CN113527523A - Recombinant protein and construction method and application thereof - Google Patents

Recombinant protein and construction method and application thereof Download PDF

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CN113527523A
CN113527523A CN202111074554.2A CN202111074554A CN113527523A CN 113527523 A CN113527523 A CN 113527523A CN 202111074554 A CN202111074554 A CN 202111074554A CN 113527523 A CN113527523 A CN 113527523A
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recombinant
fibronectin
recombinant protein
transdermal
ser
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CN113527523B (en
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李宇涵
王娟
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Meimu Beijing Technology Co ltd
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Meimu Beijing Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/004—Aftersun preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/005—Preparations for sensitive skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention discloses a recombinant protein, which is constructed by connecting transdermal peptide with fibronectin through connecting peptide to form recombinant transdermal fibronectin, and then carrying out E174D and P292G site mutation construction on an amino acid sequence of the recombinant transdermal fibronectin to obtain a recombinant transdermal fibronectin mutant, wherein the amino acid sequence of the recombinant transdermal fibronectin mutant is shown as SEQ ID No.1, and the flexible end of the recombinant transdermal fibronectin mutant is constructed by connecting the connecting peptide with elastase inhibitory peptide. The recombinant protein provided by the invention has better stability, ensures the efficacy, is simple and convenient to prepare, has lower cost, and greatly expands the application of fibronectin and collagenase inhibitory peptide in cosmetics.

Description

Recombinant protein and construction method and application thereof
Technical Field
The invention relates to a recombinant protein, in particular to a recombinant protein with high stability for preparing or adding cosmetics and a construction method thereof.
Background
Fibronectin (also known as Fn) is a high molecular weight glycoprotein in the extracellular matrix. FN is produced by liver and vascular endothelial cells, widely exists in animal tissues and tissue fluid, is a macromolecular glycoprotein, has biological functions of cell regeneration and repair, and is applied to clinical treatment at present, including treatment of spinal diseases, burns and the like. Experience proves that fibronectin has a repairing effect on sensitive skin, red blood, acne, sunburn injury and other problems of skin.
Fibronectin has excellent skin care effects, but its thermal and chemical stability is poor. Poor thermal stability is mainly observed in that the lability of fibronectin in environments above 40 ℃ causes the precipitation, precipitation or discoloration of cosmetics, with consequent effects. The poor chemical stability is mainly due to fibronectin denaturation caused by conventional additives such as surfactants. The above problems limit the use of fibronectin in the cosmetic field.
The prior art mainly utilizes a stabilizer for improving the stability of fibronectin, and the effective components of the fibronectin stabilizer are sodium alginate, collagen hydrolysate or glycerol, etc. The principle is that the stabilizer is utilized to improve the aggregation stability and the dispersion stability of the fibronectin aqueous solution in a cosmetic system, and the fibronectin aqueous solution is beneficial to maintaining the stability of the fibronectin aqueous solution for a long time. Although the stabilizer can improve the stability of fibronectin, the preparation cost of the cosmetic is increased, the components possibly influence the efficacy of other functional components, the addition proportion and the preparation process need to be strictly regulated, the stability requirement of fibronectin is met, the efficacy of other functional components is not influenced, and the research and development difficulty of the formula of the cosmetic is improved.
In addition, in cosmetic formulations, multiple protein peptides are often used in combination, such as the commonly used matrix metalloproteinase inhibiting peptides. Matrix metalloproteinases are a family of endopeptidases with the ability to degrade the extracellular matrix (ECM), including collagenases and elastases, among others. Wherein collagenase MMP-3,1,8, etc. can decompose collagen in skin; elastase (MMP-12) degrades elastin and activates the activity of other matrix metalloproteinases. By inhibiting the activities of the peptide inhibiting collagenase and elastase, the degradation of two structural proteins in the skin can be effectively reduced, the apoptosis of fibroblasts is relieved, and the aim of resisting the skin aging is fulfilled.
In the prior art, the small molecular polypeptide and fibronectin are generally compounded in a cosmetic formula by a physical mixing method, the stability of fibronectin is poor, and the small molecular polypeptide is added, so that the stability challenge is larger, and the preparation cost is higher. Meanwhile, the skin surface has a physical barrier, which prevents most of macromolecular active ingredients from acting through the skin, and because the transdermal performance of the protein polypeptide complex is often poor, the original polypeptide with high activity cannot fully exert the due effect, which further limits the application of the protein polypeptide in cosmetics.
Disclosure of Invention
The invention aims to solve the primary technical problem of providing a recombinant protein with excellent stability.
Another technical problem to be solved by the present invention is to provide a method for constructing the above recombinant protein.
The invention also aims to provide the application of the recombinant protein.
In order to achieve the purpose, the invention adopts the following technical scheme:
a recombinant protein is formed by connecting transdermal peptide with fibronectin through a connecting peptide to form recombinant transdermal fibronectin, and then E174D and P292G site mutation is carried out on an amino acid sequence of the recombinant transdermal fibronectin to construct a recombinant transdermal fibronectin mutant, wherein the amino acid sequence of the recombinant transdermal fibronectin mutant is shown as SEQ ID No.1, and the flexible end of the recombinant transdermal fibronectin mutant is connected with elastase inhibitory peptide through the connecting peptide to construct the recombinant transdermal fibronectin mutant.
Wherein preferably, the amino acid sequence of the connecting peptide is shown as SEQ ID No. 3.
Wherein preferably, the amino acid sequence of the elastase inhibitory peptide is shown as SEQ ID No. 7.
Wherein preferably, the amino acid sequence of the recombinant protein is shown as SEQ ID No. 4.
The gene sequence of the encoded recombinant protein is shown as SEQ ID No. 5.
The preparation method of the recombinant protein comprises the following steps:
(1) inserting the nucleotide sequence for coding the recombinant protein into a vector to obtain a recombinant plasmid;
(2) extracting the genome DNA of the recombinant plasmid obtained in the step, linearizing, and electrically transferring into competent pichia pastoris; adding precooled sorbitol, then coating the content on a culture medium, and incubating until cloning is generated;
(3) screening a strain capable of expressing the target recombinant protein, and expressing and purifying the screened strain.
The recombinant protein is used for preparing an external skin care product additive or a skin care preparation.
Preferably, the external skin care product additive or the skin care preparation is an external skin care product additive or a skin care preparation with an anti-aging effect.
The beneficial results of the invention are as follows:
(1) the invention connects the transdermal peptide with fibronectin, and improves the transdermal performance of fibronectin. The invention also carries out site-directed mutagenesis on the specific area of the constructed transdermal fibronectin, improves the stability of the transdermal fibronectin, does not influence the efficacy of the fibronectin while improving the stability, and ensures that the efficacy of the transdermal fibronectin is exerted more fully.
(2) The invention improves the problems of poor stability and reduced efficacy caused by physical mixing of the existing fibronectin and elastase inhibitory peptides. The invention connects the elastase inhibition peptide to the flexible end of the recombinant transdermal fibronectin through the connecting peptide, further improves the stability of the recombinant transdermal fibronectin and simultaneously improves the stability of the inhibition peptide.
(3) The invention optimizes the connecting peptide, and avoids the problem that the function of the inhibiting peptide is influenced because the connecting peptide and the inhibiting peptide form a secondary structure.
(4) The recombinant transdermal fibronectin mutant and the elastase inhibitory peptide are expressed in a yeast system after being recombined, so that the process of singly chemically synthesizing the small molecular peptide in the prior art is improved, and the cost is saved by more than 50 percent.
(5) The recombinant protein provided by the invention has better stability, the efficacy is ensured, the preparation is simple and convenient, and the cost is lower, so that the application of fibronectin and collagenase inhibitory peptide in cosmetics is greatly expanded.
Drawings
FIG. 1 is a plasmid map of a recombinant transdermal fibronectin mutant expression vector in an example of the present invention;
FIG. 2 is a graph showing the expression amount of a recombinant protein produced after purification in an example of the present invention;
FIG. 3 is a graph showing the effect of recombinant proteins on elastin in fibroblasts in an example of the present invention;
FIG. 4 is a graph showing the effect of recombinant proteins on fibroblast survival rate in examples of the present invention;
FIG. 5 is a graph showing the effect of recombinant proteins on human fibroblast adhesion in an example of the present invention;
FIG. 6 is a graph showing the results of the stability test of the recombinant protein in the examples of the present invention.
Detailed Description
The raw materials used in the example are all raw materials known in the field, can be obtained by commercial purchase, and meet the raw material standard in the field of cosmetics.
The invention utilizes the site-directed mutagenesis of fibronectin to improve the stability of fibronectin, and simultaneously utilizes the connection of inhibitory peptide at the flexible end of fibronectin to further improve the stability of fibronectin.
The sequence of the elastase inhibitory peptide used in the embodiment is GLPY shown in an artificially synthesized sequence SEQ ID No.7, the original sequence is from elastin polypeptide obtained after protease treatment of bovine arterial elastase, the optimized elastase inhibitory peptide has good efficacy and is simplified in sequence, the recombinant transdermal fibronectin mutant has the creative technical effect, the stability of recombinant protein can be further improved by increasing the elastase inhibitory peptide, and the elastase inhibitory peptide can be selected from elastase inhibitory peptides from other sources in the field, so that the realization of the final creative efficacy of the invention is not influenced.
The invention relates to two recombinant protein peptides, namely transdermal fibronectin mutant recombinant protein shown in SEQ ID No.1, and a transdermal fibronectin mutant-elastase inhibitory peptide recombinant protein shown in SEQ ID No.4 is finally obtained by connecting elastase inhibitory peptide to the flexible end of a protein mutant by using connecting peptide on the basis of SEQ ID No. 1. The construction methods of the two recombinant proteins are the same. The invention is further described below by means of detailed examples.
Example 1 development of transdermal fibronectin mutants
1. Mutant research and development thought:
the cosmetic formulation contains surfactant, thickener, essence, polyol, etc. which affect the stability of protein peptide in cosmetics. Meanwhile, cosmetics may encounter high temperature conditions during transportation and storage, accelerating degradation of protein peptides.
According to the invention, the original sequence of the transdermal peptide fibronectin is shown in SEQ ID No.6 according to the three-dimensional structure characteristics of the recombinant protein after the transdermal peptide and the fibronectin are recombined, amino acid point mutation is carried out on the surface of the transdermal peptide fibronectin, the stability of the recombinant protein can be obviously improved, active sites can be effectively avoided by selecting the mutation points, and no influence is generated on the function of the recombinant fibronectin.
Experiment design: pdb, the crystal structure file of human fibronectin, 1fnf, was downloaded from the RCSB website. The temperature factor B-facor was analyzed by PyMOL software to predict the heat-sensitive amino acid sites. The higher the B-factor value, the greater the ambiguity, the more unstable or flexible the conformation of the corresponding site. Preferred temperature sensitive sites for recombinant transdermal fibronectin are N138, E174, G230, P292, flexible regions all on the protein surface. A single point mutation or simultaneous mutation of a plurality of sites (N138Q, E174D, G230V and P292G) is carried out on the sites through PCR to construct a plurality of mutants, a surfactant SLS is added into each mutant solution, and the mutant solution is stored at 40 ℃ for 1 month to carry out a heat accelerated aging experiment so as to select a recombinant protein mutant with higher stability. The specific construction method of the recombinant protein mutant is the same as that in example 2.
2. Recombinant transdermal fibronectin mutant stability test methods:
the recombinant transdermal fibronectin mutant 100 ml 300uM solution was added with 1% SLS solution to prepare recombinant protein +1% SLS solution. The recombinant protein, recombinant protein +1% SLS solution was dispensed into 10ml vials, 10 ml/vial, and three vials were prepared for each sample. Measuring each index initial value of each sample, then storing in a constant temperature box at 40 ℃, after 1 month, taking 200 mu L of each sample, placing in a sample hole of a 96-hole plate, repeating 3 times for each bottle, taking diluent without protein as blank control, detecting the OD value of the sample under 630nm of an enzyme labeling instrument, wherein the larger the OD value is, the more turbid the sample is, and the smaller the OD value is, the clearer the sample is; turbidity characterizes the stability of the protein, the more unstable the protein is, the more easily it precipitates, and the higher the turbidity is. The mutants and specific experimental results designed in this study are shown in table 1.
TABLE 1
Encoding Sample (I) SLS content OD630 before storage OD630 (40 deg.C, 1 month) OD630 changeChemical value
C-C Blank solution without protein 1% 0.004 0.002 0.002
C-0 Recombinant transdermal fibronectin 1% 0.029 0.064 0.035
A-1 Mutant 1 (N138Q) 1% 0.031 0.050 0.019
A-2 Mutant 2 (E174D) 1% 0.029 0.039 0.010
A-3 Mutant 3 (G230V) 1% 0.029 0.049 0.020
A-4 Mutant 4 (P292G) 1% 0.030 0.046 0.011
A-5 Mutant 5 (E174D, P292G) (SEQ ID No. 1) 1% 0.030 0.036 0.006
A-6 Mutant 6 (N138Q, G230V) 1% 0.031 0.039 0.018
As shown in the table above, after the recombinant protein A-5 obtained by simultaneously mutating E174D and P292G on the recombinant transdermal fibronectin mutant is stored at 40 ℃ for 1 month, the change of OD630 value is minimum, which indicates that the mutant enhances the stability of protein structure, has better compatibility with other raw materials in cosmetics, and can better adapt to high-temperature storage conditions and transportation.
EXAMPLE 2 construction of transdermal fibronectin mutant recombinant protein plasmids
In the embodiment of the invention, the amino acid sequence of the recombinant transdermal fibronectin mutant (A-5) is shown in SEQ ID No.1, wherein ACSPPHSKSHC is a transdermal peptide sequence which can enhance the transdermal absorption performance of the protein, and GGGGS is the connection between transdermal peptide and fibronectin.
The construction method comprises the following steps:
(1) designing and selecting enzyme cutting sites EcoR I and SalI according to the position of a related sequence of a commercial vector pPICZaA; the DNA sequence of the recombinant protein for coding the transdermal fibronectin mutant is shown as SEQ ID No. 2;
(2) the 5 'end and the 3' end of the synthesized recombinant protein DNA sequence are respectively provided with a restriction enzyme site which respectively corresponds to EcoR I and SalI;
(3) inserting the recombinant protein target fragment between vector restriction enzyme sites to obtain an expression plasmid for coding the recombinant protein, wherein the structure diagram of the vector plasmid is shown in figure 1.
EXAMPLE 3 construction of transdermal fibronectin mutants-Elastase inhibitory peptide recombinant protein plasmids
In the embodiment of the invention, the transdermal fibronectin mutant-elastase inhibitory peptide recombinant protein is prepared by connecting elastase inhibitory peptide to the flexible end of the transdermal fibronectin mutant prepared in the embodiment 2 through connecting peptide, and the amino acid sequence of the recombinant protein is shown as SEQ ID No. 4.
The construction method comprises the following steps:
(1) designing and selecting enzyme cutting sites EcoR I and SalI according to the position of a related sequence of a commercial vector pPICZaA; the DNA sequence of the recombinant protein for coding the transdermal peptide-fibronectin mutant-elastase inhibitory peptide is shown as SEQ ID No. 5;
(2) the 5 'end and the 3' end of the synthesized recombinant protein DNA sequence are respectively provided with a restriction enzyme site which respectively corresponds to EcoR I and SalI;
(3) inserting the recombinant protein target fragment between the vector enzyme cutting sites to obtain the expression plasmid for coding the recombinant protein.
Example 4 recombinant protein plasmid expression purification and electrophoresis identification method
The expression purification and electrophoresis identification methods of the recombinant protein plasmids described in example 2 and example 3 are the same, and the specific methods are as follows:
(1) preparation of Yeast clones
Extracting recombinant protein genome DNA of an expression vector, carrying out enzyme digestion (namely linearization, Thermo company) on a plasmid by using restriction enzyme Fastdigest SacI, purifying, carrying out competent mixing on the linearized plasmid and Pichia pastoris (Pichia pastoris) X33 (Beijing Baiolaoblepeaceae technology Limited), carrying out ice bath for 5min, transferring into a precooled electric shock cup, carrying out electric shock according to a preset program of an electric shock instrument (2 mm electric shock cup, electric shock conditions: 2000V, 25 muF and 200 omega), immediately adding 1ml of precooled 1M sorbitol after the electric shock, transferring into a 1.5ml centrifuge tube, putting the centrifuge tube into a 30 ℃ incubator for resuscitation for 2-3h, respectively coating YPDS (Yeast Extract peptide Dextrose) plates with the Zeocin concentrations of 0.1mg/ml, 1mg/ml and 2mg/ml, and carrying out culture for 3-5 days at 30 ℃.
(2) Expression purification of recombinant proteins
Selecting single clone, transferring into 250mL BMGY (Buffered Glycerol-complex Medium) shake flask culture Medium, culturing at 30 deg.C and 200rpm until OD600 is 2.0-6.0, centrifuging the fermentation broth at 4 deg.C and 8000rpm for 10min to remove supernatant, and collecting thallus; an appropriate amount of the cells was resuspended in BMMY shake flask medium containing 0.25mM CuSO4 and 1% methanol to OD 600. apprxeq.0.6, 30 ℃ at 200 rpm. Adding the above culture into 1L shake flask, covering with two layers of sterilized gauze or cheese cloth, and placing into shaking table for continuous growth. Methanol was added at intervals of 24h to 1% (w/v, final concentration). After culturing for 96 hours, 3000g of the fermented bacterial liquid was centrifuged for 5 minutes, and the supernatant was collected and the precipitate was discarded. Since the recombinantly expressed protein carries a polyhistidine tag (His 6. tag), the target protein was isolated using nickel ion affinity chromatography.
(3) Nickel ion affinity chromatography protein purification steps:
(a) balancing: equilibrating HisTrap HP nickel ion column (1mL) with 10-fold volume of 20mM buffer (containing 5mM imidazole);
(b) loading: the sample which is processed in advance is loaded at the flow rate of 1mL min-1;
(c) and (3) elution: gradient eluting with high concentration imidazole, collecting the tube number corresponding to the peak type under the elution condition, performing SDS-PAGE protein electrophoresis to confirm that the protein liquid in the tube with the molecular weight of the recombinant protein is combined and combined on anion exchange resin, and then eluting with 150mM NaCl and 20mM phosphate 0.5M urea solution.
The purity of the obtained protein is single through SDS-PAGE electrophoresis identification, the purity is above 95%, as shown in figure 2, which is recombinant transdermal fibronectin C-0, recombinant mutant protein A-5 and C-3.
Example 5 design and study of linker peptide sequence and efficacy test of recombinant transdermal fibronectin mutant-elastase inhibitory peptide
In the present example, the recombinant transdermal fibronectin mutant and the elastase inhibitory peptide are linked by a linker peptide. The length and sequence of the linker peptide may affect the structure and viability of the recombinant transdermal fibronectin mutants and inhibitory peptides. If the length is too short, the mutant and the inhibitory peptide may cause steric hindrance, influence the combination of the mutant and the inhibitory peptide, reduce the inhibitory effect of the elastase inhibitory peptide on elastase, and simultaneously reduce the cell adhesion promoting effect of fibronectin. If the length is too long, the mutant, the linker peptide and the inhibitor peptide are likely to form secondary structures, which also affect the function of the recombinant protein. The selection of an appropriate linker peptide is important to ensure the function of the recombinant protein. Sample sequences and linker peptide designs are shown in table 2.
TABLE 2
Encoding Sample sequence Linker peptide
C-00 Mutant 5 Is free of
C-01 Elastase inhibiting peptides Is free of
C-1 Mutant 5+ linker peptide + Elastase inhibitory peptide AG
C-2 Mutant 5+ linker peptide + Elastase inhibitory peptide AGG
C-3 Mutant 5+ linker + Elastase inhibitory peptide (SEQ ID No. 4) GGGGS
C-4 Mutant 5+ linker peptide + Elastase inhibitory peptide GGGAAAA
C-5 Mutant 5+ linker peptide + Elastase inhibitory peptide GGGAAAASS
1. Fibroblast cell assay
(1) Effect of recombinant proteins on elastin in photodamaged fibroblasts
Human skin fibroblasts (HFF, chinese academy of sciences typical culture collection cell bank); DMEM high-glucose medium, fetal bovine serum, trypsin, cyan/streptomycin (Gibco, usa); MTT, DMSO, EDTA (Sigma, USA).
Taking HFF cells in logarithmic phase, inoculating the HFF cells in a 96-well plate, culturing for 24h, removing old culture solution, washing with PBS for 1 time, adding a small amount of PBS to cover the bottom of a culture dish, irradiating for 10min under an ultraviolet lamp (20 cm away from the ultraviolet lamp), and replacing fresh DMEM culture solution containing 300uM recombinant protein after irradiation to continue culturing for 24 h.
After culturing the cells, the DMEM medium containing the recombinant protein was decanted. The remaining cells were washed clean with PBS, digested with 0.25% pancreatin, gently blown down, collected and centrifuged (1000 Xg, 3 min). After that, the supernatant was discarded and 200. mu.L PBS was added to resuspend the cells. The above centrifugation step was repeated, the supernatant was discarded, and 200. mu.L of RIPA lysate was added to treat the cells for 15min to lyse the cells. Then, the cells were centrifuged at 13000 Xg at 4 ℃ for 5min to collect the supernatant lysate. The related kit was used and the elastin content of the lysate was determined according to the instructions.
The elastase content in fibroblasts increased significantly after UV irradiation, resulting in the decomposition of elastin and a rapid decrease in the content. As shown in FIG. 3, in the experimental group C-3, elastin increased significantly, and the degree of increase was very close to and slightly better than that of the control group C-01, indicating that the linker peptide GGGGS did not affect the function of the enzyme inhibitory peptide but rather promoted the increase in efficacy. The increase in the experimental groups containing other linker peptides was low, indicating that other linker peptides reduced the activity of the inhibitory peptides.
(2) After the recombinant protein is added, the protective effect on the photo-aged fibroblast HFF
After cell culture, cell viability was determined by the MTT method. UV has strong penetrating action, and can cause damage to DNA on mitochondria in fibroblasts, up-regulation of MMPs level and the like, and finally cause apoptosis of the fibroblasts. As can be seen from the results of fig. 4, the survival rate of HFF decreased after UV irradiation. When damaged fibroblasts and 300uM recombinant protein were co-cultured for 24 hours, cell viability increased to various degrees. The experimental group C-3, namely when the connecting peptide sequence is GGGGS, has more obvious effect of reducing the apoptosis of the fibroblasts induced by UV, is very close to the control group C-01 and is slightly superior to the control group C-01, and shows that the connecting peptide GGGGS has no influence on the function of the enzyme inhibiting peptide and has promotion effect on the efficacy. The effect of the experimental group containing other connecting peptides is not obvious, and the other connecting peptides are proved to reduce the activity of the inhibiting peptides.
Recombinant protein cell adhesion and growth promotion assay
Each recombinant protein was prepared at 20ug/mL, coated on a 96-well plate for 30 minutes, and washed twice with PBS. After blocking with 1% BSA at 37 ℃ for 30 minutes, human fibroblasts (cultured in serum-free medium) were added. And after 1h, gently sucking the culture medium in the hole, gently rinsing the unadsorbed cells by using PBS, detecting the number of the living cells adsorbed at the bottom of the hole plate by using a CCD8 method, and verifying the cell adhesion promoting activity of the recombinant protein. As shown in FIG. 5, the experimental groups C-3, C-4 and C-5 significantly enhanced cell adhesion with increasing concentration, and C-00 was close. In the experimental group containing the short connecting peptide, the cell adhesion of C-1 and C-2 is poor, and the short connecting peptide can cause the inhibiting peptide to form steric hindrance on fibronectin, so that the function of the fibronectin for promoting the cell adhesion is influenced.
By combining the above experimental results, the activities and effects of fibronectin and inhibitory peptides were not affected when the linker peptide selected GGGGS.
Recombinant protein stability assay
The samples were tested for thermal stability of C-0, A-5, C-1, C-2, C-3, C-4, C-5 as in the mutant A1-A5 stability screening assay. 100 ml of 300uM recombinant fibronectin solution was added to a 1% SLS solution to prepare a recombinant protein +1% SLS solution. The recombinant protein, recombinant protein +1% SLS solution was dispensed into 10ml vials, 10 ml/vial, and three vials were prepared for each sample. Measuring each index initial value of each sample, then storing in a constant temperature oven at 40 ℃, after 1 month and 3 months, putting 200 mu L of each sample into a sample hole of a 96-well plate, repeating 3 times for each bottle, using diluent without protein as blank control, and detecting the OD value of the sample under 630nm of an enzyme-labeling instrument. The larger the OD value, the more turbid the sample, and the smaller the OD value, the more clear the sample; turbidity characterizes the stability of the protein, the more unstable the protein is, the more easily it precipitates, and the higher the turbidity is.
As shown in FIG. 6, each mutant showed a smaller change in OD630 value than C-0 when stored at 40 ℃ for one month, and the difference between the mutants was not significant; after 3 months of storage, the stability of C1, C2, C3, C4, C5 were all significantly better than a-5. Therefore, on the basis of the A-5 mutant, the C terminal is connected with a segment of polypeptide, so that the flexible C terminal is stabilized, the stability of the recombinant protein is further improved, and the influence difference of each connecting peptide on the stability is not obvious.
Sequence listing
<110> Meimu (Beijing) science and technology Co., Ltd
<120> recombinant protein and construction method and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 371
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Ala Cys Ser Pro Pro His Ser Lys Ser His Cys Gly Gly Gly Gly Ser
1 5 10 15
Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys Tyr Ile Leu
20 25 30
Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu Ala Thr Ile
35 40 45
Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys Pro Gly Val
50 55 60
Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly His Gln Glu
65 70 75 80
Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr Gly Gly Ser
85 90 95
Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp
100 105 110
Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn
115 120 125
Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu
130 135 140
Leu Ser Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
145 150 155 160
Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Asp Gln His
165 170 175
Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro
180 185 190
Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His
195 200 205
Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His
210 215 220
Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser
225 230 235 240
Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val
245 250 255
Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile
260 265 270
Gly Gln Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
275 280 285
Ala Ala Thr Gly Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val
290 295 300
Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
305 310 315 320
Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile
325 330 335
Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val
340 345 350
Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn
355 360 365
Tyr Arg Thr
370
<210> 2
<211> 1113
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gcgtgcagcc cgccgcatag caaaagccat tgcggcggcg gcggcagcat tcagtggaac 60
gcgccgcagc cgagccatat tagcaaatat attctgcgct ggcgcccgaa aaacagcgtg 120
ggccgctgga aagaagcgac cattccgggc catctgaaca gctataccat taaaggcctg 180
aaaccgggcg tggtgtatga aggccagctg attagcattc agcagtatgg ccatcaggaa 240
gtgacccgct ttgattttac caccaccagc accagcaccg gcggcagcgc ggtgccgccg 300
ccgaccgatc tgcgctttac caacattggc ccggatacca tgcgcgtgac ctgggcgccg 360
ccgccgagca ttgatctgac caactttctg gtgcgctata gcccggtgaa aaacgaagaa 420
gatgtggcgg aactgagcat tagcccgagc gataacgcgg tggtgctgac caacctgctg 480
ccgggcaccg aatatgtggt gagcgtgagc agcgtgtatg atcagcatga aagcaccccg 540
ctgcgcggcc gccagaaaac cggcctggat agcccgaccg gcattgattt tagcgatatt 600
accgcgaaca gctttaccgt gcattggatt gcgccgcgcg cgaccattac cggctatcgc 660
attcgccatc atccggaaca ttttagcggc cgcccgcgcg aagatcgcgt gccgcatagc 720
cgcaacagca ttaccctgac caacctgacc ccgggcaccg aatatgtggt gagcattgtg 780
gcgctgaacg gccgcgaaga aagcccgctg ctgattggcc agcagagcac cgtgagcgat 840
gtgccgcgcg atctggaagt ggtggcggcg accggcacca gcctgctgat tagctgggat 900
gcgccggcgg tgaccgtgcg ctattatcgc attacctatg gcgaaaccgg cggcaacagc 960
ccggtgcagg aatttaccgt gccgggcagc aaaagcaccg cgaccattag cggcctgaaa 1020
ccgggcgtgg attataccat taccgtgtat gcggtgaccg gccgcggcga tagcccggcg 1080
agcagcaaac cgattagcat taactatcgc acc 1113
<210> 3
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gly Gly Gly Gly Ser
1 5
<210> 4
<211> 380
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Ala Cys Ser Pro Pro His Ser Lys Ser His Cys Gly Gly Gly Gly Ser
1 5 10 15
Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys Tyr Ile Leu
20 25 30
Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu Ala Thr Ile
35 40 45
Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys Pro Gly Val
50 55 60
Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly His Gln Glu
65 70 75 80
Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr Gly Gly Ser
85 90 95
Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp
100 105 110
Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn
115 120 125
Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu
130 135 140
Leu Ser Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
145 150 155 160
Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Asp Gln His
165 170 175
Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro
180 185 190
Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His
195 200 205
Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His
210 215 220
Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser
225 230 235 240
Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val
245 250 255
Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile
260 265 270
Gly Gln Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
275 280 285
Ala Ala Thr Gly Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val
290 295 300
Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
305 310 315 320
Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile
325 330 335
Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val
340 345 350
Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn
355 360 365
Tyr Arg Thr Gly Gly Gly Gly Ser Gly Leu Pro Tyr
370 375 380
<210> 5
<211> 1140
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gcgtgcagcc cgccgcatag caaaagccat tgcggcggcg gcggcagcat tcagtggaac 60
gcgccgcagc cgagccatat tagcaaatat attctgcgct ggcgcccgaa aaacagcgtg 120
ggccgctgga aagaagcgac cattccgggc catctgaaca gctataccat taaaggcctg 180
aaaccgggcg tggtgtatga aggccagctg attagcattc agcagtatgg ccatcaggaa 240
gtgacccgct ttgattttac caccaccagc accagcaccg gcggcagcgc ggtgccgccg 300
ccgaccgatc tgcgctttac caacattggc ccggatacca tgcgcgtgac ctgggcgccg 360
ccgccgagca ttgatctgac caactttctg gtgcgctata gcccggtgaa aaacgaagaa 420
gatgtggcgg aactgagcat tagcccgagc gataacgcgg tggtgctgac caacctgctg 480
ccgggcaccg aatatgtggt gagcgtgagc agcgtgtatg atcagcatga aagcaccccg 540
ctgcgcggcc gccagaaaac cggcctggat agcccgaccg gcattgattt tagcgatatt 600
accgcgaaca gctttaccgt gcattggatt gcgccgcgcg cgaccattac cggctatcgc 660
attcgccatc atccggaaca ttttagcggc cgcccgcgcg aagatcgcgt gccgcatagc 720
cgcaacagca ttaccctgac caacctgacc ccgggcaccg aatatgtggt gagcattgtg 780
gcgctgaacg gccgcgaaga aagcccgctg ctgattggcc agcagagcac cgtgagcgat 840
gtgccgcgcg atctggaagt ggtggcggcg accggcacca gcctgctgat tagctgggat 900
gcgccggcgg tgaccgtgcg ctattatcgc attacctatg gcgaaaccgg cggcaacagc 960
ccggtgcagg aatttaccgt gccgggcagc aaaagcaccg cgaccattag cggcctgaaa 1020
ccgggcgtgg attataccat taccgtgtat gcggtgaccg gccgcggcga tagcccggcg 1080
agcagcaaac cgattagcat taactatcgc accggcggcg gcggcagcgg cctgccgtat 1140
<210> 6
<211> 371
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ala Cys Ser Pro Pro His Ser Lys Ser His Cys Gly Gly Gly Gly Ser
1 5 10 15
Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys Tyr Ile Leu
20 25 30
Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu Ala Thr Ile
35 40 45
Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys Pro Gly Val
50 55 60
Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly His Gln Glu
65 70 75 80
Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr Gly Gly Ser
85 90 95
Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp
100 105 110
Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn
115 120 125
Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu
130 135 140
Leu Ser Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
145 150 155 160
Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His
165 170 175
Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro
180 185 190
Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His
195 200 205
Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His
210 215 220
Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser
225 230 235 240
Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val
245 250 255
Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile
260 265 270
Gly Gln Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
275 280 285
Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val
290 295 300
Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
305 310 315 320
Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile
325 330 335
Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val
340 345 350
Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn
355 360 365
Tyr Arg Thr
370
<210> 7
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Leu Pro Tyr
1

Claims (10)

1. A recombinant protein is characterized in that transdermal peptide is connected with fibronectin through a connecting peptide to form recombinant transdermal fibronectin, and then E174D and P292G site mutation construction is carried out on an amino acid sequence of the recombinant transdermal fibronectin to obtain a recombinant transdermal fibronectin mutant, wherein the amino acid sequence of the recombinant transdermal fibronectin mutant is shown as SEQ ID No.1, and the flexible end of the recombinant transdermal fibronectin mutant is connected with elastase inhibition peptide through the connecting peptide to obtain the recombinant transdermal fibronectin mutant.
2. The recombinant protein according to claim 1, wherein the amino acid sequence of the linker peptide is as set forth in SEQ ID No. 3.
3. The recombinant protein according to claim 1, wherein the amino acid sequence of the elastase inhibitory peptide is set forth in SEQ ID No. 7.
4. The recombinant protein according to claim 3, wherein the amino acid sequence of the recombinant protein is as shown in SEQ ID No. 4.
5. The recombinant protein according to claim 4, wherein the gene sequence encoding the recombinant protein is as shown in SEQ ID No. 5.
6. A method for producing a recombinant protein according to any one of claims 1 to 5, characterized in that it comprises the following steps:
(1) inserting the nucleotide sequence for coding the recombinant protein into a vector to obtain a recombinant plasmid;
(2) extracting the genome DNA of the recombinant plasmid obtained in the step, linearizing, and electrically transferring into competent pichia pastoris; adding precooled sorbitol, then coating the content on a culture medium, and incubating until cloning is generated;
(3) screening a strain capable of expressing the target recombinant protein, and expressing and purifying the screened strain.
7. Use of the recombinant protein according to any one of claims 1 to 5 for the preparation of an additive for external skin care products or skin care preparations.
8. Use according to claim 7, wherein the external skin care product additive or skin care preparation is an external skin care product additive or skin care preparation with anti-ageing effect.
9. Use of the recombinant protein prepared by the preparation method of claim 6 in preparing an additive for external skin care products or skin care preparations.
10. Use according to claim 9, wherein the external skin care product additive or skin care preparation is an external skin care product additive or skin care preparation with anti-ageing effect.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113527526A (en) * 2021-09-14 2021-10-22 美慕(北京)科技有限公司 Recombinant protein and construction method and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110204608A (en) * 2019-05-10 2019-09-06 美尔健（深圳）生物科技有限公司 One primary yeast fermentation small molecule recombination fibronectin peptide and its preparation method and application
CN110511280A (en) * 2019-07-16 2019-11-29 华南理工大学 The transdermal recombination fibronectin of one kind and its application
CN111454350A (en) * 2020-06-01 2020-07-28 广东丸美生物技术股份有限公司 Recombinant fibronectin mutant and application thereof

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CN110204608A (en) * 2019-05-10 2019-09-06 美尔健（深圳）生物科技有限公司 One primary yeast fermentation small molecule recombination fibronectin peptide and its preparation method and application
CN110511280A (en) * 2019-07-16 2019-11-29 华南理工大学 The transdermal recombination fibronectin of one kind and its application
CN111454350A (en) * 2020-06-01 2020-07-28 广东丸美生物技术股份有限公司 Recombinant fibronectin mutant and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113527526A (en) * 2021-09-14 2021-10-22 美慕(北京)科技有限公司 Recombinant protein and construction method and application thereof
CN113527526B (en) * 2021-09-14 2021-12-17 美慕(北京)科技有限公司 Recombinant protein and construction method and application thereof

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