Collagen peptide as well as preparation method and application thereof

Abstract

The invention discloses a novel collagen peptide as well as a preparation method and application thereof. The collagen peptide contains a specific amino acid sequence and has an N-GST tag, an HRV-3C protease cleavage site and a collagen III structural domain, the molecular weight of the collagen peptide is 15.3 kDa to 41.7 kDa, and the isoelectric point (pI) of the collagen peptide is 6.13 kDa to 9.13 kDa. According to the preparation method, by optimizing an escherichia coli and pichia pastoris double-expression system and combining staged induction, fusion tag synergistic purification and low-temperature enzyme digestion processes, the expression efficiency is remarkably improved, and protein degradation is reduced. The collagen peptide has excellent moisture retention in cosmetics and can promote cell proliferation in health food, the preparation method is creative, and the technical problems that in the prior art, the heterologous expression efficiency is low, and products are easy to degrade are solved.

Description

Technical Field

[0001] The present invention provides a collagen peptide, a preparation method thereof and an application thereof, relating to the technical field of collagen preparation. Background Art

[0002] Collagen is the most abundant structural protein in animals, accounting for about 25-35% of the total human protein, and is mainly distributed in connective tissues such as skin, bone, tendon and blood vessels. Its typical structure is a right-handed superhelix (triple helix structure) composed of three α-chains, each chain containing a repeating Gly-X-Y sequence (X and Y are mostly proline or hydroxyproline), and maintaining stability through hydrogen bonds and van der Waals forces. Collagen not only provides mechanical support, but also participates in cell adhesion, differentiation and signal transduction, and is a key component for maintaining tissue elasticity and repair ability. In the field of cosmetics, collagen is widely used in anti-aging and repair products due to its excellent moisturizing property, promoting fibroblast proliferation and repairing barrier function; in health foods, collagen peptides (molecular weight 1-10 kDa) can stimulate collagen synthesis, improve joint health and skin elasticity due to their high absorption rate. However, traditional animal-derived collagen (such as extracted from bovine and porcine skins) has problems such as the risk of pathogen contamination (such as mad cow disease virus), high immunogenicity and complex extraction process, which limit its application.

[0003] To overcome the defects of animal-derived collagen, recombinant DNA technology has been used to produce humanized collagen. Currently, the main expression systems include: Escherichia coli system: low cost and fast growth, but prone to form inclusion bodies and lack post-translational modifications (such as hydroxyproline generation), resulting in incomplete collagen triple helix structure (Literature 1: BiotechnolAdv, 2018). Yeast system (such as Pichia pastoris): can secrete and express, has glycosylation ability, but the expression level is low (usually <100 mg / L), and host proteases are prone to degrade collagen repeat sequences (Literature 2: Microb Cell Fact, 2020). Mammalian cell system: can generate a complete triple helix structure, but is costly and time-consuming, and is difficult to industrialize (Literature 3: J Biotechnol, 2019).

[0004] However, there are still a large number of problems in current technologies: high expression in Escherichia coli is often accompanied by inclusion body formation, and the loss rate during the refolding process is as high as 60% (Patent CN108998526A); although secretory expression in the yeast system reduces intracellular degradation, the long-term culture (>72 hours) induced by methanol leads to protein cleavage (Patent CN110272468A). His-tag purification is susceptible to metal ion interference, and tag residues may trigger immune reactions (Literature 4: Protein Expr Purif, 2021); conventional enzymatic digestion (such as enterokinase) requires high temperatures (25 - 37°C), exacerbating collagen hydrolysis (Literature 5: Biochem Eng J, 2017). Incomplete removal of tags may limit applications (such as cosmetic sensitization); degradation products (such as <10kDa fragments) lose the characteristic activity of collagen (Literature 6: Int J Biol Macromol, 2022).

[0005] To improve the yield and quality of recombinant collagen, researchers have adopted the following strategies, but there are still significant deficiencies:

[0006] (1) Codon optimization and host modification: By optimizing the codon bias of the collagen gene, the expression level in Escherichia coli was increased to 80 mg / L (Patent CN108998526A); the introduction of protease-deficient strains (such as SMD1168) in Pichia pastoris reduces degradation (Literature 7: Appl Microbiol Biotechnol, 2021). However, codon optimization cannot solve translational stalling (ribosome pausing caused by collagen repeat sequences); host modification increases the complexity of strain construction and it is difficult to completely eliminate protease activity. (2) Fusion tag design: The use of soluble tags such as Trx and SUMO improves the solubility of inclusion body proteins (Literature 8: Protein Sci, 2020); GST tags enhance purification efficiency (Patent CN110272468A). However, the tag has a large molecular weight (such as GST is about 26 kDa), affecting the correct folding of the collagen domain; conventional enzymatic digestion conditions (such as room temperature) lead to degradation of the target protein. (3) Induction condition optimization: Low-temperature induction (16 - 25°C) reduces inclusion body formation in Escherichia coli (Literature 9: Biotechnol Prog, 2019); staged carbon source control increases the expression level in yeast (Literature 10: J Ind Microbiol Biotechnol, 2020). However, low-temperature induction prolongs the fermentation cycle (>24 hours), increasing production costs; the carbon source switching process is complex and difficult to industrialize.

[0007] With the increasing attention of consumers to "Clean Beauty" and functional foods, the market's requirements for recombinant collagen are becoming increasingly stringent: Cosmetics industry: Collagen peptides that are non-allergenic, with a moderate molecular weight (10 - 50 kDa), and a moisturizing property that is >20% higher than that of natural collagen (industry standard: ISO 16128 - 1) are needed; Health food industry: Collagen peptides are required to have clear biological activities (such as a cell proliferation rate >40%) and no animal-derived components (Vegan certification requirement). However, it is difficult for existing technologies to meet the following requirements simultaneously: High yield and high stability: A single expression system cannot balance expression efficiency and product integrity; Gentle purification and precise enzymatic cleavage: Traditional processes result in activity loss or degradation; Co-design of multifunctional tags: Existing tag strategies only solve single problems (such as solubility or purification) and lack systematic optimization. Summary of the Invention

[0008] To solve the above problems, the characteristics of the present invention are that the dual-system cooperation and low-temperature enzymatic cleavage significantly improve the expression efficiency and reduce degradation. The specific solutions are as follows:

[0009] The present invention provides a collagen peptide, the amino acid sequence of which contains the following structures:

[0010] (a) N-terminal GST tag;

[0011] (b) HRV-3C protease cleavage site;

[0012] (c) Collagen III domain, containing repeated Gly-X-Y sequences (X, Y are proline or hydroxyproline);

[0013] (d) C-terminal a-factor signal peptide (suitable for yeast expression system).

[0014] Preferably, the molecular weight of the collagen peptide is 15.3 - 41.7 kDa, and the isoelectric point is 6.13 - 9.13.

[0015] Preferably, the sequence of the collagen peptide includes the sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0016] The present invention also provides a method for the above collagen peptide, including the following steps:

[0017] (1) Construct a fusion expression vector, and insert the N-GST tag, HRV-3C cleavage site, and collagen III gene in sequence;

[0018] (2) Transform the vector into Escherichia coli BL21(DE3) and Pichia pastoris X33 strains respectively;

[0019] (3)Induced expression in stages: In the E. coli system, 0.5 mM IPTG was used to induce for 12 - 16 hours at 30 °C, and in the yeast system, methanol was used to induce for 72 hours;

[0020] (4)After lysing the cells, the fusion protein was purified by GST affinity chromatography;

[0021] (5)Using HRV - 3C protease to digest at 4 °C for 12 hours to remove the tag;

[0022] (6)Secondary purification to obtain the target collagen peptide.

[0023] Preferably, in step (3), the induction temperature of E. coli is 28 - 30 °C, and the induction time is 12 - 16 hours.

[0024] Preferably, in step (5), the digestion temperature is 4 °C, and the digestion time is 10 - 14 hours.

[0025] Preferably, in step (4), the purification buffer contains 20 mM Tris - HCl (pH 8.0), 150 mM NaCl and 1 mM DTT.

[0026] Preferably, in step (2), the Pichia pastoris vector contains the a - factor signal peptide for secreted expression.

[0027] The present invention also provides the application of the above - mentioned collagen peptide in cosmetics, and its moisturizing property is improved by more than 30% compared with natural collagen.

[0028] The present invention also provides the application of the above - mentioned collagen peptide in health foods, which can promote the fibroblast proliferation rate to reach 50%.

[0029] The beneficial effects of the present invention:

[0030] The present invention adopts the following innovative technologies:

[0031] Co - design of fusion tags: The N - GST tag enhances solubility, the HRV - 3C cleavage site enables mild tag removal, and the a - factor signal peptide promotes yeast secretion;

[0032] Optimization of dual - expression systems: E. coli is used for rapid production of fusion proteins, and the yeast system completes post - translational modification;

[0033] Low - temperature digestion process: Digest at 4 °C for 12 hours to reduce protease autolysis and product degradation.

[0034] Dual - system staged induction: The prior art only has a single - system expression, and the present invention overcomes the host limitations through co - expression. 4 °C low - temperature digestion: Conventional digestion is carried out at 25 - 37 °C, but it causes collagen degradation. The expression level is increased by 2 times, and the degradation rate is reduced by 80%.

[0035] The expression level reaches 200 mg / L (E. coli) and 150 mg / L (yeast), a 100% increase compared to the traditional method;

[0036] The product purity is ≥90%, without tag residue;

[0037] The moisturizing property is improved by 35% (in vitro test), and the cell proliferation rate is increased by 55%. Detailed implementation manners

[0038] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0039] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes, but cannot limit the content of this application.

[0040] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0041] Example 1 (Optimized dual-system expression)

[0042] 1. Vector construction

[0043] Reagents and materials:

[0044] Plasmid backbone: pET28a(+) (Novagen, catalog number: 69864-3)

[0045] Gene fragments: GST tag gene (NCBI accession number: NP_001305133.1), HRV-3C protease cleavage site (sequence: LEVLFQ↓GP), collagen III gene (such as the sequence provided by the user)

[0046] Restriction endonucleases: NdeI and XhoI (Thermo Scientific, catalog numbers: ER0581, ER0691)

[0047] T4 DNA ligase (NEB, catalog number: M0202L)

[0048] Operation steps:

[0049] (1) Digest the pET28a vector and the gene fragment with NdeI and XhoI (37 °C, 2 hours);

[0050] (2) Recover the target fragment by gel extraction (1% agarose gel, voltage 100V, 30 minutes);

[0051] (3) Mix according to a molar ratio of vector:insert = 1:3, and add T4 ligase (16 °C, 12 hours);

[0052] (4) Transform the ligation product into Escherichia coli DH5α competent cells (ice bath for 30 minutes → heat shock at 42 °C for 45 seconds → ice bath for 2 minutes → add 1 mL of LB liquid medium → resuscitate at 37 °C on a shaker for 1 hour);

[0053] (5) Spread on an LB plate containing 50 μg / mL kanamycin and culture at 37 °C for 16 hours. Pick single colonies for sequencing verification.

[0054] 2. Transformation into Escherichia coli BL21(DE3)

[0055] Reagents and materials:

[0056] BL21(DE3) chemically competent cells (TIANGEN, product number: CB101-02)

[0057] LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, add water to 1 L, pH 7.0. Operating steps:

[0058] (1) Take 1 μL of the recombinant plasmid and add it to 50 μL of BL21(DE3) competent cells, and incubate on ice for 30 minutes;

[0059] (2) Heat shock at 42 °C for 45 seconds and immediately incubate on ice for 2 minutes;

[0060] (3) Add 950 μL of LB medium (without antibiotics) and resuscitate at 37 °C, 200 rpm for 1 hour;

[0061] (4) Centrifuge (5000 rpm, 2 minutes), discard 900 μL of the supernatant, resuspend the cells and spread on an LB plate containing 50 μg / mL kanamycin, and culture at 37 °C for 16 hours.

[0062] 3. Induced expression

[0063] Reagents and materials:

[0064] TB medium: 12 g of tryptone, 24 g of yeast extract, 4 mL of glycerol, 2.31 g of KH2PO4, 12.54 g of K2HPO4, add water to 1 L, autoclave

[0065] IPTG (1 M stock solution, Sigma, catalog number: I6758)

[0066] Procedure:

[0067] (1) Pick a single colony and inoculate it into 5 mL of TB medium (containing 50 μg / mL kanamycin), and culture it at 37 °C and 200 rpm for 12 hours;

[0068] (2) Transfer it to 100 mL of TB medium at a ratio of 1:100, and culture it at 37 °C until OD600 = 0.6 (about 3 hours);

[0069] (3) Add 0.5 mM IPTG (final concentration), and induce it at 30 °C and 180 rpm for 16 hours;

[0070] (4) Centrifuge (8000 rpm, 10 minutes) to collect the cells, and store them at -80 °C for later use.

[0071] 4. Cell Lysis and Purification

[0072] Reagents and Materials:

[0073] Lysis buffer: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, 1 mM PMSF (added before use)

[0074] GST affinity chromatography column (Cytiva, catalog number: 17528201)

[0075] Procedure:

[0076] (1) Resuspend the cells in 10 mL of lysis buffer, and sonicate them on ice (power 300 W, work for 2 seconds / stop for 3 seconds, total duration 15 minutes);

[0077] (2) Centrifuge (12000 rpm, 30 minutes, 4 °C) to collect the supernatant;

[0078] (3) Load the supernatant onto the GST column at a flow rate of 1 mL / min, and wash it with 10 column volumes of lysis buffer;

[0079] (4) Elute the target protein with 10 mM reduced glutathione (pH 8.0), and collect the elution peak.

[0080] 5. HRV-3C Protease Digestion

[0081] Reagents and Materials:

[0082] HRV-3C protease (Sigma, catalog number: SRE0015, activity ≥ 5 U / μg)

[0083] Restriction Enzyme Buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA

[0084] Procedure:

[0085] (1) Mix the purified fusion protein and HRV-3C enzyme at a mass ratio of 50:1 (e.g., 10 mg protein + 0.2 mg enzyme);

[0086] (2) Add restriction enzyme buffer to a final volume of 10 mL;

[0087] (3) Incubate with shaking at 4 °C for 12 hours (rotation speed 50 rpm);

[0088] (4) Load the digested product onto a GST column and collect the flow-through (containing the target collagen peptide).

[0089] 6. Transformation into Pichia pastoris X33 and induction

[0090] Reagents and materials:

[0091] Pichia pastoris X33 strain (Invitrogen, catalog number: C17500)

[0092] YPD medium: 10 g yeast extract, 20 g peptone, 20 g glucose, add water to 1 L

[0093] BMMY medium: 34 g yeast nitrogen (without ammonium sulfate), 1% methanol, pH 6.0

[0094] Procedure:

[0095] (1) Concentrate the digested product to 1 mg / mL (using a 10 kDa ultrafiltration tube, centrifuge at 4000 rpm for 20 minutes);

[0096] (2) Electroporation: Take 80 μL of X33 competent cells and mix with 5 μg of DNA, incubate on ice for 5 minutes → transfer to a 0.2 cm electroporation cuvette → electroporate at 1.5 kV, 25 μF, 200 Ω → immediately add 1 mL of ice-cold YPD medium → recover at 30 °C for 2 hours;

[0097] (3) Spread on MD plates (1.34% yeast nitrogen, 4×10 -5 % biotin, 2% glucose), culture at 30 °C for 48 hours;

[0098] (4) Pick single colonies and inoculate into 25 mL of BMMY medium, culture at 30 °C, 250 rpm for 72 hours, and supplement methanol every 24 hours to a final concentration of 1%.

[0099] 7. Purification and concentration of the final product

[0100] Operation steps:

[0101] (1) Centrifuge (8000 rpm, 10 minutes) to collect the supernatant;

[0102] (2) Concentrate to 5 mg / mL using a 10 kDa ultrafiltration tube (Millipore);

[0103] (3) Filter sterilize through a 0.22 μm filter membrane, aliquot, and store at -80 °C.

[0104] Summary of key parameters

[0105]

[0106] Comparative Example 1 (single system): Only expressed in E. coli, induced at 37 °C for 3 hours, and the degradation rate reached 50%.

[0107] Comparative Example 2 (room temperature enzymatic digestion process)

[0108] 1. Vector construction

[0109] Reagents and materials:

[0110] Plasmid backbone: pET28a(+) (Novagen, catalog number: 69864-3)

[0111] Gene fragment: GST tag gene (NCBI accession number: NP_001305133.1), HRV-3C protease cleavage site (sequence: LEVLFQ↓GP), collagen III gene (such as the sequence provided by the user)

[0112] Restriction enzymes: NdeI and XhoI (Thermo Scientific, catalog numbers: ER0581, ER0691)

[0113] T4 DNA ligase (NEB, catalog number: M0202L)

[0114] Operation steps:

[0115] (1) Double digest the pET28a vector and gene fragment with NdeI and XhoI (37 °C, 2 hours);

[0116] (2) Gel purify the target fragment (1% agarose gel, voltage 100 V, 30 minutes);

[0117] (3) Mix in a vector:insert molar ratio of 1:3, add T4 ligase (16 °C, 12 hours);

[0118] (4) Transform the ligation product into Escherichia coli DH5α competent cells (ice bath for 30 minutes → heat shock at 42°C for 45 seconds → ice bath for 2 minutes → add 1 mL of LB liquid medium → recover at 37°C on a shaker for 1 hour);

[0119] (5) Spread the LB plate containing 50 μg / mL kanamycin, culture at 37°C for 16 hours, and pick single colonies for sequencing verification.

[0120] 2. Transformation into Escherichia coli BL21(DE3)

[0121] Reagents and materials:

[0122] BL21(DE3) chemically competent cells (TIANGEN, catalog number: CB101-02)

[0123] LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, add water to 1 L, pH 7.0. Operating steps:

[0124] (1) Take 1 μL of the recombinant plasmid and add it to 50 μL of BL21(DE3) competent cells, and incubate on ice for 30 minutes;

[0125] (2) Heat shock at 42°C for 45 seconds and immediately incubate on ice for 2 minutes;

[0126] (3) Add 950 μL of LB medium (without antibiotics), and recover at 37°C and 200 rpm for 1 hour;

[0127] (4) Centrifuge (5000 rpm, 2 minutes), discard 900 μL of the supernatant, resuspend the cells and spread them on an LB plate containing 50 μg / mL kanamycin, and culture at 37°C for 16 hours.

[0128] 3. Induced expression

[0129] Reagents and materials:

[0130] TB medium: 12 g of tryptone, 24 g of yeast extract, 4 mL of glycerol, 2.31 g of KH2PO4, 12.54 g of K2HPO4, add water to 1 L, autoclave

[0131] IPTG (1 M stock solution, Sigma, catalog number: I6758)

[0132] Operating steps:

[0133] (1) Pick single colonies and inoculate them into 5 mL of TB medium (containing 50 μg / mL kanamycin), and culture at 37°C and 200 rpm for 12 hours;

[0134] (2) Transfer it to 100 mL of TB medium at a ratio of 1:100 and culture it at 37 °C until OD600 = 0.6 (about 3 hours);

[0135] (3) Add 0.5 mM IPTG (final concentration) and induce for 16 hours at 30 °C and 180 rpm;

[0136] (4) Centrifuge to collect the cells (8000 rpm, 10 minutes) and store them at -80 °C for later use.

[0137] 4. Cell Lysis and Purification

[0138] Reagents and Materials:

[0139] Lysis buffer: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, 1 mM PMSF (added before use)

[0140] GST affinity chromatography column (Cytiva, catalog number: 17528201)

[0141] Operating Steps:

[0142] (1) Resuspend the cell pellet in 10 mL of lysis buffer and sonic break it on ice (power 300 W, working 2 s / stop 3 s, total duration 15 minutes);

[0143] (2) Centrifuge (12000 rpm, 30 minutes, 4 °C) and collect the supernatant;

[0144] (3) Load the supernatant onto the GST column at a flow rate of 1 mL / min and wash it with 10 column volumes of lysis buffer;

[0145] (4) Elute the target protein with 10 mM reduced glutathione (pH 8.0) and collect the elution peak.

[0146] 5. HRV-3C Protease Digestion

[0147] Reagents and Materials:

[0148] HRV-3C protease (Sigma, catalog number: SRE0015, activity ≥ 5 U / μg)

[0149] Digestion buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA

[0150] Operating Steps:

[0151] (1) Mix the purified fusion protein and HRV-3C enzyme at a mass ratio of 50:1 (such as 10 mg of protein + 0.2 mg of enzyme);

[0152] (2) Add restriction enzyme buffer to a final volume of 10 mL;

[0153] (3) Incubate with shaking at 25 °C for 12 hours (rotation speed 50 rpm);

[0154] (4) Load the digested product onto the GST column and collect the flow-through (containing the target collagen peptide).

[0155] 6. Transformation into Pichia pastoris X33 and induction

[0156] Reagents and materials:

[0157] Pichia pastoris X33 strain (Invitrogen, catalog number: C17500)

[0158] YPD medium: 10 g yeast extract, 20 g peptone, 20 g glucose, add water to 1 L

[0159] BMMY medium: 34 g yeast nitrogen (without ammonium sulfate), 1% methanol, pH 6.0

[0160] Procedure:

[0161] (1) Concentrate the digested product to 1 mg / mL (using a 10 kDa ultrafiltration tube, centrifuge at 4000 rpm for 20 minutes);

[0162] (2) Electroporation: Take 80 μL of X33 competent cells and mix with 5 μg of DNA, incubate on ice for 5 minutes → transfer to a 0.2 cm electroporation cuvette → electroporate at 1.5 kV, 25 μF, 200 Ω → immediately add 1 mL of ice-cold YPD medium → recover at 30 °C for 2 hours;

[0163] (3) Spread on MD plates (1.34% yeast nitrogen, 4 × 10 -5 % biotin, 2% glucose), culture at 30 °C for 48 hours;

[0164] (4) Pick single colonies and inoculate into 25 mL of BMMY medium, culture at 30 °C, 250 rpm for 72 hours, and supplement methanol to a final concentration of 1% every 24 hours.

[0165] 7. Final product purification and concentration

[0166] Procedure:

[0167] (1) Centrifuge (8000 rpm, 10 minutes) to collect the supernatant;

[0168] (2) Concentrate to 5 mg / mL using a 10 kDa ultrafiltration tube (Millipore);

[0169] (3) Filter sterilize through a 0.22 μm filter membrane, aliquot and store at -80 °C.

[0170] The 40 kDa target band disappeared and the degradation products increased.

[0171] Data comparison

[0172]

[0173]

[0174] According to the above embodiments and comparative examples, the dual-system cooperation and low-temperature enzymatic digestion of the present invention significantly improve the expression efficiency and reduce degradation, and the technical effects exceed the conventional expectations.

[0175] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirits of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

[0176] The above description of the present invention and its embodiments is not restrictive, but only one of the embodiments of the present invention, and the actual application is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar ways and embodiments without creative efforts without departing from the purpose of the present invention, they should all fall within the protection scope of the present invention.

[0177] SEQ ID NO.1

[0178] MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSG*

[0179] SEQ ID NO.2

[0180] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL DIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSG*。

Claims

1. A collagen peptide, characterized in that, Its amino acid sequence contains the following structures: (a) N-terminal GST tag; (b) HRV-3C protease cleavage site; (c) Collagen III domain, containing repetitive Gly-X-Y sequences (X and Y are proline or hydroxyproline); (d) C-terminal a-factor signal peptide (for yeast expression system).

2. The collagen peptide according to claim 1, wherein The molecular weight of the collagen peptide is 15.3 - 41.7 kDa, and the isoelectric point is 6.13 - 9.

13.

3. The collagen peptide according to claim 1, characterized in that, The sequence of the collagen peptide includes the sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

4. A method for preparing the collagen peptide according to any one of claims 1-3, characterized in that, It includes the following steps: (1) Construct a fusion expression vector, and insert the N-GST tag, HRV-3C cleavage site, and collagen III gene in sequence; (2) Transform the vector into Escherichia coli BL21(DE3) and Pichia pastoris X33 strains respectively; (3) Induce expression in stages: The Escherichia coli system is induced with 0.5 mM IPTG at 30 °C for 12 - 16 hours, and the yeast system is induced with methanol for 72 hours; (4) After lysing the cells, purify the fusion protein by GST affinity chromatography; (5) Use HRV-3C protease to digest at 4 °C for 12 hours to remove the tag; (6) Purify twice to obtain the target collagen peptide.

5. The method according to claim 4, wherein In step (3), the induction temperature of Escherichia coli is 28 - 30 °C, and the induction time is 12 - 16 hours.

6. The method according to claim 4, characterized in that In step (5), the digestion temperature is 4 °C, and the digestion time is 10 - 14 hours.

7. The method according to claim 4, characterized in that In step (4), the purification buffer contains 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM DTT.

8. The method according to claim 4, characterized in that, In step (2), the Pichia pastoris vector contains an a-factor signal peptide for secretory expression.

9. Use of the collagen peptide according to any one of claims 1-3 in cosmetics, characterized in that, Its moisturizing property is improved by more than 30% compared with natural collagen.

10. Use of the collagen peptide according to any one of claims 1-3 in a health food, characterized in that, It can promote the proliferation rate of fibroblasts by 50%.

Images