Recombinant elastin-like protein as well as preparation method and application thereof

Abstract

The invention is a divisional application of an invention patent application of which the application date is November 7, 2024, the application number is 202411581372.8, and the invention name is'a recombinant elastin-like protein as well as a preparation method and application thereof '. The invention relates to a recombinant elastin-like protein as well as a preparation method and application thereof, and belongs to the technical field of biomacromolecules. The invention provides a recombinant elastin-like protein. The recombinant elastin-like protein comprises an elastin-like protein motif and a collagen III functional motif, the amino acid sequence of the elastin-like motif is VPGXG; the amino acid sequence of the III type collagen functional motif is as shown in SEQ ID NO. 1. The invention provides the recombinant elastin-like protein which is fused with the collagen motif and the elastin motif of the triple helix structural domain with the activity of promoting cell adhesion, and the recombinant elastin-like protein has the temperature-sensitive characteristic, the pH response characteristic and the gel characteristic, and has the effects of resisting aging, resisting inflammation and repairing the skin barrier.

Description

This application is a divisional application of the invention patent application filed on November 7, 2024, with application number 202411581372.8 and invention title "A recombinant elastin and its preparation method and application". Technical Field This invention relates to the field of biomacromolecule technology, and in particular to a recombinant elastin, its preparation method, and its applications. Background Technology

[0001] Sensitive skin (SS), also known as sensitive skin, was defined as a syndrome in 2016 by a special interest group of the International Forum for Research on Itch. It is characterized by unpleasant sensations (tingling, burning, pain, itching, and stinging) after stimuli that should not elicit a response, such as cold, heat, or other physical and / or chemical factors. The pathophysiology of sensitive skin remains poorly understood, but its symptoms suggest inflammation and are innervated by peripheral nerves. Once skin inflammation occurs in individuals with sensitive skin, the signaling of inflammatory factors is accelerated, and the inflammation spreads from the stratum corneum to the dermis, causing degradation of extracellular matrix components such as elastin, fibronectin, and collagen. Collagen contains a large number of polar functional groups such as hydroxyproline, glycine, and aspartic acid, while elastin is rich in glycine and proline and can form β-helices that expose hydrophilic amino acids on the outer surface. These characteristics endow collagen and elastin with moisturizing properties for the epidermis and dermis. During the inflammatory phase, the breakdown of collagen can trigger the clearance of cellular debris and bacteria by immune cells, regulating the degree of inflammatory response.

[0002] The bioprocessing of recombinant proteins involves complex purification processes. Recombinant protein purification often employs affinity chromatography techniques, such as bioaffinity chromatography, immunoaffinity chromatography, and metal affinity chromatography. However, column packing materials have limitations in efficiency and cost, sometimes requiring the use of multiple columns to purify the target protein, which significantly increases the cost of industrial production.

[0003] Therefore, there is an urgent need to develop a protein that can resist aging, soothe, reduce inflammation, and repair the skin barrier, while also utilizing the protein's own properties to reduce industrial production costs and improve production efficiency. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a recombinant elastin, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a recombinant elastin, wherein the recombinant elastin comprises an elastin-like motif and a type III collagen functional motif; The amino acid sequence of the elastin-like motif is VPGXG, where X in VPGXG is any one of H, R, K, I, F, L, W, A, M, C, N, V, G, S, Q, Y, D, E, and T; The amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1.

[0006] This invention utilizes the phase transition properties of elastin-like proteins in response to temperature, pH, and salt ion concentration to construct a recombinant elastin-like protein that integrates collagen and elastin motifs with triple-helical domains that promote cell adhesion. The resulting recombinant elastin-like protein not only exhibits the temperature-sensitive and pH-responsive characteristics of elastin-like proteins but also displays the gelatinous properties of collagen: hydrophobicity increases and a loose, porous gel precipitates when the temperature rises to approximately 25°C within the range of 4–37°C; hydrophilicity increases and the protein reversibly re-dissolves at 37°C. pH variations between 9.0 and 7.0, as well as salt ion concentration, can also trigger the phase transition of this recombinant protein from a soluble to a gel state.

[0007] In a preferred embodiment of the recombinant elastin of the present invention, the amino acid sequence repeating number of the elastin motif in the recombinant elastin is an integer of ≥5, and the amino acid sequence repeating number of the class III collagen functional motif is an integer of ≥1.

[0008] In a preferred embodiment of the recombinant elastin of the present invention, the amino acid sequence repeating number of the elastin motif in the recombinant elastin is 5 to 100, and the amino acid sequence repeating number of the class III collagen functional motif is 1 to 100.

[0009] In a preferred embodiment of the recombinant elastin of the present invention, the amino acid sequence repeating number of the elastin motif in the recombinant elastin is 10 to 60, and the amino acid sequence repeating number of the class III collagen functional motif is 5 to 50.

[0010] In a preferred embodiment of the recombinant elastin of the present invention, the amino acid sequence repeating number of the elastin motif in the recombinant elastin is 30 to 50, and the amino acid sequence repeating number of the class III collagen functional motif is 8 to 16.

[0011] In a preferred embodiment of the recombinant elastin of the present invention, the recombinant elastin further includes a histidine tag, which is connected to the amino terminus and / or carboxyl terminus of the recombinant elastin. Introducing a histidine tag at the amino or carboxyl terminus of the recombinant elastin of the present invention facilitates the purification and identification of the target protein.

[0012] As a preferred embodiment of the recombinant elastin of the present invention, the recombinant elastin includes a linker sequence, which connects the elastin motif and the class III collagen functional motif; the amino acid sequence of the linker sequence is shown in SEQ ID NO.2.

[0013] Secondly, the present invention provides a method for preparing the above-mentioned recombinant elastin, which includes the following steps: S1. The vector containing the constructed elastin-like motif, the type III collagen functional motif, the linker sequence and the histidine tag was transferred into Escherichia coli to obtain an engineered bacterium expressing recombinant elastin-like protein. S2. The engineered bacteria expressing recombinant elastin obtained in step S1 are subjected to high-density fermentation to obtain the fermentation product. S3. The fermentation product obtained in step S2 is ultrasonically disrupted and centrifuged. The supernatant is then purified by reversible phase transition to obtain the crude product. S4. Resuspend the crude product obtained in step S3 in buffer solution and incubate at 35-37℃ until the crude product dissolves. Centrifuge to obtain the supernatant. S5. Perform reversible phase transition purification on the supernatant obtained in step S4 to obtain the purified product. S6. Repeat step S4 with the purified product obtained in step S5. The resulting supernatant is the recombinant elastin.

[0014] This invention develops a method for efficiently expressing recombinant elastin-like proteins using recombinant Escherichia coli. The expressed protein content can reach over 40%, exhibiting high expression and a short fermentation cycle. Based on the phase transition characteristics of the target protein, the fermented cells undergo two rounds of precipitation-dissolution reversible phase transition cycling (ITC) purification to obtain the target protein with a purity of over 90%. Furthermore, due to the reversible phase transition characteristics of recombinant elastin-like proteins, protein preservation does not require the addition of protein protectants, reducing purification costs and simplifying the purification process.

[0015] In a preferred embodiment of the preparation method of the present invention, in step S1, the number of elastin-like motifs in the carrier is an integer of ≥5, the number of type III collagen functional motifs in the carrier is an integer of ≥1, the linker sequence in the carrier connects the elastin-like motifs and the type III collagen functional motifs, and the histidine tag connects the amino terminus and / or carboxyl terminus of the recombinant elastin.

[0016] In a preferred embodiment of the preparation method described in this invention, step S2 includes the following steps: (1) Prepare a secondary seed culture from the engineered bacteria expressing recombinant elastin obtained in step S1, inoculate the secondary seed culture into a bioreactor, add glucose with an initial concentration of 5-6 g / L and magnesium sulfate with an initial concentration of 0.6-0.7 g / L, stir at 180-200 rpm, and keep the dissolved oxygen content above 30%. (2) After culturing at 35-37℃ for 3.5-4h, feed the material until the bacterial weight is ≥40g / L, maintain the dissolved oxygen at 80-85%, add IPTG at a final concentration of 0.1-1M, and culture for 4h to obtain the fermentation product.

[0017] As a preferred embodiment of the preparation method described in this invention, in steps S3 and S5, the reversible phase change purification operation mainly involves adjusting the pH value of the supernatant to 7.0-7.5, allowing it to stand at 23-28°C until a precipitate forms, centrifuging to collect the precipitate, and the resulting precipitate is the crude product or the purified product.

[0018] Thirdly, the present invention provides the application of the above-mentioned recombinant elastin in the preparation of anti-aging, anti-inflammatory, skin barrier repair and / or soothing products.

[0019] The recombinant elastin provided by this invention has activities that repair the skin barrier, inhibit skin inflammation, soothe and resist aging, and is expected to be developed into an active raw material for use in biomedicine and cosmetics. At the same time, based on its gel properties, it can be developed into an active biomaterial as a matrix material for tissue engineering.

[0020] As a preferred embodiment of the application described in this invention, the articles include pharmaceuticals, food, health products, cosmetics, and biological products.

[0021] Fourthly, the present invention provides an anti-aging, anti-inflammatory, skin barrier repairing and soothing cosmetic, comprising the above-mentioned recombinant elastin.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a recombinant elastin-like protein that integrates collagen motifs and elastin motifs with triple helical domains that promote cell adhesion activity. It has thermosensitive properties, pH-responsive characteristics, and gel properties: it is soluble at pH=9.0 and gel at pH=7.0; its hydrophobicity increases and a loose, porous gel is precipitated when the temperature rises to about 25°C within the range of 4 to 37°C; and its hydrophilicity increases and it can reversibly redissolve at 37°C.

[0023] 2. This invention provides a simplified protein purification process based on the reversible phase transition characteristics of recombinant elastin. By utilizing temperature and pH to mediate the change in the hydrophobicity and hydrophilicity of the protein, recombinant elastin with a purity of over 90% can be obtained through two rounds of reversible phase transition cycles. Compared with affinity chromatography purification, the process is simpler, more efficient, and significantly reduces costs, making it suitable for industrial production.

[0024] 3. The recombinant elastin provided by this invention has the activities of repairing the skin barrier, inhibiting skin inflammation, soothing and anti-aging, and is expected to be developed into an active raw material for use in biomedicine and cosmetics. At the same time, based on its gel properties, it can be developed into an active biomaterial as a matrix material for tissue engineering. Attached Figure Description

[0025] Figure 1 This is a spectrum of the recombinant elastin expression vector constructed in Example 1 of the present invention; Figure 2 This is an SDS-PAGE electrophoresis image of the engineered bacteria expressing recombinant elastin in Example 1 of this invention; Figure 3 SDS-PAGE assays for optimizing the expression conditions of recombinant elastin rhELT: Figure A shows SDS-PAGE assays induced by different temperatures; Figure B shows SDS-PAGE assays induced by different concentrations of IPTG. Figure 4 This is an SDS-PAGE electrophoresis image of recombinant elastin expressed by high-density fermentation at different induction times in Example 1 of the present invention. The bands indicated by the arrows in the image are recombinant elastin. Figure 5 The following are the SDS-PAGE and WB results of the recombinant elastin purified in Example 1 of the present invention. Figure A is an SDS-PAGE electrophoresis diagram. In Figure A, lane 1 is the first round of ITC product, lane 2 is the supernatant after centrifugation at pH 7.0, lane 3 is the reconstitution solution of the precipitate after centrifugation at pH 7.0, and lane 4 is the supernatant product after the second round of ITC centrifugation. Figure B is the WB result. Figure 6 The phase transition observation results of recombinant elastin in Example 1 of the present invention are shown in Figure A, which shows the appearance of the protein sample during the process of adjusting the pH from 9.0 to 7.0; Figure B shows the cross-sectional electron microscopy scanning results of the precipitate obtained at pH=7.0; and Figure C shows the longitudinal section electron microscopy scanning results of the precipitate obtained at pH=7.0. Figure 7 The results of the detection of degranulation of P815 mast cells by recombinant elastin in Example 2 of the present invention are shown in Figure A, which shows the results of neutral red staining in the cells, and Figure B shows the statistics of cell degranulation rate. *P<0.05, **P<0.01, ***P<0.001 vs. model group. Figure 8The results of the detection of the effect of recombinant elastin on the expression of matrix metalloproteinases (MMPs) in fibroblasts in Example 3 of the present invention are shown. *P<0.05, **P<0.01, ***P<0.001 vs. control group (-UV group); Figure 9 The results of the detection of the effect of recombinant elastin on the expression of collagen and fibronectin in the extracellular matrix of fibroblasts in Example 3 of the present invention are shown. *P<0.05, **P<0.01, ***P<0.001 vs. control group (-UV group); Figure 10 The results of the detection of the effect of recombinant elastin on the expression of HNEK cell barrier protein in Example 3 of the present invention are shown. *P<0.05, **P<0.01, ***P<0.001 vs. blank group.

[0026] Figure 11 The results of the detection of the effect of recombinant elastin on the transcriptional level of the capsaicin receptor TRPV1 gene in Example 3 of the present invention. *P<0.05, **P<0.01, ***P<0.001 vs. model group.

[0027] Figure 12 The results of the detection of the inflammatory factor TNF-α released by RAW264.7 cells by recombinant elastin in Example 3 of the present invention are shown in Figure A, which shows the content of inflammatory factor TNF-α, and Figure B shows the statistical inhibition rate of TNF-α production by recombinant elastin rhELT. *P<0.05, ***P<0.001 vs. model group, ns indicates no significant difference.

[0028] Figure 13 The results show the effect of recombinant elastin on ROS content in fibroblasts in Example 3 of this invention. *P<0.05, **P<0.01, ***P<0.001 vs. model group; ### P < 0.001 vs. model group.

[0029] In the above figure, "rhELT" refers to recombinant elastin. Detailed Implementation

[0030] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0031] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0032] Unless otherwise specified, in the following examples, comparative examples, and effect examples, the feed consisted of 5 g / L sterile glucose solution and 0.65 g / L sterile magnesium sulfate solution.

[0033] The mouse mast cell tumor cells P815 were provided by ATCC, catalog number TIB-64.

[0034] The human immortalized keratinocytes HaCat were provided by BeiNa Biotechnology, with the catalog number BNCC101683.

[0035] The mouse mononuclear macrophages RAW264.7 were provided by ATCC, catalog number TIB-71.

[0036] The techniques not described in detail in the following examples and effect examples are all commonly used techniques in the field. For reference, please refer to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Publishing House), "Molecular Biology Experiment (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiment" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0037] In the following examples and effect examples, "V" refers to valine (Val), "P" refers to proline (Pro), "G" refers to glycine (Gly), "H" refers to histidine (His), "R" refers to arginine (Arg), "K" refers to lysine (Lys), "I" refers to isoleucine (Ile), "F" refers to phenylalanine (Phe), "L" refers to leucine (Leu), "W" refers to tryptophan (Trp), "A" refers to alanine (Ala), "M" refers to methionine (Met), "C" refers to cysteine ​​(Cys), "N" refers to asparagine (Asn), "S" refers to serine (Ser), "Q" refers to glutamine (Gln), "Y" refers to tyrosine (Tyr), "D" refers to aspartic acid (Asp), "E" refers to glutamate (Glu), and "T" refers to threonine (Thr).

[0038] Example 1 This embodiment provides a recombinant elastin and its preparation method, the preparation method comprising the following steps: 1. Construction of engineered bacteria expressing recombinant elastin 1.1 In the class elastin motif VPGXG, "X" represents I, and the amino acid sequence of VPGIG is repeated 50 times, denoted as (VPGIG)50; the amino acid sequence of the class III collagen functional motif is repeated 16 times, and the amino acid sequence of the class III collagen functional motif is shown in SEQ ID NO.1, denoted as (aa)16, and named rhELT; 1.2 The (VPGIG)50 and (aa)16 obtained in step 1.1 are linked together using a linker sequence, and a histidine tag (HHHHHH) is introduced at the carboxyl terminus of the target protein. The amino acid sequence of the linker is shown in SEQ ID NO.2, and the amino acid sequence of the resulting recombinant elastin is shown in SEQ ID NO.3. The amino acid sequence of the recombinant elastin is codon optimized, and NdeⅠ is introduced at the amino terminus and HindⅢ restriction enzyme site sequences are introduced at the carboxyl terminus. The resulting optimized nucleic acid sequence with restriction enzyme sites is shown in SEQ ID NO.4. 1.3 The recombinant elastin-like gene fragment (SEQ ID NO.4) obtained in step 1.2 and incorporating restriction enzyme sites was synthesized by Suzhou Genewise Biotechnology Co., Ltd. The synthesized recombinant elastin-like gene fragment was ligated into the vector pET20b via NdeⅠ and HindⅢ restriction enzyme sites, transformed into *E. coli* DH5α, and positive clones were screened using LB agar plates. The positive clones were verified by colony PCR and sequencing to obtain the recombinant plasmid rhELT / pET20b (plasmid map shown). Figure 1 ); 1.4 The recombinant plasmid rhELT / pET20b obtained in step 1.3 was transformed into Escherichia coli BL21(DE3). Positive clones were screened and identified according to the method in step 1.3. After identification, it was confirmed that the Escherichia coli carried the recombinant vector rhELT / pET20b, that is, the obtained recombinant Escherichia coli was an engineered bacterium expressing recombinant elastin.

[0039] 2. Identification of the expression of recombinant elastin Pick the engineered bacteria expressing recombinant elastin obtained in step 1.4 (hereinafter referred to as engineered bacteria) and add them to 5 mL of LB broth medium with antibiotic resistance. Incubate at 37°C until OD500. 600 =0.6-0.8, add IPTG to a final concentration of 1mM, induce culture for 4h, collect bacterial cells, and detect the expression level of the target protein by SDS-PAGE. The SDS-PAGE results are shown in the figure. Figure 2 .

[0040] like Figure 2 As shown, all seven selected recombinant engineered bacteria rhELT / pET20b-BL21 expressed the target protein, with an apparent molecular weight consistent with the theoretical molecular weight of approximately 80.8 kDa. Gray-scale analysis results showed that the background protein content was low, and the target protein proportions of bacteria 1-7 were 40%, 45%, 47%, 40%, 45%, 42%, and 45%, respectively, all of which were highly expressed. This indicates that the present invention successfully and efficiently expressed recombinant elastin-like proteins through the E. coli system.

[0041] 3. Optimization of inducing expression conditions To determine the optimal induction conditions for high expression of the target protein, engineered bacteria with high expression levels were selected for experiments involving the three factors of induction temperature, time, and inducer concentration. Induction temperatures were set at 20℃, 30℃, and 37℃, corresponding to induction times of 4h, 6h, and 18h, respectively. After determining the optimal induction temperature and time, the optimal inducer concentration was screened. IPTG was used as the inducer, with concentrations set at 0.1, 0.25, 0.5, and 1.0 mM. The expression level of the target protein under different induction conditions was detected by SDS-PAGE electrophoresis.

[0042] like Figure 3 As shown, the expression levels of the target protein at different induction temperatures and times were 20℃, 4h > 37℃, 18h > 30℃, and 6h. Different IPTG concentrations and induction times had no significant effect on the expression levels of the target protein.

[0043] 4. Production and purification of recombinant elastin 4.1 The engineered bacteria that highly express recombinant elastin obtained in step 2 were inoculated into 50 mL of resistant LB medium and cultured at 37 °C with shaking for 12-16 h to obtain the primary seed culture; 4.2 Inoculate 50 mL of the primary seed culture obtained in step 4.1 into 2 L of resistant LB medium and culture at 37 °C with shaking for 12-16 h to obtain the secondary seed culture; 4.3 Inoculate 2L of the secondary seed culture obtained in step 4.3 into a 20L bioreactor, add sterile glucose solution with an initial concentration of 5g / L and sterile magnesium sulfate solution with an initial concentration of 0.65g / L, stir at 200rpm, aerate at 5L / min, maintain dissolved oxygen at above 30%, and incubate for 3.5h. 4.4 Feed the culture until the bacterial weight is ≥40g / L, maintain the dissolved oxygen level at 80%, add 7mL of 1M IPTG, and induce fermentation at 30℃ for 4h to obtain the fermentation product; 4.5 The fermentation product obtained in step 4.4 was centrifuged at 30,000 g for 10 min at 4 °C to collect the cell bodies. The cell bodies were resuspended in 50 mM Tris buffer (pH = 9.0) (cell bodies: Tris buffer = 1:20). The cell bodies were homogenized once at 4 °C and atmospheric pressure, and twice at 4 °C and 600 Pa. The supernatant was obtained by centrifugation. 4.6 Adjust the pH of the supernatant obtained in step 4.5 to 7.5, let it stand at 23-28℃ for 0.5h to precipitate, centrifuge at 4℃ and 18000rpm for 20min to collect the precipitate, and the precipitate obtained is the crude product; 4.7 Add an equal volume of 50 mM Tris buffer to the precipitate obtained in step 4.6, incubate in a 37°C water bath until the precipitate dissolves, and centrifuge at 25°C and 18000 rpm for 20 min to collect the supernatant. 4.8 Repeat the operation of step 4.6 with the supernatant obtained in step 4.7 to obtain a precipitate, which is the purified product; 4.9 Repeat the operation of step 4.7 on the precipitate obtained in step 4.8 to obtain the supernatant, which is the recombinant elastin solution.

[0044] Example 2 This embodiment provides a recombinant elastin-like protein and its preparation method. The preparation method is similar to that of Example 1, except that in step 1.1, "X" in the elastin-like protein motif VPGXG is V, the amino acid sequence of VPGVG is repeated 30 times, and it is denoted as (VPGVG)30; the amino acid sequence of the type III collagen functional motif is repeated 1 time, and the amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1, and it is denoted as (aa)1. The remaining steps and parameters remain unchanged, and it is named rhELT-1.

[0045] Example 3 This embodiment provides a recombinant elastin-like protein and its preparation method. The preparation method is similar to that of Example 1, except that in step 1.1, "X" in the elastin-like protein motif VPGXG is I, the amino acid sequence of VPGIG is repeated 30 times, and is denoted as (VPGIG)30; the amino acid sequence of the type III collagen functional motif is repeated 1 time, and the amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1, and is denoted as (aa)8. The remaining steps and parameters remain unchanged, and the product is named rhELT-2.

[0046] Example 4 This embodiment provides a recombinant elastin-like protein and its preparation method. The preparation method is similar to that of Example 1, except that in step 1.1, "X" in the elastin-like protein motif VPGXG is Y, the amino acid sequence of VPGYG is repeated 30 times, and it is denoted as (VPGYG)30; the amino acid sequence of the type III collagen functional motif is repeated 1 time, and the amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1, and it is denoted as (aa)8. The remaining steps and parameters remain unchanged, and it is named rhELT-3.

[0047] Example 5 This embodiment provides a recombinant elastin-like protein and its preparation method. The preparation method is similar to that of Example 1, except that in step 1.1, "X" in the elastin-like protein motif VPGXG is Y, the amino acid sequence of VPGYG is repeated 50 times, and it is denoted as (VPGYG)50; the amino acid sequence of the type III collagen functional motif is repeated 1 time, and the amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1, and it is denoted as (aa)16. The remaining steps and parameters remain unchanged, and it is named rhELT-4.

[0048] Example 1 The recombinant elastin obtained in Example 1 was characterized using the following specific method: 1. The fermentation product obtained in step 4.4 was analyzed by SDS-PAGE. The recombinant elastin-like protein from steps 4.7 and 4.9 was analyzed by SDS-PAGE and Western Blot (primary antibody: histidine antibody; secondary antibody: mouse anti-histidine antibody). The results are shown in the table below. Figure 4-5 .

[0049] like Figure 4 As shown, the expression level of recombinant elastin gradually increased after induction. Gray-scale analysis results showed that the target protein accounted for 27% after 3.5 hours, and the cell density reached 52 g / L after fermentation.

[0050] like Figure 5 As shown in Figure A, the effects of pH and temperature on the phase transition of recombinant elastin were used to purify it. After two rounds of ITC purification, a protein with high purity was obtained. Figure 5 As shown in Figure B, the immunoblot bands are single and consistent with the molecular weight in the SDS-PAGE. These results demonstrate that the preparation method of this invention can effectively prepare and purify recombinant elastin-like proteins.

[0051] 2. Phase transition observation and scanning electron microscopy (SEM) observation were performed on the recombinant elastin obtained in Example 1. Phase transition observation included the following steps: adjusting the pH of the recombinant elastin solution from 9.0 to 7.0 and observing the state of the recombinant elastin during the pH change. A small amount of the protein precipitated at pH 7.0 was freeze-dried. The freeze-dried protein sample was then adhered to conductive tape, sputtered with gold, and observed under a scanning electron microscope. The results are shown in [Figure number missing]. Figure 6 .

[0052] like Figure 6 As shown, the changes in hydrophobicity and hydrophilicity of recombinant elastin are directly related to temperature and pH of the buffer solution. When the pH of the buffer solution is adjusted from 9.0 to 7.0, the target protein gradually changes from a soluble state to a loose and porous gel.

[0053] Example 2 The soothing efficacy of the recombinant elastin obtained in Examples 1-5 was compared and tested, and the specific procedures are as follows: Logarithmic growth phase mouse mast cell tumor cells (P815) were counted and seeded into 24-well plates. After 24 hours of incubation, the original culture medium was discarded, and the medium containing the drug was replaced for another 24 hours. The positive control group received 1 mg / mL sodium cromoglycate, while the recombinant elastin-like proteins rhELT, rhELT-1, rhELT-2, rhELP-3, and rhELT-4 were incubated at 200 nM. The original culture medium was then discarded and replaced with medium containing C48 / 80 stimulant (20 μg / mL) to stimulate P815 cells for 1 hour, with the drug present simultaneously. The blank control group received an equal volume of basal culture medium. The supernatant was discarded, and 200 μL of 0.33% neutral red staining solution was added to cover the cells. After staining for 5 minutes, the cells were diluted with an equal volume of PBS, observed and photographed under a microscope, and the degranulation rate was statistically analyzed. The results are shown in the table below. Figure 7 .

[0054] like Figure 7 As shown in the neutral red staining results, at the same concentration, rhELT alleviated the degranulation of P815 hypersensitive cells better than rhELT-1, rhELT-2, rhELT-3, and rhELT-4, indicating that the recombinant elastin obtained in this invention has a better soothing effect.

[0055] Example 3 The function of the recombinant elastin obtained in Example 1 was tested, and the specific method is as follows: 1. Gene expression analysis of matrix metalloproteinases (MMP1, MMP3, MMP9) in fibroblasts Healthy human fibroblasts were seeded into 24-well plates and cultured in a 5% CO2 incubator for 24 hours. The original culture medium was discarded and replaced with pre-cooled PBS. The plates were then placed in a UV curing chamber for UVA irradiation (30 min, dose pre-set to 3.33 mW / cm²). 2 That is, 6J / cm 2 After irradiation, PBS was aspirated, and 2 mL of basal culture medium was added to each well of the blank and model groups. The drug group received culture medium containing 250 nM recombinant elastin. Each group was divided into three replicates, and cultured for another 72 h. Using GAPDH as a reference gene, the transcriptional levels of MMP1, MMP3, and MMP9 protein-related genes were detected by qRT-PCR (CFX384 Touch). The results are shown in the figure. Figure 8 .

[0056] Matrix metalloproteinases (MMPs) play a crucial role in aging. MMPs are a group of enzymes that degrade the extracellular matrix (ECM), functioning in tissue remodeling, repair, and cell migration. MMP1, MMP3, and MMP9 are members of the MMP family, and their roles in aging and longevity have attracted considerable attention. Studies have shown that reducing MMP expression or inhibiting their activity may help reduce extracellular matrix damage, thereby maintaining tissue structure and function and delaying aging. Figure 8 As shown, the recombinant elastin of the present invention can significantly inhibit the expression of MMPs under the administration condition of 250 nM (P≤0.05), indicating that the recombinant elastin of the present invention can reduce the degradation of the extracellular matrix induced by ultraviolet light, thereby achieving the effect of anti-aging.

[0057] 2. Gene expression analysis of extracellular matrix proteins (COL1A1, COL3A1, COL4A1, COL17A1, and FN1) in fibroblasts Healthy human fibroblasts were seeded into 24-well plates and cultured in a 5% CO2 incubator for 24 hours. The original culture medium was discarded and replaced with pre-cooled PBS. The plates were then placed in a UV curing chamber for UVA irradiation (30 min, dose pre-set to 3.33 mW / cm²). 2 That is, 6J / cm 2 After irradiation, PBS was aspirated, and 2 mL of basal culture medium was added to each well of the blank and model groups. The drug group received culture medium containing 250 nM recombinant elastin. Each group was divided into three replicates, and cultured for another 72 h. Using GAPDH as the reference gene, the transcriptional levels of COL1A1, COL3A1, COL4A1, COL17A1, and FN1 protein-related genes were detected by qRT-PCR (CFX384 Touch). The results are shown in the figure. Figure 9 .

[0058] The extracellular matrix (ECM) plays a crucial role in anti-aging. The ECM is a complex network composed of various macromolecules, including structural proteins (such as collagen and elastin), connective proteins (such as fibronectin and laminin), glycosaminoglycans, proteoglycans, and various growth factors. With age, the composition, structure, and function of the ECM change, and these changes are associated with decreased cellular activity, ultimately leading to tissue aging. Anti-aging strategies primarily involve reducing external damage to the ECM (such as UV exposure) and directly supplementing the ECM or stimulating its secretion. Collagen and fibronectin are key proteins in the ECM, playing a vital role in maintaining skin elasticity, resilience, and overall appearance. With age, the levels of collagen and fibronectin in the skin gradually decrease, leading to signs of aging such as sagging, drooping, and wrinkles. Figure 9 As shown, the recombinant elastin of the present invention can significantly promote the expression of type I, type III, type IV and type XVII collagen and fibronectin under the administration condition of 250 nM (P≤0.05), reduce the damage of ultraviolet radiation to ECM, and has anti-aging effects.

[0059] 3. NHEK cell barrier protein expression analysis Healthy human epidermal keratinocytes (NHEK, PCS-200-010, ATCC) were seeded into 12-well plates and cultured in a 5% CO2 incubator until the cell layering rate reached 50%–60%. The original culture medium was discarded. 2 mL of basal medium was added to each well of the blank control group, and equal volumes of medium containing 50 nM and 200 nM recombinant elastin were added to each well of the drug group, respectively. Three replicates were made for each group. After 24 h of further culture, the original culture medium was discarded, and the cells were washed twice with PBS and collected by centrifugation. Using GAPDH as a reference gene, the transcriptional levels of FLG and LOR protein-related genes were detected by qRT-PCR. The results are shown in [Figure number missing]. Figure 10 .

[0060] like Figure 10 As shown, the recombinant elastin of the present invention can significantly promote the transcription level of FLG and LOR protein-related genes under the administration condition of 200 nM, indicating that the recombinant elastin of the present invention can promote the expression of HNEK cell barrier-related proteins to improve the skin barrier.

[0061] 4. Analysis of TRPV1 gene expression in keratinocytes Human immortalized keratinocytes (HaCat) were stimulated with capsaicin to construct an in vitro efficacy evaluation model for relieving nerve pain. The experiment consisted of five groups: a blank control group, a model group, a positive control group, a low-dose group, and a high-dose group, which were treated as follows: The blank control group received no treatment. Model group, with 10μM capsaicin added; Positive group, with the addition of 10 μM capsaicin and 50 μM 4-tert-butylcyclohexanol; The low-dose group was supplemented with 10 μM capsaicin and 250 nM recombinant elastin. The high-dose group was supplemented with 10 μM capsaicin and 750 nM recombinant elastin.

[0062] The specific procedure is as follows: HaCat cells in good growth condition are seeded into 12-well plates and cultured in a 5% CO2 incubator until the cell deposition rate reaches 50%–60%. The original culture medium is discarded, and 300 μL of basal culture medium, basal culture medium containing 4-tert-butylcyclohexanol, or basal culture medium containing recombinant elastin are added respectively according to the above treatment. After culturing for 24 hours, the culture medium is discarded.

[0063] Following the above treatment, 300 μL of basal medium, basal medium containing capsaicin, basal medium containing recombinant elastin and capsaicin, or basal medium containing 4-tert-butylcyclohexanol and capsaicin were added respectively, and the cells were cultured for another 24 h. Cells were then collected by centrifugation, and the mRNA levels of TRPV1 receptor on the cell membranes of the blank control group, model group, positive group, and recombinant elastin group were detected by quantitative real-time PCR. Results are shown in [Figure 1]. Figure 11 .

[0064] like Figure 11 As shown, at a drug concentration of 750 nM, the recombinant elastin can effectively reduce the mRNA expression level of TRPV1, indicating that the recombinant elastin of the present invention can relieve nerve pain and has a certain soothing and calming effect on the skin.

[0065] 5. Expression analysis of inflammatory factors released by RAW264.7 macrophages RAW264.7 mouse mononuclear macrophages in good growth condition were seeded into 24-well plates and cultured in a 5% CO2 incubator for 24 h. The original culture medium was discarded. 2 mL of basal medium was added to each well of the control group, the model group to medium containing 1 μg / mL LPS, the positive control group to medium containing 1 μg / mL LPS and 100 μg / mL dexamethasone, and the drug group to medium containing 1 μg / mL LPS and 100 nM or 250 nM recombinant elastin. Each group was divided into three replicates, and the cells were cultured for another 24 h. Cells were then collected, and the levels of the inflammatory factor TNF-α were detected using the Mouse TNF-α (Tumor Necrosis Factor Alpha) ELISA Kit (E-EL-M3063, Elabscience). Results are shown below. Figure 12 .

[0066] like Figure 12 As shown, the recombinant elastin of the present invention has a significant inhibitory effect on the release of inflammatory factor TNF-α from LPS-stimulated RAW264.7 cells under a drug administration condition of 250 nM.

[0067] 6. Analysis of ROS content generated by fibroblasts Human fibroblasts in good growth condition were seeded into 6-well plates, 2 mL of cell suspension per well, and cultured in a 5% CO2 incubator for 24 h. The original culture medium was discarded and replaced with pre-cooled PBS, then the plates were placed in a UV curing chamber for UVA irradiation (30 min, dose pre-set to 3.33 mW / cm²). 2 That is, 6J / cm 2After irradiation, the PBS was aspirated, and 2 mL of basal culture medium was added to each well of the blank group and the model group. The positive group was given a culture medium containing 100 μg / mL ascorbic acid, and the drug group was given a culture medium containing 0.2%, 0.5%, or 1% recombinant elastin. Each group was divided into 3 replicates and cultured for another 1 hour.

[0068] Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μM. After cell collection, suspend the cells in the diluted DCFH-DA at a concentration of 1 million to 20 million cells / mL and incubate at 37°C for 20 minutes. During incubation, invert the cells every 3-5 minutes to ensure thorough contact between the probe and cells. Wash the cells three times with serum-free cell culture medium to remove any unextracted DCFH-DA. Analyze the results using flow cytometry (Beckman, CytoFlex), acquire and analyze images (Beckman, CytExpert). See attached table. Figure 13 .

[0069] Free radicals generated by mitochondria during electron transport can attack cell membranes, proteins, and even DNA, leading to cellular aging and even death, and are closely related to various skin diseases. In daily life, skin is frequently exposed to ultraviolet radiation, causing living cells to produce a large number of free radicals. These free radicals further damage living cells, leading to excessive ROS production in skin cells, resulting in cellular toxicity and oxidative stress damage. Figure 13 As shown, the recombinant elastin of the present invention significantly reduced the ROS content in a UV-induced fibroblast senescence model under administration conditions of 0.2% and 0.5%. This indicates that the recombinant elastin may enhance the skin's resistance to UV radiation damage by reducing ROS levels, thus exhibiting anti-photoaging and anti-aging effects.

[0070] In summary, the recombinant elastin of this invention has the effects of improving and repairing the skin barrier, soothing, anti-inflammatory and anti-aging, and has good prospects for pharmaceutical and cosmetic applications.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A recombinant elastin, characterized in that, The recombinant elastin-like protein comprises an elastin-like motif and a collagen type III functional motif; The amino acid sequence of the elastin-like motif is VPGXG, wherein X is any one of V and Y; and the amino acid sequence of the collagen type III functional motif is shown in SEQ ID NO.

1.

2. The recombinant elastin of claim 1, wherein, The number of repetitions of the amino acid sequence of the elastin-like motif in the recombinant elastin-like protein is an integer greater than or equal to 5, and the number of repetitions of the amino acid sequence of the collagen type III functional motif is an integer greater than or equal to 1.

3. The recombinant elastin of claim 1, wherein The recombinant elastin-like protein further comprises a histidine tag, which is connected to the amino terminal end and / or the carboxyl terminal end of the recombinant elastin-like protein.

4. The recombinant elastin of claim 1, wherein The recombinant elastin-like protein comprises a linker sequence, which connects the elastin-like motif and the collagen type III functional motif; and the amino acid sequence of the linker sequence is shown in SEQ ID NO.

2.

5. The method of producing a recombinant elastin according to any one of claims 1 to 4, wherein The method comprises the following steps: S1, transferring a vector containing an elastin-like motif, a collagen type III functional motif, a linker sequence and a histidine tag into E. coli to obtain an engineered bacterium expressing a recombinant elastin-like protein; S2, performing high-density fermentation on the engineered bacterium expressing the recombinant elastin-like protein obtained in step S1 to obtain a fermentation product; S3, performing crushing and centrifugation on the fermentation product obtained in step S2, and performing reversible phase transition purification on the obtained supernatant to obtain a crude product; S4, resuspending the crude product obtained in step S3 in a buffer, incubating at 35-37℃ until the crude product is dissolved, and then centrifuging to obtain a supernatant; S5, performing reversible phase transition purification on the supernatant obtained in step S4 to obtain a purified product; S6, repeating the operation of step S4 on the purified product obtained in step S5, and the obtained supernatant is the recombinant elastin-like protein.

6. The production method according to claim 5, wherein In step S1, the number of elastin-like motifs in the vector is an integer greater than or equal to 5, the number of collagen type III functional motifs in the vector is an integer greater than or equal to 1, the linker sequence in the vector connects the elastin-like motif and the collagen type III functional motif, and the histidine tag is connected to the amino terminal end and / or the carboxyl terminal end of the recombinant elastin-like protein.

7. The production method according to claim 5, wherein In step S2, the operation of high-density fermentation comprises the following steps: (1) preparing the engineered bacterium expressing the recombinant elastin-like protein obtained in step S1 into a secondary seed solution, inoculating the secondary seed solution into a bioreactor, adding glucose with an initial concentration of 5-6 g / L and magnesium sulfate with an initial concentration of 0.6-0.7 g / L, stirring at a speed of 180-200 rpm, and maintaining the dissolved oxygen content at more than 30%; (2) after culturing at 30-37℃ for 3.5-4 h, supplementing the bacteria to a weight of ≥40 g / L, maintaining the dissolved oxygen content at 80-85%, adding IPTG with a final concentration of 0.1-1 M, and culturing for 4 h to obtain the fermentation product.

8. The production method according to claim 5, wherein In steps S3 and S5, the operation of reversible phase transition purification mainly comprises adjusting the pH value of the supernatant to 7.0-7.5, standing at 23-28℃ until the precipitate is separated out, and collecting the precipitate by centrifugation, and the obtained precipitate is the crude product or the purified product.

9. Use of a recombinant elastin as claimed in any of claims 1 to 4 in the manufacture of a soothing preparation.

10. Use according to claim 9, wherein the compound is ###0002### The preparations include pharmaceuticals, foodstuffs, nutraceuticals, cosmetics and biologicals.

Images