain of recombinant collagen

[0066] To improve the transdermal effect of recombinant collagen, the transmembrane peptides SPACE (amino acid sequence as shown in SEQ ID NO.3), TAT (amino acid sequence as shown in SEQ ID NO.4), and ANTP (amino acid sequence as shown in SEQ ID NO.5) were sequence-optimized according to the codon preference of Pichia pastoris and then ligated to the N-terminus of the Col4 sequence to obtain three recombinant collagen proteins: SPACE-Col4 (nucleotide sequence as shown in SEQ ID NO.6), TAT-Col4 (nucleotide sequence as shown in SEQ ID NO.7), and ANTP-Col4 (nucleotide sequence as shown in SEQ ID NO.8).

[0067] The encoding genes for three recombinant collagen proteins were cloned into the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K, respectively, to obtain the recombinant expression vectors pPIC9K-SPACE-Col4, pPIC9K-TAT-Col4, and pPIC9K-ANTP-Col4. DNA sequencing confirmed the correct sequence of the recombinant expression vectors. After linearization with SalI rapid digestion, the recombinant expression plasmids were electroporated into P. pastoris GS115 expression host cells. The recombinant transformants were selected using genimycin G418 to obtain high-copy recombinant Pichia pastoris GS115 / pPIC9K-SPACE-Col4, P. pastoris GS115 / pPIC9K-TAT-Col4, and P. pastoris GS115 / pPIC9K-ANTP-Col4.

[0068] SDS-PAGE protein electrophoresis was performed on the fermentation supernatant of the recombinant engineered bacteria and the fermentation supernatant of the control bacteria. The electrophoresis results are shown in Figure 1A. Near the theoretical protein molecular weight of 35 kDa, an additional protein band (indicated by the arrow) appeared in the fermentation supernatant (lane 2) of the recombinant engineered bacteria P. pastoris GS115 / pPIC9K-SPACE-Col4. Further Western blotting analysis, as shown in Figure 1B, confirmed that the band at this location was recombinant collagen SPACE-Col4.

[0069] Example 5: Preparation of recombinant collagen Col4 and SPACE-Col4

[0070] Recombinant bacteria P. pastoris GS115 / pPIC9K-Col4 and P. pastoris GS115 / pPIC9K-SPACE-Col4 were cultured in shake flasks for fermentation. The fermentation supernatant was purified by hollow fiber clarification, ultrafiltration membrane concentration and replacement, ion exchange chromatography, ultrafiltration membrane desalting, and lyophilization (for specific purification steps, please refer to CN118359703A) to obtain pure recombinant collagen. The protein purified by the above steps had a purity greater than 96% and a yield greater than 55%.

[0071] Example 6: Antioxidant Experiment of Recombinant Collagen

[0072] Take 2.5 mL of the purified stock solution of recombinant collagen with a concentration of 1 mg / mL from Example 5 and place it in a test tube. Add 2.5 mL of DPPH free radical solution (0.1 mmol / L) dissolved in 95% ethanol. Shake the mixture vigorously for 10 s and then let it react at room temperature for 30 min. After the reaction is complete, measure the absorbance of the reaction mixture at 517 nm. Use distilled water instead of sample solution as a blank control. Comparative experiments were conducted with commercially available and self-developed recombinant collagen (Comparative Example 1: the recombinant collagen was purchased from Hebei Naco Biotechnology Co., Ltd., model number 3CH-FD05; Comparative Example 2: the recombinant collagen was independently developed by the company, namely the recombinant transdermal collagen SPACE-Col7 disclosed in CN118359703A).

[0073] DPPH free radical scavenging activity = (OD blank - OD sample) / OD blank

[0074] Among them, OD blank is the absorbance value of the blank control group, and OD sample is the absorbance value of recombinant humanized collagen or commercially available recombinant collagen group.

[0075] As shown in Figure 2, the recombinant collagen SPACE-Col4 of the present invention has a significant DPPH free radical scavenging ability compared with the blank control, and its DPPH free radical scavenging rate is higher than that of commercially available recombinant collagen. Therefore, the recombinant collagen provided in this embodiment has a significant antioxidant effect.

[0076] Example 7: Transdermal Performance Test of Recombinant Collagen

[0077] This test used piglet skin tissue to conduct a permeability experiment on the samples. The accumulation of fluorescently labeled substances in the isolated skin tissue at different time points was observed using a fluorescence microscope to evaluate the skin permeability behavior of the samples. The test was conducted according to the test protocol in Table 1.

[0078] Table 1 Test Plan

[0079] The test results are shown in Figures 3 and 4. In all three experimental groups, the accumulation of fluorescent substances increased significantly with increasing test time, and the fluorescence intensity of the experimental groups was greater than that of the commercially available recombinant collagen in the control group. Furthermore, the fluorescence intensity of recombinant collagen SPACE-Col4 was significantly greater than that of recombinant collagen Col4 and SPACE-Col7. These results indicate that, compared to commercially available recombinant collagen, recombinant collagen Col4, and SPACE-Col7, recombinant collagen SPACE-Col4 with membrane-penetrating peptides exhibits the best transdermal performance.

[0080] Matters not covered in this invention are common knowledge.

[0081] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

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

1.

2. A recombinant collagen, characterized in that, The recombinant collagen is further modified with a membrane-penetrating peptide, which modifies the amino terminus of the recombinant collagen; the membrane-penetrating peptide is SPACE, and its amino acid sequence is shown in SEQ ID NO.3; the nucleotide sequence encoding the gene of the recombinant collagen is shown in SEQ ID NO.

6.

3. A polynucleotide, characterized in that, The polynucleotide encodes the recombinant collagen of claim 1 or 2; the polynucleotide is selected from: (b1) A nucleotide sequence as shown in either SEQ ID NO.2 or SEQ ID NO.6; (b2) and (b1) are nucleotide sequences that encode proteins with the same amino acid sequence, but are different in sequence due to the degeneracy of the genetic code.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide of claim 3.

5. A host bacterium, characterized in that, The host bacterium contains the recombinant expression vector of claim 4 or integrates the polynucleotide of claim 3 or expresses the recombinant collagen of any one of claims 1-2.

6. The host bacterium as described in claim 5, characterized in that, The host bacteria are bacteria, fungi, and actinomycetes.

7. The host bacterium as described in claim 6, characterized in that, The fungus is yeast.

8. The host bacterium as described in claim 7, characterized in that, The yeast is Pichia pastoris.

9. A method for preparing recombinant collagen as described in claim 1 or 2, characterized in that, The preparation method includes: culturing the host bacteria according to any one of claims 5-8 to express the recombinant collagen; and isolating and purifying the recombinant collagen.

10. The use of the recombinant collagen according to claim 1 or 2 in the preparation of cosmetics or medical aesthetic products.

11. A cosmetic product, characterized in that, The cosmetic product contains the recombinant collagen as described in claim 1 or 2.

12. The cosmetic product as described in claim 11, characterized in that, The cosmetics in question are skincare products.

Images