Method for preparing high-moisture-activity lea protein fragments and its application in promoting hyaluronic acid synthesis

Abstract

This invention discloses a method for preparing a highly moisturizing active LEA protein fragment and its application in promoting hyaluronic acid synthesis, belonging to the field of bioactive peptide technology. The method for preparing the highly moisturizing active lotus seed LEA protein NnLEA-3 includes the following steps: obtaining the NnLEA-3 nucleotide sequence and introducing Nde I and Xho I restriction enzyme sites; ligating it into a pET-24a(+) vector after PCR amplification and double enzyme digestion; transforming it into E. coli BL21(DE3); and obtaining the NnLEA-3 protein through IPTG induction, ultrasonic disruption, and purification. The NnLEA-3 protein prepared by this invention exhibits extremely high biocompatibility, showing no cytotoxicity at concentrations up to 1000 µg / mL, with stable cell viability between 95-105%. Simultaneously, this protein can improve the survival rate of human immortalized keratinocytes under extremely dry conditions and upregulate the expression levels of FLG, AQP3, and HAS1 genes, actively promoting the synthesis of endogenous hyaluronic acid. The NnLEA-3 protein of this invention can be safely added at high concentrations, making it suitable for developing highly safe moisturizing cosmetics that deeply promote hyaluronic acid production.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to a method for preparing highly moisturizing active LEA protein fragments and their application in promoting hyaluronic acid synthesis. Background Technology

[0002] As the skin's first line of defense against external stimuli, adequate hydration of the stratum corneum is fundamental to maintaining its barrier function. In prolonged drought or low-temperature environments, the stratum corneum loses moisture rapidly, leading to a decline in barrier function. Late embryonic abundant proteins (LEAs), with their inherent disordered structure, exhibit a certain water-binding capacity and are being explored for use in skincare products to slow down moisture evaporation.

[0003] However, the mechanisms of action of currently available moisturizing ingredients and nascent LEA-type components are mostly limited to epidermal occlusion or basic water replacement, failing to penetrate to the cellular metabolic level to activate the skin's own water-producing system. Specifically, hyaluronic acid synthase 1 (HAS1) is a key rate-limiting enzyme driving the de novo synthesis of endogenous hyaluronic acid, but existing active ingredients generally have weak regulatory effects on this target, making it difficult to achieve the leap from passive water retention to active water production. Furthermore, recombinant biological proteins often face concentration-dependent cytotoxicity issues in cosmetic development. Increasing the dosage to achieve better efficacy often results in decreased cell viability, limiting their practical application in high-drug-load, fast-acting, deep moisturizing formulations. Therefore, we propose a method for preparing highly moisturizing active LEA protein fragments and their application in promoting hyaluronic acid synthesis to alleviate or solve the above problems.

[0004] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for preparing highly moisturizing active LEA protein fragments and their application in promoting hyaluronic acid synthesis. This addresses the technical issues that existing moisturizing ingredients struggle to actively upregulate HAS1 to promote de novo synthesis of endogenous hyaluronic acid, and that high-concentration additions can easily induce cytotoxicity.

[0006] To achieve the above objectives, this invention provides a method for preparing highly moisturizing active lotus seed LEA protein NnLEA-3, comprising the following steps:

[0007] (1) Obtain the nucleotide sequence encoding NnLEA-3, and introduce Nde I and XhoI restriction endonuclease sites at the 5' and 3' ends of the sequence, respectively;

[0008] (2) The synthesized sequence was amplified by PCR. The purified PCR product and the empty vector pET-24a(+) were simultaneously digested with Nde I and Xho I. The recombinant expression vector was constructed by directional ligation at 16℃ under the action of T4 DNA ligase. The double digestion reaction system was reacted at 37℃ for 2h.

[0009] (3) The recombinant expression vector was transformed into E.coli BL21(DE3) competent cells and seeded into LB liquid medium containing 50 µg / mL kanamycin. The cells were then cultured at 37°C and 200 rpm.

[0010] (4) Add 1 mM IPTG solution and induce expression for 6 h at 37℃ and 200 rpm. Collect the precipitate by ultrasonic disruption of the bacterial cells and separate and purify the NnLEA-3 protein.

[0011] Compared with the prior art, the beneficial effects of the present invention are:

[0012] The NnLEA-3 protein prepared by this invention has high biocompatibility. Experimental data show that the cell viability of each group is stable between 95% and 105% in the concentration range of 25-1000 µg / mL. No obvious cytotoxicity was observed at the highest concentration of 1000 µg / mL. This characteristic allows the protein to be used in cosmetic formulations without being limited by the upper limit of safe addition at low concentrations.

[0013] In this invention, the NnLEA-3 protein can actively regulate the hydration metabolism of skin cells. RT-qPCR results showed that NnLEA-3 significantly upregulated the transcriptional levels of aquaporin 3 (AQP3) and hyaluronic acid synthase 1 (HAS1). In particular, the activation of HAS1 indicates that it can promote the de novo synthesis of endogenous hyaluronic acid in keratinocytes, making it suitable for developing moisturizing cosmetics with high concentrations of added, deeply hyaluronic acid-promoting ingredients.

[0014] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0015] Figure 1 The image shows the results of double enzyme digestion identification and analysis of the NnLEA-3 recombinant plasmid in Example 2 of this invention.

[0016] Figure 2 This is a graph showing the effect of NnLEA-3 protein on cell viability in Example 3 of the present invention.

[0017] Figure 3 This is a graph showing the effect of NnLEA-3 protein on the morphology of dried cells in Example 4 of the present invention.

[0018] Figure 4 This is a graph showing the moisturizing effect of NnLEA-3 protein on dried cells in Example 4 of the present invention.

[0019] Figure 5 This is a diagram illustrating the effect of NnLEA-3 protein on the expression of skin barrier-related functional genes in Example 5 of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. It should be noted that the drawings are schematic and not illustrated to scale. For clarity and convenience, the relative sizes and proportions of the parts shown in the drawings have been exaggerated or reduced in size. Any size is only exemplary and not limiting.

[0021] Example 1: Obtaining the NnLEA-3 sequence of the LEA protein

[0022] The genome sequence file of *Nelumbonucifera*, an ancient Chinese lotus, was obtained from the Nelumbo Genome Database. Combined with the sequenced and assembled genome data (Genome assembly ASM303368v1) of *Nelumbo nucifera* (Cultivar: ChinaAntique) retrieved from NCBI, the selected gene sequences were imported into the NBCI database for BLAST gene alignment analysis. To screen for candidate genes with high potential for skin protection, the following screening principles were established:

[0023] (1) Amino acid composition and functional orientation: The number of hydrophilic amino acids in the amino acid sequence, and their hydration ability.

[0024] (2) Physicochemical property evaluation: The candidate protein sequences were preliminarily analyzed using online tools such as ProtParam, and sequences with high content of hydrophilic amino acids and significantly negative average hydrophilicity index (GRAVY) were given priority screening;

[0025] (3) Structural feature screening: Focus on the proportion of inherently disordered sequences in the sequence.

[0026] Based on the above principles, BLAST was used for gene homology comparison analysis, which ultimately determined the complete CDS sequence of the target gene, clarified its location on the chromosome and species origin, and obtained the NnLEA-3 protein coding sequence. (See Table 1.)

[0027] The amino acid sequence of this protein is as follows:

[0028] MARSLSNAKLFFALVDGVSVAISRRGYSAAAAASSSVVR

[0029] GGARSGIGNQGMEKVMMKEESAASSWIPDPITGYYRPEN

[0030] HGEEIDVAELRRVLLNQKIRPQN

[0031] Example 2: Induced expression of LEA protein NnLEA-3

[0032] Based on the Asian lotus (N. nucifera) genome database and BLAST results, the full-length sequence of NnLEA-3 was obtained. The target fragment was synthesized artificially, and Nde I and Xho I restriction endonuclease sites were introduced at the 5' and 3' ends of the sequence, respectively. The synthesized sequence was amplified by high-fidelity DNA polymerase. The amplified product was identified by agarose gel electrophoresis and purified to obtain the gene fragment with specific restriction sites at the ends. The purified PCR product and the empty vector pET-24a(+) were simultaneously double-digested with Nde I and Xho I at 37°C for 2 hours. The digested target fragment was then mixed with the linearized vector at a specific molar ratio and ligated directionally at 16°C using T4 DNA ligase.

[0033] Through the above steps, the target gene was precisely integrated into the multiple cloning site of pET-24a(+), placing it downstream of the T7 promoter. 5-10 µL of the ligation product pET-24a(+)-NnLEA-3 was placed in 50-100 µL of E. coli BL21(DE3) competent cells and transformed to obtain E. coli BL21(DE3) glycerol bacteria containing the correct recombinant plasmid. This was inoculated into 25 mL of LB broth containing kanamycin (50 µg / mL) and cultured overnight at 37°C and 200 rpm for scale-up. 1 mM IPTG solution was added, and expression was induced for 6 h at 37°C and 200 rpm. The bacterial cells were then sonicated to collect the precipitate, and the protein was separated and purified to obtain a large amount of NnLEA-3 protein.

[0034] Example 3: The extremely high biocompatibility and high concentration safety of NnLEA-3 protein

[0035] To assess the biosafety of NnLEA-3 protein at high concentrations, this study used the CCK-8 assay to detect its effect on HaCaT cell proliferation. Logarithmic growth phase human immortalized keratinocytes were cultured at 1×10⁻⁶ concentrations. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated at 37°C and 5% CO2 for 24 hours to ensure complete cell adhesion. The original culture medium was discarded, and 100 µL of fresh complete culture medium containing different concentrations of NnLEA-3 protein was added (concentration gradients were set at 25, 50, 100, 200, 300, 400, 500, 750, and 1000 µg / mL). Cell-free blank wells and control wells containing only standard culture medium were also included. After incubation for another 24 hours, the drug-containing medium was discarded, and 100 µL of fresh culture medium and 10 µL of LCK-8 reagent were added to each well. The plates were then incubated in the dark for 3 hours. The absorbance (OD) value at 450 nm was then read using a microplate reader.

[0036] The experimental results are attached. Figure 2 As shown, within a broad concentration range of 25-1000 µg / mL, cell viability in all treatment groups closely adhered to the baseline, remaining stably within the range of 95-105%, with no concentration-dependent toxicity observed. This confirms that NnLEA-3 possesses an extremely wide safety window, providing a safety guarantee for the development of high-drug-load, fast-acting moisturizing products.

[0037] Example 4: Moisturizing activity of NnLEA-3 protein on dried cells

[0038] To simulate the extreme dehydration of the epidermal stratum corneum under harsh climate conditions, this experiment constructed an in vitro cellular drought stress model. Log-phase HaCaT cells were used at a rate of 1×10⁻⁶ cells / year. 4 Cells were seeded per well in 96-well plates and allowed to adhere overnight. The supernatant was then discarded, and the cell surface was gently washed with warm, sterile PBS. The cells were then pretreated for 24 hours with culture medium containing different concentrations of NnLEA-3 protein (25-200 µg / mL) to allow the protein to fully act on the cell membrane and exert its biological effects.

[0039] Subsequently, the remaining liquid in the wells was aspirated and transferred to a sealed, temperature-controlled incubator where the relative humidity was pre-adjusted to 89-90% using a saturated barium chloride (BaCl2) aqueous solution. The cells were exposed to this low-humidity environment for 18 hours to induce severe dehydration damage. After the stress period, 100 µL of fresh complete culture medium was rapidly added to each well to terminate the damage and restore nutrient supply. Then, 10 µL of CCK-8 solution was added and the cells were incubated for 3 hours. The absorbance at 450 nm was measured to assess cell viability.

[0040] As attached Figure 3 Appendix Figure 4 As shown, the cell survival rate in the drought model control group dropped sharply, while the cell survival rate in the experimental group pretreated with NnLEA-3 significantly recovered and was protected. Microscopic observation also showed that NnLEA-3 effectively alleviated cell shrinkage and morphological damage caused by dehydration. This indicates that NnLEA-3, with its excellent hydrophilic and water-binding properties, constructs a protective hydration microenvironment during cell dehydration stress.

[0041] Example 5: Effects of NnLEA-3 protein on the expression levels of genes promoting hyaluronic acid production and moisturizing.

[0042] This embodiment aims to explore the underlying mechanism by which NnLEA-3 promotes endogenous hyaluronic acid synthesis and enhances hydration metabolism at the molecular level. HaCaT cells were seeded in 6-well plates, and after reaching a suitable confluence level, they were incubated for 24 hours with culture medium containing NnLEA-3 protein.

[0043] Cells were collected, and total RNA was isolated and extracted using TransZol Up reagent. The procedure was as follows: after washing with PBS, lysis buffer was added and the RNA was collected by pipetting. The RNA precipitate was purified by chloroform phase separation, isopropanol precipitation, and washing with 75% ethanol. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (reaction program: incubation at 50℃ for 5 min, followed by heating at 85℃ for 2 min to inactivate). Subsequently, qPCR amplification was performed using cDNA as a template with specific primers (see Table 2) and PerfectStart Green qPCR SuperMix, with GADPH as an internal control, and the relative expression levels of each gene were calculated.

[0044] The test results are attached. Figure 5 As shown, NnLEA-3 not only upregulated the transcriptional level of filaggrin (FLG), but also significantly activated the transcriptional levels of aquaporin 3 (AQP3) and hyaluronic acid synthase 1 (HAS1) genes. In particular, the significant increase in the expression level of HAS1, a key rate-limiting enzyme in the de novo synthesis of endogenous hyaluronic acid, demonstrates that NnLEA-3 breaks through the limitations of conventional moisturizers that rely solely on epidermal occlusion to lock in moisture. It can act as a bioactive signaling factor to deeply awaken the cell's own water-making engine, achieving source hydration from the inside out. Furthermore, no abnormal fluctuations in apoptosis-related genes such as CASP14 were observed in the test group, further confirming its extremely high safety even at high concentrations.

[0045] Table 1. Information on NnLEA-3 Protein

[0046]

[0047] Table 2 Primer sequences for skin barrier-related genes

[0048]

[0049] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0050] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a highly moisturizing active lotus seed LEA protein NnLEA-3, characterized in that, Includes the following steps: (1) Obtain the nucleotide sequence encoding NnLEA-3, and introduce Nde I and Xho I restriction endonuclease sites at the 5' and 3' ends of the sequence, respectively; (2) The synthesized sequence was amplified by PCR. The purified PCR product and the empty vector pET-24a(+) were simultaneously digested with Nde I and Xho I. The recombinant expression vector was constructed by directional ligation at 16℃ under the action of T4 DNA ligase. (3) The recombinant expression vector was transformed into E.coli BL21(DE3) competent cells and seeded into LB liquid medium containing 50 µg / mL kanamycin. The cells were then cultured at 37°C and 200 rpm. (4) Add 1mM IPTG solution and induce expression for 6h at 37℃ and 200rpm. Collect the precipitate by ultrasonic disruption of the bacterial cells and separate and purify the NnLEA-3 protein.

2. The preparation method according to claim 1, characterized in that, In step (2), the double enzyme digestion reaction system is reacted at 37°C for 2 hours.

3. A highly moisturizing active lotus seed LEA protein NnLEA-3 prepared by the preparation method described in claim 1 or 2, the amino acid sequence of which is shown in SEQ ID NO.

1.

4. A nucleic acid molecule encoding the lotus seed LEA protein NnLEA-3 as described in claim 3, the nucleotide sequence of which is shown in SEQ ID NO.

2.

5. A highly moisturizing cosmetic composition, characterized in that, The composition comprises a safe and effective amount of lotus seed LEA protein NnLEA-3 as described in claim 3, and cosmetically acceptable excipients, wherein the concentration of lotus seed LEA protein NnLEA-3 in the cosmetic composition is 25-1000 µg / mL.

6. The use of the cosmetic composition according to claim 5 in the preparation of deep-regenerating hyaluronic acid or highly safe moisturizing skin care products.

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