Water stress protein WSP derived from nymphaea alba and its application in skin protection

Abstract

The application discloses a water stress protein WSP derived from Nostoc commune and application of the water stress protein WSP in skin protection, and belongs to the technical field of biotechnology and skin care. The amino acid sequence of the water stress protein WSP is shown as SEQ ID NO: 2, and the DNA sequence for coding the WSP protein is shown as SEQ ID NO: 1. The WSP protein of the application can significantly improve the survival rate of skin cells (such as HaCaT cells) under extreme dehydration conditions, protect the activity of key metabolic enzymes (such as lactate dehydrogenase), and effectively reduce the level of intracellular reactive oxygen species, and the comprehensive protection efficiency is superior to that of the existing moisturizing agent hyaluronic acid. The WSP protein provided by the application can be applied to skin care products such as face cream, essence and spray as an active ingredient, and is used for relieving dry skin, repairing skin barrier and antioxidation.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and skin care technology, and in particular to the water stress protein WSP derived from kudzu root and its application in skin protection. Background Technology

[0002] As the largest organ in the human body, the skin is not only a barrier for physiological metabolism but also the first line of defense against external environmental damage (such as low humidity, ultraviolet radiation, and pollution). The balanced distribution of moisture in the various layers of the skin is crucial for maintaining stratum corneum metabolism, enzyme activity, and skin elasticity. However, with changes in the modern living environment and aging, dry skin has become a common sub-health condition, manifesting as a damaged barrier, fine lines, sensitivity, and irritability, and can even lead to chronic skin inflammation.

[0003] Currently, mainstream moisturizing ingredients on the market are mainly divided into three categories: occlusive agents (such as petrolatum and silicone oil), humectants (such as glycerin and butylene glycol), and biomimetic repair agents (such as hyaluronic acid and ceramides). Hyaluronic acid (HA), as a leader among them, is widely used in cosmetics due to its excellent water-holding capacity (each gram can bind several liters of water). However, existing research shows that in high-salt, extremely dry, or extremely low-humidity environments, simple humectants often fail to maintain the conformational stability of intracellular proteins. In some extreme cases, high molecular weight HA, due to its extremely strong osmotic pressure and "water-grabbing" effect, may even accelerate the denaturation of sensitive proteins. Therefore, finding a natural active ingredient that can both retain water and act as a "molecular chaperone" to protect biomolecules has become a hot topic in the field of dermatology.

[0004] Nostoc commune is a terrestrial alga widely distributed in paddy fields, wetlands, or extremely arid surfaces. It can tolerate prolonged drought and strong ultraviolet radiation, and rapidly recovers its metabolic activity after rehydration. This remarkable resilience is inseparable from the large amounts of extracellular polysaccharides (EPS) and extreme desiccation stress proteins (WSP) it secretes. WSP, a hydrophilic acidic protein, is synthesized in large quantities and secreted into the extracellular matrix when Nostoc commune encounters water deficit.

[0005] Studies have shown that WSP proteins (such as WspA) in *Gnaphalium affine* not only participate in regulating the rheological properties of the extracellular polysaccharide matrix, maintaining its flexibility during dehydration and preventing disintegration, but also bind to key enzymes through their unique amino acid sequences, preventing enzyme aggregation and inactivation. Furthermore, WSP proteins contain a high proportion of polar residues such as glycine and serine, which can replace the function of water molecules through hydrogen bonds (water replacement hypothesis), providing a hydration-like shell for biomembranes and proteins in a dry state.

[0006] Although preliminary explorations have been made into the mechanisms of cyanobacterial WSP proteins in stress biology, their potential application as a protectant for human skin cells has not yet been systematically explored. In particular, data on the gene sequence, recombinant expression process, and protective kinetics of human keratinocytes under extreme dryness stress for the WSP subtype specific to Nostoc sphaeroides str. Hefeng are currently lacking. Summary of the Invention

[0007] Addressing the limitations of existing anti-dryness ingredients, such as insufficient protection of key intracellular enzymes under extreme environments and inability to effectively quench high ROS levels induced by dryness, this invention aims to provide WSP2, an extreme dryness stress protein derived from Nostoc sphaeroides str. Hefeng. This invention aims to achieve efficient heterologous expression of this protein through genetic engineering and establish a complete experimental verification system to demonstrate the outstanding efficacy of WSP2 in protecting HaCaT cell viability, maintaining the catalytic stability of endogenous enzymes, and regulating cellular redox balance. This provides technical support for its application in high-end functional skincare products and bio-repair agents, namely, proposing the water stress protein WSP derived from Nostoc sphaeroides str. Hefeng and its application in skin protection.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a water stress protein WSP derived from Nostoc sphaeroides, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0009] Preferably, the DNA sequence encoding the water stress protein WSP is shown in SEQ ID NO: 1.

[0010] A second aspect of the present invention provides a method for preparing the above-mentioned water stress protein WSP, comprising the following steps: (1) Synthesize a DNA fragment having the sequence shown in SEQ ID NO: 1; (2) The DNA fragment is cloned into an expression vector to construct a recombinant expression vector; (3) The recombinant expression vector is transformed into host cells and induced to express, thereby obtaining the water stress protein WSP.

[0011] This invention first screened and identified a key WSP gene of 909 bp in length from the genomic library of *Nostoc sphaeroides* str. Hefeng, and named it NS-WSP002. This gene sequence was optimized for high-abundance preparation using an *E. coli* expression system.

[0012] According to bioinformatics analysis, the protein encoded by the NS-WSP002 gene exhibits typical acidic and hydrophilic characteristics. To ensure that the expression product carries a histidine tag that facilitates purification, this invention uses pET-24a(+) as the expression vector. This vector contains a strong T7 promoter and a kanamycin resistance gene, which can support high-level induced expression of the target protein in E. coli. The specific steps are as follows: (1) WSP protein was retrieved from NCBI. The source species of the obtained WSP protein was determined by BLAST gene alignment analysis. The DNA sequence was adapted according to the selected expression plasmid and engineered bacteria to ensure that it could produce WSP protein efficiently. The DNA sequence of the obtained NS-WSP002 protein is shown in SEQ ID NO: 1. (2) The above DNA sequences were synthesized by gene synthesis and Nde I and Xho I restriction endonuclease sites were added to the 5' and 3' ends, respectively. Gene fragments with Nde I and Xho I restriction endonuclease sites were obtained by PCR amplification. The PCR product and pET-24a(+) vector were double digested with restriction endonucleases Nde I and Xho I to ensure directional ligation. The reaction conditions were set at 37℃ for 1-2 h.

[0013] (3) Electrophoresis and gel recovery of the double digestion products: The single WSP gene and the linearized pET-24a(+) fragment after double digestion were recovered using a gel recovery kit. The digested vector products and the WSP fragment were ligated with the linearized pET-24a(+) fragment using T4 ligase to form the pET-24a(+)-WSP recombinant plasmid. After the system was prepared, it was incubated at 22℃ for 0.5-1h. After the ligation reaction was completed, it was inactivated by heat at 65℃ for 10-20min.

[0014] (4) Take 5-10 μL of the ligation product and place it in 50-100 μL of E.coli BL21(DE3) competent cells. The transformation process is as follows. Pick single clones the next day: ① Remove E. coli BL21(DE3) competent cells from the -80℃ ultra-low temperature freezer and thaw them on ice; ② In a clean bench, add 300-500 ng of plasmid to 50-100 μL of E.coli BL21(DE3) competent cells, gently tap to mix, and distribute evenly to the competent cells. Place on ice for 30-60 min. The following operations are all performed in a clean bench: ③ Place competent cells with added plasmids in a pre-opened 42℃ water bath and heat shock for 90s; ④ Remove the competent cells and place them on ice for 3-5 minutes; ⑤ Add 750-900 μL of antibiotic-free LB medium to the tube containing competent cells and incubate on a shaker at 200-220 rpm for 30-60 min; ⑥ Centrifuge at room temperature at 2000-3000 rpm for 3-5 minutes; ⑦ After centrifugation, discard most of the culture medium, leaving approximately 100-150 μL of liquid culture medium. Mix this liquid medium with the bacterial precipitate by blowing and aspiration. Add the suspension dropwise to a solid culture medium containing kanamycin resistance, spread it evenly with a spreader until it feels rubbed against the surface of the medium; ⑧ Seal the culture dish with sealing film, incubate it upside down at 37℃ overnight, and observe the growth status of the plaques the next day; ⑨ The next day, pick a single colony and add it to 5-10 mL of LB liquid medium, and add an appropriate proportion of 100 mg / mL kanamycin stock solution according to the ratio of 50-100 μg / mL kanamycin resistance, and incubate at 37℃ and 200-220 rpm; ⑩ Store the bacterial culture at a ratio of 300 μL bacterial culture to 300 μL 50% glycerol, label it with the name and date, and store it in an ultra-low temperature freezer at -80℃; Perform plasmid mini-extraction according to the Tiangen plasmid mini-extraction kit, and measure the plasmid concentration using a Nanodrop 2000 nucleic acid quantification instrument after plasmid extraction.

[0015] (5) Single colony selection and culture after transformation: After overnight culture in a 37℃ incubator, remove the plate and observe the number and morphology of colonies. Select single colonies and prepare them for inoculation with liquid culture medium. Take out a bottle of sterilized liquid culture medium, add 50-100 μg / mL kanamycin, mix well, take out a sterilized clean test tube, use a sterile pipette tip to aliquot 5 mL of liquid culture medium into the test tube, use a sterile pipette tip to pick a single colony and inoculate it into the test tube, write a mark on the test tube wall, tilt the test tube and place it in a shaker, 37℃, 200-220 rpm, shake and culture overnight. After the culture is completed, take out the test tube and check the cell concentration. When the cell OD reaches 0.7-1.0, add 0.25-0.5 mM IPTG and continue to induce at 37℃ for 10-15 hours. After induction, centrifuge to collect the cells and perform ultrasonic lysis (ultrasonic volume is 3-5 mL, ultrasonic conditions are 2-5 seconds on, 2-5 seconds off, total ultrasonic time is 5-10 minutes). After centrifugation at 10,000-12,000 rpm, the supernatant was filtered through a 0.45 μm pore size filter membrane to collect the total bacterial protein. The total protein was then purified using Ni-NTA agarose gel permeation resin to purify the recombinant histidine-tagged WSP protein. The purified protein was concentrated and desalted using ultrafiltration to obtain concentrated WSP protein. SDS-PAGE was used for electrophoresis, and the total protein concentration was determined using the BCA method.

[0016] (6) The protective effect of WSP2 on human epidermal cells was evaluated by simulating an extremely dry microenvironment. The protective effect of WSP protein against desiccation damage in HaCaT cells was assessed using CCK-8 cell viability assays. The physical protective ability of WSP2 protein against individual biomolecules was verified by an in vitro LDH enzyme stability protection experiment. The level of reactive oxygen species in cells after drying was monitored using the fluorescent probe DCFH-DA, and the effect of WSP protein was evaluated by assessing the level of cellular oxidative stress (ROS).

[0017] A third aspect of the invention provides the use of the above-described water stress protein WSP in the preparation of topical skin compositions for protecting skin cells or intracellular enzymes against dryness damage.

[0018] Preferably, protecting skin cells from dryness damage includes improving the survival rate of skin cells after dehydration. More preferably, the skin cells are keratinocytes.

[0019] Preferably, the protection of intracellular enzymes against desiccation damage includes maintaining the activity of lactate dehydrogenase.

[0020] Preferably, the protection of skin cells against dryness damage also includes reducing intracellular reactive oxygen species levels.

[0021] A fourth aspect of the present invention provides a skin repair composition comprising an effective amount of the aforementioned water-stress protein WSP and cosmetically acceptable excipients. Preferably, the water-stress protein WSP is added to the composition at an amount of 0.1%-0.3% (w / w). The composition may be in the form of a cream, serum, spray, or medical cooling patch.

[0022] The extreme dryness stress protein WSP derived from *Ge Xian Mi* (a type of rice) and its application scheme provided by this invention have the following significant advantages compared with existing technologies: (1) Excellent molecular stability protection: Under extreme dehydration conditions, WSP exhibits significantly better activity retention of key metabolic enzymes (such as LDH) than hyaluronic acid. It can exert a protective efficacy of over 65% at a low concentration of 250 μg / mL, providing a novel solution for the preservation of bioactive ingredients and the repair of damaged skin barrier under extremely dry conditions.

[0023] (2) Multidimensional cell protection effect: WSP can not only increase the viability of damaged HaCaT cells by nearly 3 times, but also show excellent antioxidant stress resistance, reducing the ROS level in a unit cell by about 64%. This characteristic of integrating "physical protection, chemical repair and biological balance" makes it an ideal raw material for developing all-around skin care products.

[0024] (3) Natural, safe, and highly biocompatible: It is derived from Ge Xian Mi, which has a long history of being used for food and medicine. As a mature stress molecule, WSP protein has good compatibility with human skin matrix proteins in its amino acid composition. It can not only perform its function, but also is not likely to induce an immunogenic response.

[0025] (4) Highly efficient and stable heterologous expression system: Through codon optimization and vector modification, this invention achieves high-level soluble expression of WSP protein in the E. coli system. The gene length is 909 bp, and the expression product has high purity, clearing the way for subsequent industrial production and efficacy promotion.

[0026] (5) This invention clarifies the difference in the mechanism of action between WSP protein and conventional moisturizer HA through comparative experiments, and reveals its hormesis effect at specific concentrations, providing detailed reference data for precision skin care and dermatopathology research. Attached Figure Description

[0027] Figure 1 This is an agarose gel electrophoresis image of the DNA of the target gene Ns-WSP002 in this invention; Figure 2 The purity of the purified NsWSP002 protein sample for this invention was determined by SDS-PAGE gel electrophoresis. Figure 3 The bar chart shows the protective effect of the NWSP protein of this invention against desiccation damage in HaCaT cells. Figure 4 This is a bar chart showing the protective effect of the NWSP protein against LDH in this invention. Figure 5 This is a bar chart showing the ROS (reactive oxygen species) scavenging results of the NWSP protein in this invention. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with existing known technologies. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0029] Example 1: Acquisition of the 5-NS-WSP002 gene, codon optimization, and construction of the recombinant vector This embodiment details the origin and molecular cloning process of the WSP2 gene, the core active ingredient of this invention. Nostoc sphaeroides str. Hefeng, a resource-rich algae, contains several unexplored desiccation stress genes in its genome. Through deep sequencing and BLAST alignment of the whole genome of Nostoc sphaeroides, we identified an ORF sequence with high homology to WspA from Nostoc coli but with unique variant sites. This gene is 909 bp in length. After codon optimization using E. coli, a full-length DNA fragment was synthesized, and the resulting DNA sequence is shown in SEQ ID NO: 1, with the corresponding amino acid sequence shown in SEQ ID NO: 2.

[0030] (2) Synthesize the above sequence and ligate it into the pET-24a(+) vector. The Ns-WSP002 DNA sequence was synthesized using gene synthesis techniques, and Nde I and Xho I restriction endonuclease sites were added to the 5' and 3' ends, respectively. The WSP gene fragment containing Nde I and Xho I restriction sites was obtained by PCR amplification. The PCR product and pET-24a(+) vector were double-digested with Nde I and Xho I restriction endonucleases to ensure directional ligation. The reaction conditions were set at 37℃ for 1-2 hours. The double digestion system is shown in the table below. Table 1. Formulation of the double enzyme digestion system (3) Electrophoresis and gel recovery of double-digested products: The double-digested products were subjected to 2.5% agarose gel electrophoresis. Weigh 1g of agarose into an Erlenmeyer flask, add 40mL of 1×TAE, heat in a microwave oven, shake after boiling, repeat this operation until the solution becomes clear and transparent without particles, add 2 drops of nucleic acid dye and pour into the tank until solidification. Spot the double-digested pUC57-WSP plasmid into the well and spot a marker on one side. Connect the apparatus correctly and electrophoresis for 20min at 120V / 30mA. Place the gel under a gel cutter and use a blade to recover the single gel vector fragment, removing as much of the excess as possible, and place it in a pre-weighed 1.5mL EP tube. Weigh the gel. Use a gel recovery kit to recover the single WSP gene and the linearized pET-24a(+) fragment after digestion. The vector digestion product and WSP fragment were ligated with linearized pET-24a(+) using T4 ligase to construct the pET-24a(+)-WSP recombinant plasmid. After preparing the system, it was incubated at 22℃ for 30 min. The ligation system is shown below. After the ligation reaction was completed, the plasmid was inactivated at 65℃ for 10 min to obtain the recombinant expression vector pET-24a(+)-WSP.

[0031] Table 2. Electrophoresis System Formulation Verification by 2.5% agarose gel electrophoresis showed clear bands at approximately 3500 bp (linearized vector) and approximately 900 bp (target fragment), proving successful cloning.

[0032] Example 2: High Abundance Expression and Quality Control of WSP2 Recombinant Protein Based on Example 1, the pET-24a(+)-WSP recombinant plasmid was introduced into E. coli BL21(DE3). After single-clone screening and large-scale culture, the WSP2 protein was found to exhibit excellent expression stability under 37℃ and 0.25 mM IPTG induction. The induced bacterial cells were collected, sonicated, and purified using Ni-NTA affinity chromatography. The purity of the obtained protein was greater than 95% as determined by SDS-PAGE. The purified WSP2 protein showed a band of approximately 33-35 kDa on the gel. The final concentrated protein solution had a concentration as high as 3.5 mg / mL, with endotoxin content controlled below 100 EU / mg, fully meeting the requirements for cell experiments.

[0033] Figure 1 DNA agarose gel electrophoresis results confirmed that the target gene Ns-WSP002 had been successfully inserted into the pET-24a(+) vector through Nde I and Xho I restriction sites, and the correct recombinant expression plasmid pET-24a-NsWSP was constructed.

[0034] Lane 1 contains the recombinant plasmid pET-24a-NsWSPDNA, digested with Nde I and Xho I restriction endonucleases. This lane shows two clear DNA bands: one around 3500 bp, corresponding to the linearized fragment of the pET-24a(+) vector (3385 bp in size); and the other around 900 bp, corresponding to the inserted target gene fragment Ns-WSP002 (909 bp in length). The two fragments obtained after digestion are highly consistent with the expected size, and the clear bands indicate complete digestion.

[0035] Lane 2 contains the undigested recombinant plasmid pET-24a-NsWSP DNA. A bright and diffuse band with a molecular weight greater than 3000 bp is visible above the gel, consistent with the characteristics of supercoiled and linear isomers of undigested plasmids in agarose gel.

[0036] like Figure 2As shown, on a Tris-Glycine 20% gel, the NsWSP002 purified sample exhibited a significant and clear main band between approximately 33 kDa and 35 kDa, with a molecular weight consistent with the theoretical molecular weight of the WSP protein of this invention.

[0037] Marker: Protein molecular weight standard (kDa); Lanes 1 & 3: Escherichia coli (E. coli) whole protein samples after introduction of recombinant plasmids; Lane 2 & 4: Purified recombinant protein WSP2 samples (expected molecular weight approximately 33-35 kDa). Lane 5: Blank control whole protein sample of E. coli without plasmid introduction.

[0038] Example 3: Dose-dependent protection of HaCaT cells from desiccation damage by WSP2 protein (1) Seed 200 μL of HaCaT cells (concentration 8-10×10⁵) in each well of a 96-well plate and incubate with DMEM complete medium for 24 hours to allow the cells to adhere. (2) After aspirating and washing the culture medium, add different concentrations of WSP or hyaluronic acid (HA) as pretreatment solution and continue incubation for 24 hours.

[0039] (3) Drying treatment: After cleaning the pretreatment solution, place the orifice plate in the drying chamber and dry for 1.5 hours.

[0040] (4) Proliferation assay (CCK-8): Cells were rehydrated using DMEM medium. CCK-8 reagent was added and incubated for 30 minutes. The absorbance at 450 nm was measured to assess the recovery of cell proliferation.

[0041] Figure 3 The study demonstrated the protective effect of NWSP protein against drying damage in HaCaT cells. In the control group, the proliferation recovery rate of untreated cells was only 14% after 1.5 hours of drying. The protective effect of NWSP showed a clear concentration-dependent and biphasic dose-response relationship. As the NWSP concentration increased from 500 μg / mL to 2000 μg / mL, the cell viability steadily increased, reaching a maximum of 40%. However, compared with hyaluronic acid (HA), at the same or lower concentrations, HA's cell protective effect (57%) was significantly better than NWSP2. In addition, low concentrations of WSP (500 μg / mL) actually led to a slight decrease in survival rate, exhibiting a "hormesis effect."

[0042] Example 4: LDH enzyme activity protection assay This experiment investigated the protective effect of WSP on protein molecular structure using in vitro testing. First, the enzyme solution was prepared by adding 5 μL of pure LDH enzyme solution (0.1 mg / mL) to the bottom of a 96-well plate and mixing in 5 μL of different concentrations of treatment solution (WSP or HA). A simulated drying process was then performed by placing the plate in an airtight container filled with absorbent silica gel and incubating at 37°C for 24 hours. After 24 hours, enzyme activity was measured. The dried enzyme was rehydrated with PBS, and a colorimetric enzyme activity assay solution was added. The absorbance at 490 nm was measured to calculate the activity recovery rate. The experiment showed that WSP protein has a protective effect on enzyme activity.

[0043] Figure 4 The NWSP protein demonstrated a protective effect against LDH activity. Contrary to cell experiments, NWSP showed superior performance in protecting individual protein molecules, far outperforming HA. NWSP achieved a peak recovery rate of 66% at 250 μg / mL. HA's peak recovery rate was only 39%, possibly because its large molecular weight and strong hygroscopic properties cause it to compete with the enzyme for water, leading to enzyme structural collapse.

[0044] Example 5: ROS (Reactive Oxygen Species) Scavenging Experiment (1) Probe loading: DCFH-DA solution was added to the pretreated and dried cells.

[0045] (2) Detection: After incubation for 30 minutes, the fluorescence value was measured (excitation / emission: 488 / 525 nm).

[0046] (3) Normalization: The raw ROS data and CCK-8 values ​​were normalized to ensure that the data reflected the ROS level of healthy living cells, thus proving that WSP protein has a scavenging effect on ROS generated under dry conditions and exhibits anti-inflammatory activity.

[0047] Figure 5 The NWSP protein ROS (reactive oxygen species) scavenging experiment showed that NWSP could significantly reduce the increase in ROS caused by drying, thereby inhibiting the oxidative stress response of cells, maintaining cell stability, and as the concentration of NWSP increased, the ROS level gradually decreased and tended to the baseline value.

[0048] The present invention has been described in detail above through preferred embodiments and specific examples. However, those skilled in the art should understand that the scope of the present invention is not limited thereto, and any modifications or alterations that do not depart from the present invention are within the scope of the present invention.

Claims

1. A water stress protein WSP derived from *Gnaphalium affine*, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

2.

2. The water stress protein WSP according to claim 1, characterized in that, The DNA sequence encoding the water stress protein WSP is shown in SEQ ID NO:

1.

3. The application of the water stress protein WSP as described in claim 1 or 2 in skin protection, characterized in that, The water stress protein WSP is added to a topical skin composition for protecting skin cells or intracellular enzymes against dryness damage.

4. The application according to claim 3, characterized in that, Protecting skin cells from dryness damage includes increasing the survival rate of skin cells after dehydration.

5. The application according to claim 4, characterized in that, The skin cells are keratinocytes.

6. The application according to claim 3, characterized in that, The protection of intracellular enzymes against desiccation damage includes maintaining the activity of lactate dehydrogenase.

7. The application according to claim 3, characterized in that, The protection of skin cells against dryness damage also includes reducing intracellular reactive oxygen species levels.

8. A skin repair composition, characterized in that, Contains an effective amount of the water stress protein WSP as described in claim 1 or 2 and cosmetic-grade excipients.

9. The skin repair composition according to claim 8, characterized in that, The water stress protein WSP is added to the composition at an amount of 0.1wt%-0.3wt%.

10. The skin repair composition according to claim 8 or 9, characterized in that, The composition is available in the form of a face cream, serum, spray, or medical cold compress.

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