Method for improving soluble expression and stability of recombinant collagen

Abstract

The invention discloses a method for improving soluble expression and stability of recombinant collagen, and belongs to the technical field of genetic engineering. According to the invention, a part of proline in a repetitive sequence (GPP) 10 is transformed into aspartic acid and lysine. The repetitive sequence is suitable for various collagen egg domains, the purity of the constructed collagen reaches 90% or above, the yield is increased by 23.6% or above compared with that of unmodified collagen (V-P10BP10) composed of the repetitive sequence (GPP) 10, heat stability and solubility are improved, and meanwhile good salt tolerance is kept. According to the invention, the yield of produced collagen is improved, and the method has wide application prospects in the field of biological materials.

Description

Technical Field

[0001] The present invention relates to a method for improving the soluble expression and stability of recombinant collagen, belonging to the technical field of genetic engineering. Background Art

[0002] Collagen is the most abundant protein in mammals, widely distributed in skin, bones, tendons, vascular systems, muscle connective tissues, etc. So far, 28 types have been discovered, mainly divided into fibrous collagen, reticular collagen, beaded filamentous collagen, anchoring fibril protein, etc. Among them, type I, type II, and type III collagen in fibrous collagen account for 80% - 90% of human collagen.

[0003] The collagen sequence is composed of repeating peptide triplets (Gly-Xaa-Yaa), where Xaa and Yaa are usually proline and hydroxyproline. The structural basis of collagen is a left-handed helical conformation of polyproline type II formed by the interlacing of 3 α-chains, namely the triple helix structure. The correctly folded triple helix structure is crucial for collagen to achieve cell-cell interaction in the extracellular matrix. Research on the collagen sequence shows that the close packing of the three chains of collagen, the hydrogen bonds formed between the chains, the extensive hydration network, and the high content of proline and hydroxyproline are important factors affecting the stability of the collagen triple helix structure. Due to the good biological properties of collagen, it has a wide range of applications in tissue engineering, clinical medicine, food industry, packaging materials, cosmetics, medical beauty, biomaterials, and medical devices.

[0004] Currently, according to the source, collagen is mainly divided into animal-extracted collagen and recombinant collagen. Animal-derived collagen is mainly sourced from terrestrial animals and marine animals, and the extraction process is complex and has safety hazards. Recombinant collagen is a protein obtained by cloning the collagen gene into a selected expression vector, transforming it into an expression cell, and finally through purification technology. Recombinant collagen has higher purity, can be mass-produced, has small differences between batches, has good water solubility, strong processability, no virus hazards, has a lower rejection reaction, and more importantly, can be modified by modifying the gene sequence in recombinant collagen to endow it with more biological functions, thus having a wide application prospect in the fields of tissue engineering and regenerative medicine.

[0005] Currently, the commonly used expression systems for recombinant collagen are Escherichia coli. As the most well-studied prokaryotic bacterium, Escherichia coli has the advantages of clear genetic background, convenient genetic manipulation, short fermentation cycle, and high expression level, and is the most widely used host bacterium for heterologous protein expression. However, due to the lack of post-translational modification, recombinant collagen often has difficulty in correctly folding into a triple helix structure.

[0006] In the previous research, the inventor team designed the sequence of collagen so that the recombinant collagen could be correctly folded to form a triple helix structure. Qi Jingjing's master's thesis "Design and heterologous expression of biomimetic microfibril collagen" analyzed the α1 chain of type I collagen through protein computational analysis and thermal stability prediction, and selected the predicted T m The value is close to 37°C, the sequence has a high triple helix tendency, and (GPP) is added to the N and C termini of the sequence 10 The sequence forms a ribbon-like fiber with periodic light and dark stripes of different dark stripe lengths. However, the yield of this sequence is low and its thermal stability needs to be improved. Hu Jinyuan's paper "Recombinant Expression and Thermal Stability of Proline-Rich Collagen" adds (GPP) at both ends of the collagen sequence. n (n=5, 10, 15), it was found that the two ends of the collagen region were embedded with a certain length of (GPP) n Short peptides can promote the folding of collagen regions, but too long (GPP) 15 This will cause the collagen region to be unable to fold correctly to form a triple helix structure; CN111333715B discloses a method for preparing type I collagen fibers, with N and C termini (GPP) 10 The sequence is based on the collagen sequence, and a continuous Gly-Xaa-Yaa triplet is inserted in the middle to form a ribbon fiber with periodic light and dark stripes. However, the existing technologies all have problems such as low protein expression and poor stability.

[0007] Therefore, it is necessary to design a method that can increase the expression level of recombinant collagen, has high stability, and can promote the correct folding of recombinant collagen to form a triple helix structure. Summary of the invention

[0008] In order to solve the problems of low collagen expression and poor stability in the prior art, the present invention provides a method for synthesizing collagen in a repetitive sequence (GPP). 10 On the basis of the above, part of the proline in the sequence is transformed into aspartic acid and lysine. The method and the repeat sequence improvement method of the present invention are universal and applicable to a variety of collagen domains. The collagen constructed by the improved repeat sequence is relatively (GPP) 10 The constructed collagen has the advantages of increased expression, maintained or improved thermal stability, and increased solubility, while also maintaining good salt tolerance.

[0009] The first object of the present invention is to provide a collagen single chain, wherein the single chain comprises a structure such as a first repeating sequence-collagen domain-second repeating sequence (such as Figure 10as shown); wherein, the amino acid sequence of at least one of the first repeat sequence and the second repeat sequence contains the sequence shown in SEQ ID NO.4 (abbreviated as KD2) or SEQ ID NO.5 (abbreviated as KD3); and when the first repeat sequence is SEQ ID NO.4, the second repeat sequence is SEQ ID NO.4 or SEQ ID NO.3.

[0010] In one embodiment, in the first repeat sequence and the second repeat sequence of the single-chain collagen, the amino acid sequence of one of them is as shown in SEQ ID NO.3 (abbreviated as P10).

[0011] In one embodiment, the amino acid sequence of the first repeat sequence of the single-chain collagen is as shown in SEQ ID NO.5, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.3 (abbreviated as P10);

[0012] Or, the amino acid sequence of the first repeat sequence of the single-chain collagen is as shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.4;

[0013] Or, the amino acid sequence of the first repeat sequence of the single-chain collagen is as shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.5;

[0014] Or, both the first repeat sequence and the second repeat sequence of the single-chain collagen are as shown in SEQ ID NO.4.

[0015] The core point of the present invention lies in the design of the repeat sequence, which can be applied to different collagen domains and can produce any one or more of the effects of increased expression level, increased stability, and increased solubility. Optionally, the collagen domain can be any collagen domain sequence from human collagen or bacterial collagen; for example, the collagen domain sequence can be the collagen domain sequence disclosed in CN111333715A or the collagen domain sequence disclosed in CN 117186210 A.

[0016] In one embodiment, the amino acid sequence of the collagen domain is as shown in SEQ ID NO.16-22; or is obtained by combining any two amino acid sequences among SEQ ID NO.16-18; or is obtained by repeating any one of the amino acid sequences shown in SEQ ID NO.16-22 2-3 times. Optionally, the amino acid sequence of the collagen domain is obtained by splicing SEQ ID NO.16 and SEQ ID NO.17 in sequence, or by splicing 2 SEQ ID NO.17.

[0017] In one embodiment, the amino acid sequence of the collagen domain is as shown in SEQ ID NO.2.

[0018] In one embodiment, the N-terminus of the first repeat sequence further contains a folding domain with a structure as Figure 9 shown.

[0019] In one embodiment, the folding domain and the first repeat sequence are connected by a cleavage site; optionally, the cleavage site is LVPRGSP.

[0020] In one embodiment, the amino acid sequence of the folding domain is as shown in SEQ ID NO.1.

[0021] In one embodiment, a glycine can be added to the end of the amino acid sequence of the single-chain collagen.

[0022] In one embodiment, the amino acid sequence of the single-chain collagen is as shown in any one of SEQ ID NO.6 to SEQ ID NO.9.

[0023] The second object of the present invention is to provide a gene encoding any one of the above single-chain collagens.

[0024] The present invention also provides a plasmid or a cell carrying the above gene.

[0025] In one embodiment, it includes: plasmids of the pColdIII series or pET series.

[0026] In one embodiment, the plasmid is pColdIII.

[0027] In one embodiment, it is an Escherichia coli cell, including E.coli BL21, E.coli BL21(DE3), E.coli JM109, E.coli DH5α or E.coli TOP10.

[0028] In one embodiment, the host cell is E.coli BL21(DE3).

[0029] The third object of the present invention is to provide a method for improving the stability of collagen. A first repeat sequence is connected to the N-terminus of the collagen domain, and a second repeat sequence is connected to the C-terminus to construct a single-chain collagen with a structure of first repeat sequence - collagen domain - second repeat sequence (as Figure 10 shown); wherein the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0030] The present invention also provides a method for improving the solubility of collagen, which involves connecting a first repeat sequence to the N-terminus of the collagen domain and a second repeat sequence to the C-terminus to construct a single-chain collagen with the structure of first repeat sequence - collagen domain - second repeat sequence (as shown in Figure 10 ); wherein the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0031] The present invention also provides a method for increasing the yield of collagen, which involves connecting a first repeat sequence to the N-terminus of the collagen domain and a second repeat sequence to the C-terminus to construct a single-chain collagen with the structure of first repeat sequence - collagen domain - second repeat sequence (as shown in Figure 10 ), and expressing it through a host cell; wherein the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0032] The fourth object of the present invention is to provide a collagen composed of three single-chain collagens with the structure of first repeat sequence - collagen domain - second repeat sequence (as shown in Figure 10 ); wherein the amino acid sequence of at least one of the first repeat sequence and the second repeat sequence further contains the sequence as shown in SEQ ID NO.4 (abbreviated as KD2) or SEQ ID NO.5 (abbreviated as KD3).

[0033] In one embodiment, the single-chain collagen coils around a common central axis to form a triple helix structure.

[0034] In one embodiment, the collagen is obtained by expressing the above single-chain collagen in a host and then enzymatically removing the folding domain.

[0035] In one embodiment, the amino acid sequences of both the first repeat sequence and the second repeat sequence of the single-chain collagen are as shown in SEQ ID NO.4; or, the amino acid sequence of the first repeat sequence of the single-chain collagen is as shown in SEQ ID NO.5, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.3; or, the amino acid sequence of the first repeat sequence of the single-chain collagen is as shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.4; or the amino acid sequence of the first repeat sequence of the single-chain collagen is as shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.5.

[0036] The present invention also provides collagen fibers formed by the self-assembly of the above collagen.

[0037] In one embodiment, the folding domain and the first repeating sequence are connected via a restriction site. Alternatively, the folding domain and the first repeating sequence are connected via LVPRGSP.

[0038] The present invention also provides the use of the above-mentioned collagen single chain or collagen or collagen fiber in the preparation of collagen products.

[0039] The present invention also provides a collagen product, which contains any of the above-mentioned collagen single chains or collagen or collagen fibers.

[0040] In one embodiment, the collagen product also contains vitamins, minerals, hyaluronic acid, natural polysaccharides, essential oils, polyphenols, natural plant extracts, and the like.

[0041] The present invention also provides the use of the above-mentioned collagen single chain or collagen or collagen fiber in the preparation of collagen products.

[0042] In one embodiment, the collagen product is a cosmetic, a food or a medicine.

[0043] In one embodiment, the collagen product can be a drug carrier, a medical product or a cosmetic product.

[0044] Beneficial effects:

[0045] The present invention improves the repetitive sequence in collagen expression and transforms part of the proline in the sequence into aspartic acid and lysine.

[0046] The method of the present invention is versatile and applicable to a variety of collagen domains. The present invention uses the modified repeat sequence and different collagen domains to construct multiple collagen single chains and achieve the expression of collagen single chains. The results show that the expression levels are all improved; the pure collagen obtained after purification and enzyme digestion has the characteristics of maintaining or improving stability, improving solubility, and maintaining good salt tolerance.

[0047] The present invention utilizes the method for modifying the repeating sequence to obtain multiple collagens, and the purity of the constructed collagens reaches more than 90%, which is higher than that of the unmodified (GPP) 10 Compared with the collagen (V-P10BP10) composed of the raw materials, the yield is increased by more than 23.6%, the heat resistance temperature is increased by 2°C, the solubility is improved, and good salt tolerance is maintained. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1SDS-PAGE diagram of the supernatant collected by centrifugation after cell disruption; in the figure, 1-5 correspond to the sequences V-P10BP10, V-KD3BP10, V-P10BKD2, V-KD2BKD2, V-P10BKD3 respectively. The arrow represents the target band; M: protein marker.

[0049] Figure 2 SDS-PAGE diagram of the target protein eluted with 400 mM imidazole concentration; in the figure, 1-5 correspond to the sequences V-P10BP10, V-KD3BP10, V-P10BKD2, V-KD2BKD2, V-P10BKD3 respectively. The arrow represents the target band; M: protein marker.

[0050] Figure 3 Yield diagram of collagens with different sequences.

[0051] Figure 4 SDS-PAGE diagram of the collagen with V-domain after trypsin digestion; in the figure, 1-5 correspond to the sequences P10BP10, KD3BP10, P10BKD2, KD2BKD2, P10BKD3 respectively. The arrow represents the target band; M: protein marker.

[0052] Figure 5 To verify whether it is the molecular weight of the target protein by MALDI-TOF, A-D are KD3BP10, P10BKD2, KD2BKD2, P10BKD3.

[0053] Figure 6 Full wavelength spectrum, thermal denaturation curve and first derivative graph of the thermal denaturation curve of the designed type I collagen.

[0054] Figure 7 Full wavelength spectrum, thermal denaturation curve and first derivative graph of the thermal denaturation curve of the designed type I collagen at 0 mM, 10 mM and 100 mM, A-D are KD3BP10, P10BKD2, KD2BKD2, P10BKD3.

[0055] Figure 8 The sample concentration was prepared to be 2 mmol / L, dissolved with 10 mM sodium phosphate buffer, and photographed and recorded after standing at 4°C for 1 day.

[0056] Figure 9 Schematic diagram of the collagen structure.

[0057] Figure 10 Schematic diagram of the collagen structure after enzymatic cleavage to remove the folding domain. Detailed implementation method

[0058] Culture medium:

[0059] LB medium (g / L): Tryptone 10, yeast extract powder 5, NaCl 10, pH 7.0;

[0060] TB medium (g / L): Tryptone 12, yeast powder 24, glycerol 4 mL, KH2PO4 2.31, K2HPO4 12.54 Culture method (shake flask fermentation):

[0061] Absorb 50 μL of bacterial liquid from the glycerol tube storing the target gene into 5 mL of LB (Amp resistant), and culture overnight at 37°C and 200 r / min. Transfer 1% to 100 mL of TB fermentation broth (Amp resistant), culture at 37°C and 200 r / min for 24 h, then add IPTG to a final concentration of 1 mmol / L, and ferment and culture at 25°C and 200 r / min for 10 h, and then transfer to 15°C for fermentation for 14 h.

[0062] Protein purification method:

[0063] After fermentation, collect the bacterial liquid, centrifuge at 4°C and 10000 rpm for 5 min, discard the supernatant, collect the bacterial cell precipitate, break it and centrifuge at 4°C and 10000 rpm for 20 min, and filter through a 0.45 μm aqueous filter membrane. Then use His Trap TM HP 5 mL affinity purification. First, equilibrate with 5 column volumes of binding buffer A (20 mmol / L Na2HPO4, 20 mmol / L NaH2PO4, 500 mmol / L NaCl, 10 mmol / L Imidazole, pH 7.4), and then load the sample at a flow rate of 2.5 mL / min. After loading the sample, perform gradient elution with elution buffer B (20 mmol / L Na2HPO4, 20 mmol / L NaH2PO4, 500 mmol / L NaCl, 500 mmol / L Imidazole, pH 7.4) to obtain the target protein, and analyze the purification situation using SDS-PAGE.

[0064] Trypsin digestion:

[0065] Dissolve the purified collagen in water to a concentration of 2 mg / mL, add trypsin with a concentration of 2.5 g / L according to a molar ratio of 10:1, and digest with a shaker at 25°C for 20 h, and verify the purity by SDS-PAGE analysis.

[0066] Desalting and freeze-drying treatment method:

[0067] The digested reactants were desalted using HiTrap Desalting with ultrapure water as the mobile phase at a flow rate of 5 mL / min. The peak samples were collected and vacuum freeze-dried at -50°C after verification by SDS-PAGE.

[0068] Identification of the triple helix structure and stability of the sample:

[0069] Circular dichroism was used for identification. The specific steps were as follows: The freeze-dried sample was dissolved in a 10 mmol / L sodium phosphate buffer solution with pH 7.4 to form a 1 mg / mL solution, and after equilibration at 4°C for 24 h, circular dichroism was performed. The full wavelength was measured at 4°C at 1 nm intervals for the CD spectrum from 190 - 260 nm, with an average scan time of 5 s. The thermal denaturation curve was obtained by monitoring the CD signal at 225 nm and increasing the temperature from 10°C to 80°C at a heating rate of 10°C / h, with equilibration for 8 s at each temperature. The melting temperature (T m ) was obtained by taking the median of the absorbance values corresponding to 10°C and 80°C in the fitted thermal denaturation curve, and this data represents the stability of the sample.

[0070] Determination of protein purity:

[0071] ImageJ was used to analyze the SDS-PAGE electrophoresis pattern, and the ratio of the gray value of the target band to the gray value of the lane where the target band is located was calculated to obtain the protein purity.

[0072] Method for determining the molecular weight using a MALDI-TOF-MS (ultrafleXtreme) mass spectrometer:

[0073] The freeze-dried sample was dissolved in water to form a 1 mg / mL solution respectively, and the molecular weight was determined using a MALDI-TOF-MS (ultrafleXtreme) mass spectrometer. The matrix used was DHAP (2-acetylresorcinol, used in combination with ethanol and diammonium hydrogen citrate), and it was operated in linear mode.

[0074] Example 1: Design of the collagen sequence

[0075] Design the collagen amino acid sequence as Figure 9 shown. Among them, the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0076] The introduction of the folding domain is to assist the folding of collagen to form a triple helix structure. Optionally, the folding domain is the V-domain.

[0077] The introduction of the collagen domain (abbreviated as B) was achieved by analyzing the α1 chain of type I collagen through protein computational analysis and thermal stability prediction, and selecting sequence B with a predicted T m value close to 37 °C and a high triple helix propensity.

[0078] The introduction of the repeat sequence module can assist in the folding of the collagen triple helix and improve its thermal stability. The repeat sequences are selected from P10, KD2, and KD3; where P10 is (GPP) 10 (amino acid sequence is SEQ ID NO.3); KD2 is obtained by mutating 4 prolines in (GPP) 10 to 2 aspartic acids and 2 lysines, and the amino acid sequence of KD2 is as shown in SEQ ID NO.4; KD3 is obtained by mutating 6 prolines in (GPP) 10 to 3 aspartic acids and 3 lysines, and the amino acid sequence of KD3 is as shown in SEQ ID NO.5; considering that proline at the sequence end may be unfavorable for protein expression, an additional glycine is added at the end of the collagen amino acid sequence.

[0079] According to the position of the repeat sequence, it is divided into the first repeat sequence and the second repeat sequence. An enzyme cleavage site (such as LVPRGSP) sequence is added between the folding domain and the first repeat sequence to facilitate subsequent enzymatic cleavage to remove the folding domain.

[0080] Specifically, in the collagen single chain as Figure 9 shown, the naming rule is as follows:

[0081] Taking V-KD3BP10 as an example, "V" refers to the folding domain (amino acid sequence is SEQ ID NO.1); "B" is the collagen domain (amino acid sequence is as shown in SEQ ID NO.2); "KD3" before the collagen domain "B" represents the amino acid sequence of the first repeat sequence, and "P10" after the collagen domain "B" represents the second repeat sequence, and P10 is (GPP) 10 , and the amino acid sequence is as shown in SEQ ID NO.3.

[0082] Replace the corresponding first repeat sequence and / or second repeat sequence of V-KD3BP10 to construct the corresponding collagen single chain sequences V-P10BKD2, V-KD2BKD2, and V-P10BKD3. Among them, the amino acid sequences of V-KD3BP10, V-P10BKD2, V-KD2BKD2, V-P10BKD3, and V-P10BP10 are as shown in SEQ ID NOs.6-10 respectively.

[0083] Example 2: Construction of recombinant plasmid and recombinant bacteria

[0084] (1) Construction of recombinant plasmid

[0085] Starting from the nucleotide sequences of the repetitive amino acid sequences V-KD3BP10, V-P10BKD2, V-KD2BKD2, V-P10BP10, and V-P10BKD3 designed in Example 1 (the nucleotide sequences are shown in SEQ ID NO.11-15), when synthesizing the nucleotide sequence of the single-chain protein, the base GC was introduced at the 5' flanking end, and Nco I and Bam HI restriction enzyme sites were introduced at the 5' and 3' ends respectively. The nucleotide sequence of the Nco I restriction enzyme site is CCATGG, and the nucleotide sequence of the Bam HI restriction enzyme site is GGATCC. The above synthesized genes were respectively inserted between Nco I and Bam HI of the pColdIII plasmid (purchased from Genewiz), and recombinant collagen protein plasmids expressing different repetitive amino acid sequences were obtained, and the correct recombinant plasmids were obtained by sequencing.

[0086] (2) Construction of recombinant strains

[0087] The correctly sequenced recombinant plasmids in step (1) were respectively transformed into E. coli BL21(DE3) competent cells, spread on LB plates containing ampicillin, cultured and screened, and stored in glycerol tubes to obtain recombinant bacteria containing recombinant collagen, named E. coli BL21(DE3)-V-KD3BP10, E. coli BL21(DE3)-V-P10BKD2, E. coli BL21(DE3)-V-KD2BKD2, E. coli BL21(DE3)-V-P10BKD3, and E. coli BL21(DE3)-V-P10BP10 respectively.

[0088] Example 3: Expression, purification and enzymatic cleavage of collagen sequences

[0089] The recombinant bacteria E. coli BL21(DE3)-V-KD3BP10, E. coli BL21(DE3)-V-P10BKD2, E. coli BL21(DE3)-V-KD2BKD2, E. coli BL21(DE3)-V-P10BKD3, and E. coli BL21(DE3)-V-P10BP10 obtained in Example 2 were cultured by shake-flask fermentation. After collecting the bacteria, breaking them and centrifuging, the SDS-PAGE results of the supernatant are as Figure 1 shown. The supernatant was taken and affinity purified with His Trap TM HP 5mL, and the samples at imidazole concentrations of 175 mmol / L and 400 mmol / L were collected. The samples were verified as the target protein by SDS-PAGE, and the results are as Figure 2 shown.

[0090] The verified protein was desalted, freeze-dried and weighed to obtain its yield, and the results are as Figure 3 shown. The yields of recombinant collagen V-P10BP10, V-KD3BP10, V-P10BKD2, V-KD2BKD2, and V-P10BKD3 were 55.02 mg / L, 83.25 mg / L, 68.02 mg / L, 73.51 mg / L, and 91.98 mg / L, respectively. The yields of the modified collagens V-KD3BP10, V-P10BKD2, V-KD2BKD2, and V-P10BKD3 were increased by 23.6% - 67.18% compared with V-P10BP10, and among them, the yield of V-P10BKD3 was the highest.

[0091] To remove the folding domain, a trypsin cleavage site LVPRGSP sequence was introduced between the repeat sequence and the folding domain during sequence design. The purified collagen was dissolved in water to a concentration of 2 mg / mL and digested with trypsin (purchased from Shanghai Titan Technology Co., Ltd.) at a molar ratio of 10:1 and a concentration of 2.5 g / L to obtain a pure collagen domain structure.

[0092] Under the action of trypsin, the V-domain will be digested into multiple short peptides containing 2 - 20 amino acid residues. If the collagen domain correctly folds into a rigid triple helix structure under the action of the V-domain, it will not be digested by trypsin in a short time. After digestion, desalting was carried out to remove the short peptides. Finally, the collagens P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3 were verified by SDS-PAGE as Figure 4 shown, and the purity was greater than 90%.

[0093] Example 4: MALDI-TOF Identification

[0094] The collagens KD3BP10, P10BKD2, KD2BKD2, and P10BKD3 after enzymatic digestion and desalting in Example 3 were freeze-dried. The freeze-dried samples were respectively dissolved in water to a solution of 1 mg / mL and used for molecular weight determination with a MALDI-TOF-MS (ultrafleXtreme) mass spectrometer. The matrix used was DHAP (2-acetylresorcinol, used in combination with ethanol and diammonium hydrogen citrate), and the operation was carried out in linear mode. It was verified that the molecular weight of the obtained collagen was the same as the theoretical value, and the results are as Figure 5 shown.

[0095] Example 5: Circular Dichroism Characterization of Collagen and Sequence Structure

[0096] To confirm the secondary structure of the collagen domain, the freeze-dried samples of collagen P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3 after enzymatic digestion and desalting in Example 3 were prepared into a 1 mg / mL solution with 10 mmol / L sodium phosphate buffer and equilibrated at 4 °C for 24 h. After the equilibration, full-wavelength scanning was performed using circular dichroism spectroscopy.

[0097] The results are as Figure 6 shown. Characteristic positive absorption peaks appeared at 225 nm for P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3, indicating that all six types of collagen were correctly folded into a triple helix structure with the assistance of the V-domain. The thermal denaturation curves of the collagen from 4 °C to 80 °C at 225 nm were monitored using circular dichroism spectroscopy, and the T m value of the collagen was obtained by taking the first derivative of the thermal denaturation curve. The T m value of P10BP10 was 35 °C, and the T m values of the other sequences were all 37 °C.

[0098] The above results indicate that the collagen sequences designed in the present invention can all be correctly folded into a triple helix structure, and the T m value is increased by 2 °C compared to the P10BP10 protein sequence. The designed sequences can all improve the thermal stability of the protein.

[0099] Example 6: Detection of the salt tolerance of collagens with different repeat sequences

[0100] To verify the stability of different collagen sequences in a high-concentration salt solution, the freeze-dried samples of collagen KD3BP10, P10BKD2, KD2BKD2, and P10BKD3 prepared in Example 3 were dissolved in 10 mmol / L sodium phosphate buffer to prepare a 1 mg / mL solution. On this basis, 100 mmol and 200 mmol of sodium chloride were added to the solution. The results are as Figure 7 shown. The thermal denaturation curves of the collagen from 4 °C to 80 °C at 225 nm were monitored using circular dichroism spectroscopy, and the T m value of the collagen was obtained by taking the first derivative of the thermal denaturation curve. The results showed that the T m values of each sequence were still 37 °C in a high-salt environment. Thus, it can be seen that the modified collagen still has good salt tolerance.

[0101] Example 7: The repeat sequences improve the solubility of the collagen domain protein

[0102] To verify the solubility of different collagen sequences, the freeze-dried samples of collagen P10BP10, KD3BP10, P10BKD2, KD2BKD2, and P10BKD3 prepared in Example 3 were dissolved in 10 mmol / L sodium phosphate buffer to prepare a solution with a sample concentration of 2 mmol / L. The solution was allowed to stand at 4°C for 1 day, and the results were as follows Figure 8 shown. There was precipitation in P10BP10, and no precipitation in the other proteins. This indicates that the modified collagen has a lower turbidity and higher molecular solubility than the sequence P10BP10 at the same concentration.

[0103] Example 8: Application of repetitive sequences in improving the yield, heat resistance, and salt tolerance of collagen in different collagen domains

[0104] Based on Example 1, the collagen domains with the amino acid sequence shown in SEQ ID NO.2 were respectively replaced with the collagen domains with the amino acid sequences shown in SEQ ID NOs. 16 - 22 (denoted as B’), and multiple collagen single chains were obtained, expressed, and purified to analyze the generality of the repetitive sequence modification method of the present invention in improving the yield, heat resistance, and salt tolerance of collagen. At the same time, recombinant collagen was prepared and purified by referring to the methods of Examples 2 - 3, and the yield, heat resistance, and salt tolerance of different recombinant collagens were detected by the methods of Examples 4 - 7. The results showed that the yields of KD3B’P10, P10B’KD2, KD2B’KD2, and P10B’KD3 with the replaced collagen domains were more than 15% higher than that of (GPP) during recombinant expression 10 The heat resistance temperature T of the purified collagen m increased by about 2°C or remained at about 37 - 38°C, and at the same time had good salt tolerance.

[0105] In addition, a new collagen domain (abbreviated as HC1 - 12) obtained by splicing SEQ ID NO.16 and SEQ ID NO.17 in sequence, and a new collagen domain (abbreviated as HC1 - 22) obtained by splicing 2 SEQ ID NO.17s were also used to construct collagen single chains, express, and purify collagen by the above method. The results showed that the yield during recombinant expression was also more than 18% higher than that of (GPP) 10 The heat resistance temperature T of the purified collagen m increased by about 2°C or remained at about 37 - 38°C, and at the same time had good salt tolerance.

[0106] Sequences involved in the present invention:

[0107] SEQ ID NO.1: Amino acid sequence of the V - domain

[0108] ADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD

[0109] SEQ ID NO.2: Amino acid sequence of collagen domain B

[0110] GFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGA AGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPS

[0111] SEQ ID NO.3: Amino acid sequence of (GPP) 10 of

[0112] GPPGPPGPPGPPGPPGPPGPPGPPGPPGPP

[0113] SEQ ID NO.4: Amino acid sequence of KD2

[0114] GPPGPPGPKGDPGPPGPPGPKGDPGPPGPP

[0115] SEQ ID NO.5: Amino acid sequence of KD3

[0116] GPPGPKGDPGPPGPKGDPGPPGPKGDPGPP

[0117] SEQ ID NO.6: Amino acid sequence of V-KD3BP10

[0118] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPKGDPGPPGPKGDPGPPGPKGDPGPPGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPG

[0119] SEQ ID NO.7: Amino acid sequence of V-P10BKD2

[0120] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPPGPKGDPGPPGPPGPKGDPGPPGPPG

[0121] SEQ ID NO.8: Amino acid sequence of V-KD2BKD2

[0122] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPKGDPGPPGPPGPKGDPGPPGPPGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPPGPKGDPGPPGPPGPKGDPGPPGPPG

[0123] SEQ ID NO.9: Amino acid sequence of V-P10BKD3

[0124] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPKGDPGPPGPKGDPGPPGPKGDPGPPG

[0125] SEQ ID NO.10: Amino acid sequence of V-P10BP10

[0126] HHHHHHHHGGGGSADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPG

[0127] SEQ ID NO.11: Nucleotide sequence of V-KD3BP10

[0128] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTAAAGGTGATCCTGGTCCTCCGGGTCCTAAAGGTGATCCTGGTCCTCCTGGTCCTAAAGGTGATCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTCCTGGTCCACCTGGTCCTCCGGGTCCTCCGGGCCCTCCTGGCCCGCCTGGTCCGCCTGGTCCACCGGGTCCTCCTGGT

[0129] SEQ ID NO.12: Nucleotide sequence of V-P10BKD2

[0130] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTCCTGGCCCTCCTGGCCCTCCGGGTCCTCCAGGTCCGCCTGGTCCTCCTGGCCCTCCAGGTCCGCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTCCTGGTCCAAAAGGTGATCCGGGTCCTCCGGGCCCTCCTGGCCCGAAAGGTGATCCTGGTCCACCGGGTCCTCCTGGT

[0131] SEQ ID NO.13: Nucleotide sequence of V-KD2BKD2

[0132] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTCCTGGCCCTAAAGGTGATCCGGGTCCTCCAGGTCCGCCTGGTCCTAAAGGTGATCCAGGTCCGCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTCCTGGTCCAAAAGGTGATCCGGGTCCTCCGGGCCCTCCTGGCCCGAAAGGTGATCCTGGTCCACCGGGTCCTCCTGGT

[0133] SEQ ID NO.14: Nucleotide sequence of V - P10BP10

[0134] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTCCTGGCCCTCCTGGCCCTCCGGGTCCTCCAGGTCCGCCTGGTCCTCCTGGCCCTCCAGGTCCGCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTCCTGGTCCACCTGGTCCTCCGGGTCCTCCGGGCCCTCCTGGCCCGCCTGGTCCTCCTGGTCCACCGGGTCCTCCTGGT

[0135] SEQ ID NO.15: Nucleotide sequence of V-P10BKD3

[0136] CATCATCATCATCATCATCATCATGGTGGTGGTGGTTCAGCAGATGAACAGGAAGAAAAGGCAAAAGTGCGTACAGAACTGATCCAGGAACTGGCACAGGGCCTGGGTGGTATCGAAAAGAAGAACTTTCCGACTCTGGGTGATGAAGATCTGGATCATACCTATATGACAAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCAGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGTATTCAGGATCATGCTCTGGATCTGGTGCCTCGTGGTTCACCGGGTCCTCCTGGTCCTCCTGGCCCTCCTGGCCCTCCGGGTCCTCCAGGTCCGCCTGGTCCTCCTGGCCCTCCAGGTCCGCCAGGTCCGCCGGGTTTTCCTGGTGAACGTGGTGTTCAGGGTCCTCCTGGTCCGGCAGGTCCTCGTGGTGCAAATGGTGCTCCTGGTAATGATGGTGCCAAAGGTGATGCCGGTGCACCGGGTGCACCTGGTAGTCAGGGTGCACCTGGCCTGCAGGGTATGCCTGGTGAACGCGGTGCTGCTGGTCTGCCTGGTCCTAAAGGCGATCGTGGTGATGCAGGTCCGAAAGGTGCAGATGGTAGTCCGGGTAAAGATGGTGTTCGTGGTCTGACCGGTCCGATTGGTCCGCCTGGCCCTGCAGGTGCACCTGGTGATAAAGGTGAAAGCGGTCCGAGCGGTCCGCCAGGCCCTAAAGGTGATCCTGGTCCTCCGGGTCCTAAAGGCGATCCTGGCCCGCCTGGTCCTAAAGGTGATCCGGGTCCTCCTGGT

[0137] SEQ ID NO.16: Amino acid sequence of HC1-1

[0138] GARGLPGTAGLPGMKGHRGFPGERGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGP RGPQGPPGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKR

[0139] SEQ ID NO.17: Amino acid sequence of HC1-2

[0140] GFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGA AGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPS

[0141] SEQ ID NO.18: Amino acid sequence of HC1-3

[0142] GPAGFAGPPGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGFPGERGAPGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQ

[0143] SEQ ID NO.19: Amino acid sequence of HC2A

[0144] GLTGPAGEPGREGSPGADGPPGRDGAAGVKGDRGETGAVGAPGAPGPPGDRGEAGA QGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPS

[0145] SEQ ID NO.20: Amino acid sequence of HC3A

[0146] GFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGSPGP KGDKGEPGPPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEP

[0147] SEQ ID NO.21: Amino acid sequence of HC3B

[0148] GFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGAKGE PGPRGERGEAGIPGVPGAKGEDGKPGEPGPKGDAGAPGAPGPKGDAGAPGER

[0149] SEQ ID NO.22: Amino acid sequence of HC3C

[0150] GFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGAKGE PGPRGERGEAGIPGVPGAKGEDGRDGNPGSDGLPGRDGSPGPKGDRGENGSP

[0151] Although the present invention has been disclosed above in the preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A single-chain collagen, characterized in that, The single-chain structure is a first repeat sequence - collagen domain - second repeat sequence; wherein, the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.

5.

2. The single-chain collagen according to claim 1, wherein The amino acid sequence of the first repeat sequence of the single-chain collagen is as shown in SEQ ID NO.5, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.3; or, the amino acid sequence of the first repeat sequence of the single-chain collagen is as shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.4; or, the amino acid sequence of the first repeat sequence of the single-chain collagen is as shown in SEQ ID NO.3, and the amino acid sequence of the second repeat sequence is as shown in SEQ ID NO.5; or, both the first repeat sequence and the second repeat sequence of the single-chain collagen are as shown in SEQ ID NO.

4.

3. The single-chain collagen according to claim 1, wherein The N-terminus of the first repeat sequence further contains a folding domain; optionally, the amino acid sequence of the folding domain is as shown in SEQ ID NO.1; optionally, the folding domain is connected to the first repeat sequence through a cleavage site; optionally, the cleavage site is LVPRGSP.

4. A gene encoding the single-chain collagen according to any one of claims 1 to 3, a plasmid carrying the gene encoding the single-chain collagen according to any one of claims 1 to 3, or a cell.

5. A method for improving the stability, solubility or yield of collagen, characterized in that, Connect the first repeat sequence to the N-terminus of the collagen domain and the second repeat sequence to the C-terminus to construct a single-chain collagen with the structure of first repeat sequence - collagen domain - second repeat sequence, and express it through a host cell; wherein the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.

5.

6. A collagen, characterized in that, It consists of a protein single chain with the structure of first repeat sequence - collagen domain - second repeat sequence; wherein, the amino acid sequence of at least one of the first repeat sequence or the second repeat sequence is as shown in SEQ ID NO.4 or SEQ ID NO.

5.

7. The collagen fiber formed by the self-assembly of the collagen according to claim 6.

8. Use of the single-chain collagen according to any one of claims 1 to 3, the collagen according to claim 6, or the collagen fiber according to claim 7 in the preparation of a collagen product.

9. A collagen product, characterized in that, The product contains the single-chain collagen according to any one of claims 1 to 3, the collagen according to claim 6, or the collagen fiber according to claim 7.

10. The collagen product according to claim 8, wherein, The collagen product is a cosmetic, a food, or a drug; optionally, the collagen product is a drug carrier, a medical supply, or a beauty product; optionally, the collagen product further contains one or more of vitamins, minerals, hyaluronic acid, natural polysaccharides, essential oils, polyphenolic substances, and natural plant extracts.

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