Extraction method of micromolecular protein compound and application of micromolecular protein compound in skin care field

Abstract

The invention relates to an extraction method of a micromolecular protein compound and application of the micromolecular protein compound in the field of skin care. According to the method, a goat embryo is taken as a raw material, and a protein compound taking Ubiquitin as a main material and EFS, ALB, BIN1, H2AJ, FBLN1 and KPNA3 as auxiliary materials is obtained through tissue crushing, chymotrypsin hydrolysis and hydrophobic chromatography enrichment. The compound has hydrophobic and hydrophilic amphoteric characteristics and is used in skin care products to realize skin barrier penetration and activity exertion.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and cosmetics, specifically to a method for extracting small molecule protein complexes and their application in skincare. Background Technology

[0002] Current protein extraction methods often employ conventional chromatography and centrifugation, which are insufficient for achieving efficient extraction of specific small-molecule protein complexes. Raw material sources are limited, resulting in the extraction of only a single type of protein or proteins with low activity, failing to meet the needs of the skincare industry. Protein components in skincare products, due to their molecular structure, struggle to penetrate the skin barrier, leading to insufficient activity. Furthermore, there is a lack of application solutions for clearly defined protein complexes, primarily ubiquitin, and their sequences.

[0003] Due to limitations in molecular structure and properties, protein components in existing skincare products struggle to penetrate the skin barrier and reach the dermis, resulting in limited activity and unsatisfactory skincare effects. Furthermore, the lack of clearly defined protein complex application protocols based on composition and sequence makes it difficult to guarantee efficacy stability. Common raw material sources are limited, and extraction methods are rudimentary, resulting in either complex mixtures or single components with low activity, making them unsuitable for skincare applications. Traditional extraction methods, often employing conventional chromatography and centrifugation, fail to specifically target small-molecule protein complexes for efficient extraction, hindering precise enrichment and activity preservation, leading to low extraction efficiency. Protein raw materials are currently used in skincare products primarily for basic functions such as moisturizing, lacking small-molecule proteins capable of transmembrane skincare effects; particularly lacking raw materials with specific complexes that synergistically enhance transmembrane activity, primarily based on ubiquitin and supplemented by six enzymes such as EFS.

[0004] Ubiquitin is a small protein widely found in eukaryotes, composed of 76 amino acids with a molecular weight of approximately 8.451 kDa, and is highly conserved across different species. Its core function is to maintain intracellular protein homeostasis by labeling proteins to be degraded, enabling them to be recognized and hydrolyzed by the 26S proteasome. It also participates in various physiological processes such as transmembrane protein labeling, signal transduction, and DNA damage repair. The 2004 Nobel Prize in Chemistry was awarded to scientists who elucidated the mechanism of ubiquitin-regulated protein degradation, a mechanism that provides a crucial foundation for understanding the precise regulation of proteins within cells.

[0005] From an anti-aging perspective, aging is often accompanied by protein homeostasis imbalance and the accumulation of misfolded proteins. Studies have shown that ubiquitination levels generally decrease during aging, leading to the inability of abnormal proteins to be effectively degraded and their accumulation within cells. Maintaining normal ubiquitination levels can reduce such accumulation and slow down the aging process, providing an important approach for the development of anti-aging related ingredients.

[0006] In the field of bioactive proteins, various proteins play crucial roles in cell physiology and metabolism: EFS, as a adaptor protein, participates in cell signal transduction and regulates cell proliferation and differentiation; ALB (albumin) maintains osmotic pressure and transports nutrients, providing support for cell metabolism; BIN1 participates in cell membrane vesicle formation and cytoskeleton regulation; KPNA3, as a nuclear transporter, mediates internuclear-cytoplasmic transport; FBLN1 maintains tissue stability by binding to extracellular matrix components; and H2AJ, as a histone variant, participates in chromatin regulation and DNA damage repair. These proteins, in synergy with ubiquitin, can regulate cell function from multiple dimensions.

[0007] Currently, the demand for natural active protein complexes in the skincare industry is increasing. However, existing technologies lack extraction and application methods for small molecule protein complexes with ubiquitin as the main component and the aforementioned six proteins as auxiliary components. Furthermore, conventional extraction methods struggle to achieve efficient enrichment and activity retention of such complexes. Therefore, developing an extraction method for these complexes and applying it to the skincare field is of significant practical importance. Summary of the Invention

[0008] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0009] In view of the problems existing in the prior art, the inventors have proposed the present invention.

[0010] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for extracting small molecule protein complexes and their application in the field of skincare.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for extracting small molecule protein complexes, comprising the following steps: using 45-90 day old goat embryos as raw materials, tissue disruption is performed to obtain a supernatant; the supernatant is hydrolyzed with chymotrypsin; and a protein complex is obtained by hydrophobic chromatography enrichment, with Ubiquitin as the main component and EFS, ALB, BIN1, H2AJ, FBLN1, and KPNA3 as auxiliary components.

[0012] As a preferred embodiment of the extraction method for small molecule protein complexes described in this invention, the tissue disruption includes: rinsing the embryo with pre-cooled physiological saline, adding 2-3 times the volume of cell lysis buffer, the buffer consisting of 50mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA, and 1% Triton X-100, homogenizing under ice bath conditions, centrifuging at 5000g for 20 minutes at 4°C, and collecting the supernatant.

[0013] As a preferred embodiment of the extraction method for a small molecule protein complex according to the present invention, the chymotrypsin hydrolysis includes: determining the protein content of the supernatant using a BCA protein quantification kit, adding chymotrypsin at a protein to chymotrypsin ratio of 250:1, adjusting the temperature to 55°C, pH to 8.0, stirring speed to 150 rpm, reacting for 30 minutes, and terminating the reaction in an ice bath.

[0014] As a preferred embodiment of the extraction method for small molecule protein complexes described in this invention, the hydrophobic chromatography enrichment includes: using phenyl-Sepharose 6FF medium, equilibrating the column with 20mM Tris-HCl pH 7.5 and 0.5M NaCl equilibration buffer, loading enzyme digestion solution, rinsing with equilibration buffer until the absorbance at 280nm is close to the baseline, collecting the target peak by linear gradient elution, concentrating by 3kDa ultrafiltration, and lyophilizing for storage.

[0015] As a preferred embodiment of the extraction method for a small molecule protein complex according to the present invention, the relative contents of each protein in the protein complex are: Ubiquitin 30%, EFS 24%, ALB 16%, BIN 110%, KPNA 310%, FBLN 16%, and H2AJ 4%.

[0016] As a preferred embodiment of the extraction method for a small molecule protein complex according to the present invention, the protein complex contains Ubiquitin, which includes an enzymatically cleaved ubiquitin with the sequence AGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSD TIENVKAKIQDKEGIPPDQQRLIF, and its content is more than 5 times that of natural ubiquitin.

[0017] It also includes the use of a small molecule protein complex in the preparation of skin care products.

[0018] As a preferred embodiment of the application of the small molecule protein complex described in this invention in the preparation of skin care products, the skin care product is an emulsion containing 0.1% small molecule protein complex, 0.5% sodium hyaluronate, 5% glycerin, 1% vitamin C, 0.1% retinol, 3% emulsifier, appropriate amount of preservative, and the balance being deionized water and oil phase raw materials.

[0019] As a preferred embodiment of the application of the small molecule protein complex described in this invention in the preparation of skin care products, the preparation of the skin care products includes: dissolving the small molecule protein complex in deionized water, adding water-soluble components and stirring to dissolve; dissolving oil-soluble components in the oil phase, adding water phase and stirring at high speed to emulsify, adjusting the pH to 5.5-6.5, and then bottling and packaging.

[0020] As a preferred embodiment of the application of the small molecule protein complex described in this invention in the preparation of skin care products, the following are observed: after a 4-week human trial, improvements in skin moisture, elasticity, and wrinkle depth are detected; and cell experiments are conducted to detect increased collagen synthesis and antioxidant enzyme activity.

[0021] The beneficial effects of this invention are as follows: using goat embryos as raw materials provides a source of highly expressed proteins; improving the purity and activity of the complex through specific hydrolysis and precise condition control; achieving efficient enrichment through hydrophobic chromatography and enhancing skin care efficacy; the amphipathic characteristics of the complex promote skin penetration and achieve synergistic regulation of skin function; disclosing the composition and sequence of the complex supports further applications; and providing a sufficient raw material basis for extracting highly active complexes, which is superior to traditional low-expression raw materials, by using goat embryos with high expression of 7 target proteins (6 proteins, including EFS, are highly expressed throughout the embryo, and ALB is highly expressed in the fetal liver).

[0022] Furthermore, through specific hydrolysis with chymotrypsin and precise enzymatic cleavage conditions, the purity and activity of the target protein fragments were enhanced, with a 5-fold increase in the yield of enzymatically cleaved ubiquitin. After enrichment by hydrophobic chromatography, the content of effective ingredients significantly increased, reducing impurity interference. The complex exhibits high skin-care activity and efficiency due to enrichment. Its amphipathic properties allow it to bind to and penetrate cell membranes, reaching deep into the dermis to exert its effects, solving the problem of traditional proteins' inability to penetrate the skin. In particular, enzymatically cleaved ubiquitin exhibits higher transmembrane activity than natural ubiquitin. Seven proteins work synergistically to comprehensively regulate skin cell function. The composition, protein sequence, and relative content of the ubiquitin-based complex are disclosed for the first time, along with the differences between enzymatically cleaved ubiquitin and natural ubiquitin, providing a clear basis for related research and product development. The highly efficient enriched product is a small-molecule protein with hydrophobic properties, working synergistically to exert skin-care effects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0024] Figure 1 This is a hydrophilic and hydrophobic analysis diagram of the natural ubiquitin of this invention.

[0025] Figure 2 This is a hydrophilic and hydrophobic analysis diagram of the enzymatic cleavage of ubiquitin in this invention.

[0026] Figure 3 This is a transmembrane trend analysis diagram of the natural ubiquitin of this invention.

[0027] Figure 4 This is a transmembrane trend analysis diagram of ubiquitin cleavage by the enzyme in this invention. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single embodiment or an embodiment selectively excluded from other embodiments.

[0031] Example 1

[0032] This embodiment provides a method for extracting small molecule protein complexes and their application in the field of skincare.

[0033] Specifically, 45-day-old goat embryos were selected, collected under sterile conditions, and transferred to the laboratory in a pre-cooled sterile preservation solution.

[0034] The tissue was rinsed with pre-cooled physiological saline to remove impurities, and then 2 volumes of cell lysis buffer were added. The buffer consisted of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100. The tissue was homogenized and lysed under ice bath conditions to release intracellular proteins.

[0035] The homogenate was centrifuged at 5000g for 20 minutes at 4℃, and the supernatant was collected for later use. The supernatant contained proteins such as Ubiquitin, EFS, ALB, BIN1, H2AJ, FBLN1, and KPNA3. Among them, six proteins, including EFS, were highly expressed throughout the embryo, and ALB was highly expressed in the fetal liver.

[0036] The protein content in the supernatant was determined using the BCA protein quantification kit. The protein to chymotrypsin ratio was slowly added at 250:1 and gently stirred until homogeneous.

[0037] Place the mixture in a constant temperature water bath, adjust the temperature to 55℃ and the pH to 8.0, and stir at 150 rpm for 30 minutes. Immediately after the reaction, stop the enzymatic digestion by placing it on ice to obtain a solution containing the target protein fragment.

[0038] During this process, ubiquitin production increased fivefold, and the ubiquitin cleavage sequence is as follows:

[0039] AGKQLEDGRTLSDY-NIQKESTLHLVLRLRGG-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF;

[0040] With natural ubiquitin sequences:

[0041] Unlike MQIFVKTLTGKTITLEVEPSDTIENVKVKIQEKEGIPPDQQRLIF-AGKQ LEDGRTLSDY-NIQKESTLHLVLRLRGG, it has enhanced hydrophobicity; such as Figure 2 As shown.

[0042] Furthermore, the natural ubiquitin sequence is divided into three segments, arranged in an ABC pattern: MQIFVKTLTGKTITLEVEPSDTIENVKVKIQEKEGIPPDQQRLIF-AGKQLED GRTLSDY-NIQKESTLHLVLRLRGG;

[0043] The ubiquitin sequence is also divided into 3 segments, which are arranged in BCA: AGKQLEDGRTLSDY-NIQKESTLHLVLRLRGG-MQIFVKTLTGKTITLEVEPS DTIENVKAKIQDKEGIPPDQQRLIF.

[0044] This change in arrangement leads to a change in hydrophilicity and hydrophobicity. Enzymes that cleave ubiquitin have better hydrophobicity, which is more conducive to cell membrane penetration.

[0045] Furthermore, phenyl-Sepharose 6FF was selected as the hydrophobic medium. After swelling and equilibration, the column was packed and equilibrated with equilibration buffer (20mM Tris-HCl pH 7.5, 0.5M NaCl) until the effluent parameters were consistent.

[0046] Adjust the pH and conductivity of the enzyme digestion solution to match the loading buffer, and load the sample slowly. After adsorption, wash with equilibration buffer until the absorbance at 280 nm is close to the baseline.

[0047] Linear gradient elution was employed. Solution A consisted of 20 mM Tris-HCl pH 7.5 and 0.5 M NaCl, while solution B consisted of 20 mM Tris-HCl pH 7.5. The target elution peak was collected. The eluent was concentrated using a 3 kDa ultrafiltration membrane, and the purity was determined by HPLC and MS before lyophilization and storage. The relative contents of each protein in the complex were: Ubiquitin 30%, EFS 24%, ALB 16%, BIN 1 10%, KPNA 3 10%, FBLN 16%, and H2AJ 4%.

[0048] The protein sequence is as follows:

[0049] Ubiquitin:AGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGK TITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF;

[0050] EFS: DVPLLGPETPPSPEPLGASASSDMDTLALLLARSPPAPHRPRLPSAES LSRRPLPALPVPEAPSPSPAPSPAAGRKGSIQDRPLPPPPPRLPGYGGPKVEGD PEGREVEDHPAGHHNEY;

[0051] ALB:GERALKAWSVARLSQKFPKADFTDVTKIVTDLTKVHKECCHGDL LECADDRADLAKY;

[0052] BIN1:TVKAQPSDSAPAKGNKSPSPPPDGSPAATPEIRVNHEPEPAGAATP GAALPKSPSQLRKGPPVPPPPKHTPSKEVKQEQILSLFDDTFVPEISVTTPSQF;

[0053] KPNA3:KAQNVTLEAILQNATSDNPVVQLSAVQAARKLLSSDRNPPIDDLIKSGILPILVKCLERDDNPSLQF;

[0054] FBLN1:TRPEEIIFLRAITPAYPANHADIIFDITDGNLRDSFDIIKRY;

[0055] H2AJ:LTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESQKTKSK.

[0056] This complex exhibits both hydrophobic and hydrophilic properties, and its transmembrane activity in cleaving ubiquitin is higher than that of natural ubiquitin. Figure 4 As shown.

[0057] Furthermore, the emulsion formulation is designed to contain 0.1% small molecule protein complex, 0.5% sodium hyaluronate, 5% glycerin, 1% vitamin C, 0.1% retinol, 3% emulsifier, appropriate amount of preservative, and the remainder is deionized water and oil phase raw materials.

[0058] The complex was dissolved in deionized water, and the water-soluble components were added and stirred until dissolved. The oil-soluble components were dissolved in the oil phase, and the water phase was added and emulsified by high-speed stirring. The pH was adjusted to 5.5-6.5, and then packaged. After 4 weeks of human trials, skin hydration increased by 20% and elasticity improved by 15%. Cell experiments showed a 25% increase in collagen synthesis.

[0059] Example 2

[0060] This embodiment provides a method for extracting small molecule protein complexes and their application in the field of skincare.

[0061] Specifically, 60-day-old goat embryos were selected, collected under aseptic conditions, and transferred to the laboratory in pre-cooled sterile preservation solution. Impurities were removed by rinsing with pre-cooled physiological saline, and 2.5 times the volume of cell lysis buffer was added. This buffer consisted of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100.

[0062] Tissue was homogenized and disrupted under ice bath conditions to release intracellular proteins. The homogenate was centrifuged at 5000g for 20 minutes at 4°C, and the supernatant was collected for later use. This supernatant contained proteins such as Ubiquitin, EFS, ALB, BIN1, H2AJ, FBLN1, and KPNA3, among which ALB was highly expressed in fetal liver.

[0063] The protein content in the supernatant was determined using the BCA protein quantification kit. Chymotrypsin was slowly added at a protein to chymotrypsin ratio of 250:1, and the mixture was gently stirred until homogeneous. The mixture was placed in a constant temperature water bath, and the temperature was adjusted to 55℃ and the pH to 8.0. The stirring speed was 150 rpm, and the reaction was carried out for 30 minutes.

[0064] Immediately after the reaction, the enzyme digestion was terminated by placing the sample on ice to obtain a solution containing the target protein fragment. During this process, ubiquitin production increased fivefold. The ubiquitin digestion sequence is as follows:

[0065] AGKQLEDGRTLSDY-NIQKESTLHLVLRLRGG-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF, has enhanced hydrophobicity, such as Figure 2 As shown, the hydrophobic sites are concentrated.

[0066] The natural ubiquitin sequence is divided into three segments, arranged in an A, B, and C order:

[0067] MQIFVKTLTGKTITLEVEPSDTIENVKVKIQEKEGIPPDQQRLIF-AGKQ LEDGRTLSDY-NIQKESTLHLVLRLRGG;

[0068] The ubiquitin cleavage sequence is also divided into 3 segments, arranged in BCA sequence:

[0069] AGKQLEDGRTLSDY-NIQKESTLHLVLRLRGG-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF.

[0070] This change in arrangement leads to a change in hydrophilicity and hydrophobicity. Enzymes that cleave ubiquitin have better hydrophobicity, which is more conducive to cell membrane penetration.

[0071] Furthermore, phenyl-Sepharose 6FF was selected as the hydrophobic medium. After swelling and equilibration, the column was packed and equilibrated with equilibration buffer (20mM Tris-HCl pH 7.5, 0.5M NaCl) until the effluent parameters were consistent.

[0072] Adjust the pH and conductivity of the enzyme digestion solution to match the loading buffer, and load the sample slowly. After adsorption, wash with equilibration buffer until the absorbance at 280 nm is close to the baseline. Use a linear gradient elution: Solution A is 20 mM Tris-HCl pH 7.5 and 0.5 M NaCl, and Solution B is 20 mM Tris-HCl pH 7.5. Collect the target elution peak. Concentrate the eluent using a 3 kDa ultrafiltration membrane, and after purity detection by HPLC and MS, freeze-dry for storage.

[0073] The relative contents of each protein in the complex are: Ubiquitin 30%, EFS 24%, ALB 16%, BIN 110%, KPNA 310%, FBLN 16%, and H2AJ 4%.

[0074] The protein sequence is as follows:

[0075] Ubiquitin:AGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGK TITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF;

[0076] EFS: DVPLLGPETPPSPEPLGASASSDMDTLALLLARSPPAPHRPRLPSAES LSRRPLPALPVPEAPSPSPAPSPAAGRKGSIQDRPLPPPPPRLPGYGGPKVEGD PEGREVEDHPAGHHNEY;

[0077] ALB:GERALKAWSVARLSQKFPKADFTDVTKIVTDLTKVHKECCHGDL LECADDRADLAKY;

[0078] BIN1:TVKAQPSDSAPAKGNKSPSPPPDGSPAATPEIRVNHEPEPAGAATP GAALPKSPSQLRKGPPVPPPPKHTPSKEVKQEQILSLFDDTFVPEISVTTPSQF;

[0079] KPNA3:KAQNVTLEAILQNATSDNPVVQLSAVQAARKLLSSDRNPPIDDLIKSGILPILVKCLERDDNPSLQF;

[0080] FBLN1:TRPEEIIFLRAITPAYPANHADIIFDITDGNLRDSFDIIKRY;

[0081] H2AJ:LTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESQKTKSK.

[0082] This complex exhibits both hydrophobic and hydrophilic properties, and its tendency to cleave ubiquitin across membranes is higher than that of natural ubiquitin. Figure 4 As shown.

[0083] Furthermore, the emulsion formula is designed to contain 0.1% small molecule protein complex, 0.5% sodium hyaluronate, 5% glycerin, 1% vitamin C, 0.1% retinol, 3% emulsifier, appropriate amount of preservative, and the remainder is deionized water and oil phase raw materials.

[0084] The complex was dissolved in deionized water, and the water-soluble components were added and stirred until dissolved. The oil-soluble components were dissolved in the oil phase, and the water phase was added and emulsified by high-speed stirring. The pH was adjusted to 5.5-6.5, and then packaged. After 4 weeks of human trials, skin elasticity improved by 18% and wrinkle depth decreased by 12%. Cell experiments showed a 30% increase in antioxidant enzyme activity.

[0085] Example 3

[0086] This embodiment provides a method for extracting small molecule protein complexes and their application in the field of skincare.

[0087] Specifically, 90-day-old goat embryos were selected, collected under sterile conditions, and transferred to the laboratory in pre-cooled sterile preservation solution. Impurities were removed by rinsing with pre-cooled physiological saline, and three volumes of cell lysis buffer were added. This buffer consisted of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100.

[0088] Tissue was homogenized and disrupted under ice bath conditions to release intracellular proteins. The homogenate was centrifuged at 5000g for 20 minutes at 4°C, and the supernatant was collected for later use. This supernatant contained proteins such as Ubiquitin, EFS, ALB, BIN1, H2AJ, FBLN1, and KPNA3, among which six proteins, including EFS, were highly expressed throughout the embryo.

[0089] The protein content in the supernatant was determined using the BCA protein quantification kit. Chymotrypsin was slowly added at a protein to chymotrypsin ratio of 250:1, and the mixture was gently stirred until homogeneous. The mixture was placed in a constant temperature water bath, and the temperature was adjusted to 55℃ and the pH to 8.0. The stirring speed was 150 rpm, and the reaction was carried out for 30 minutes.

[0090] Immediately after the reaction, the enzyme digestion was terminated by placing the sample on ice to obtain a solution containing the target protein fragment. During this process, ubiquitin production increased fivefold. The ubiquitin digestion sequence is as follows:

[0091] AGKQLEDGRTLSDY-NIQKESTLHLVLRLRGG-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF, has enhanced hydrophobicity, such as Figure 2 As shown.

[0092] The natural ubiquitin sequence is divided into three segments, arranged in an A, B, and C order:

[0093] MQIFVKTLTGKTITLEVEPSDTIENVKVKIQEKEGIPPDQQRLIF-AGKQ LEDGRTLSDY-NIQKESTLHLVLRLRGG;

[0094] The ubiquitin cleavage sequence is also divided into 3 segments, arranged in BCA sequence:

[0095] AGKQLEDGRTLSDY-NIQKESTLHLVLRLRGG-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF.

[0096] This change in arrangement leads to a change in hydrophilicity and hydrophobicity. Enzymes that cleave ubiquitin have better hydrophobicity, which is more conducive to cell membrane penetration.

[0097] Furthermore, phenyl-Sepharose 6FF was selected as the hydrophobic medium. After swelling and equilibration, the column was packed and equilibrated with equilibration buffer (20mM Tris-HCl pH 7.5, 0.5M NaCl) until the effluent parameters were consistent.

[0098] Adjust the pH and conductivity of the enzyme digestion solution to match the loading buffer, and load the sample slowly. After adsorption, wash with equilibration buffer until the absorbance at 280 nm is close to the baseline. Use a linear gradient elution: Solution A is 20 mM Tris-HCl pH 7.5 and 0.5 M NaCl, and Solution B is 20 mM Tris-HCl pH 7.5. Collect the target elution peak. Concentrate the eluent using a 3 kDa ultrafiltration membrane, and after purity detection by HPLC and MS, freeze-dry for storage.

[0099] The relative contents of each protein in the complex are: Ubiquitin 30%, EFS 24%, ALB 16%, BIN 110%, KPNA 310%, FBLN 16%, and H2AJ 4%.

[0100] The protein sequence is as follows:

[0101] Ubiquitin:AGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGK TITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF;

[0102] EFS: DVPLLGPETPPSPEPLGASASSDMDTLALLLARSPPAPHRPRLPSAES LSRRPLPALPVPEAPSPSPAPSPAAGRKGSIQDRPLPPPPPRLPGYGGPKVEGD PEGREVEDHPAGHHNEY;

[0103] ALB:GERALKAWSVARLSQKFPKADFTDVTKIVTDLTKVHKECCHGDL LECADDRADLAKY;

[0104] BIN1:TVKAQPSDSAPAKGNKSPSPPPDGSPAATPEIRVNHEPEPAGAATP GAALPKSPSQLRKGPPVPPPPKHTPSKEVKQEQILSLFDDTFVPEISVTTPSQF; KPNA3:KAQNVTLEAILQNATSDNPVVQLSAVQAARKLLSSDRNPPIDDLIKS GILPILVKCLERDDNPSLQF;

[0105] FBLN1:TRPEEIIFLRAITPAYPANHADIIFDITDGNLRDSFDIIKRY;

[0106] H2AJ:LTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESQKTKSK.

[0107] This complex exhibits both hydrophobic and hydrophilic properties, and its transmembrane activity in cleaving ubiquitin is higher than that of natural ubiquitin. Figure 3 and Figure 4 As shown in the comparison.

[0108] The emulsion formula is designed to contain 0.1% small molecule protein complex, 0.5% sodium hyaluronate, 5% glycerin, 1% vitamin C, 0.1% retinol, 3% emulsifier, and appropriate amount of preservatives, with the remainder being deionized water and oil phase raw materials. The complex is dissolved in deionized water, and water-soluble components are added and stirred until dissolved. Oil-soluble components are dissolved in the oil phase, and water phase is added and emulsified at high speed. The pH is adjusted to 5.5-6.5, and then packaged. After 4 weeks of human trials, skin hydration increased by 22%, elasticity improved by 16%, and wrinkle depth decreased by 10%. Cell experiments showed a 28% increase in collagen synthesis and a 32% increase in antioxidant enzyme activity.

[0109] Through the above procedures, a small-molecule protein complex was extracted from goat embryos. This complex has a purity exceeding 95%, and its enzymatically cleaved ubiquitin content is more than five times that of natural ubiquitin. Seven proteins work synergistically to regulate skin cell function. In skincare applications, this complex promotes skin barrier penetration, achieving deep-layer activity. Experimental data show that this method has higher extraction efficiency than traditional methods, exhibits good product stability, and supports the verification of skincare efficacy.

[0110] Three examples, based on variations in different developmental stages of goat embryos (45 days, 60 days, and 90 days) and the addition ratio of cell lysis buffer (2x, 2.5x, and 3x), demonstrate the robustness and adaptability of the extraction method. They also verify the purity, yield, and skincare application effects of the complex under different conditions. Through optimization of the raw material development stage and lysis ratio, a consistent 5-fold increase in ubiquitin yield and a complex purity exceeding 95% were achieved. Specific efficacy, including moisturizing, anti-wrinkle, and comprehensive skincare, was validated, demonstrating the method's versatility and precise control capabilities, surpassing the limitations and inefficiencies of traditional extraction methods.

[0111] Simultaneously, this extraction method achieves enrichment of the target complex by precisely controlling enzymatic digestion and chromatographic conditions, reducing impurity interference and improving activity retention. The addition of the complex to the emulsion achieves uniform distribution and synergistic effects, suitable for various skincare needs. The overall solution provides a complete pathway from raw material processing to product application, driving development in related fields.

[0112] 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 it. 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 spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for extracting small molecule protein complexes, characterized in that: Includes the following steps: Using 45-90 day old goat embryos as raw material, tissue disruption was performed to obtain the supernatant; The supernatant was hydrolyzed with chymotrypsin; the protein complex was enriched by hydrophobic chromatography to obtain a protein complex with Ubiquitin as the main component and EFS, ALB, BIN1, H2AJ, FBLN1, and KPNA3 as auxiliary components.

2. The method for extracting small molecule protein complexes as described in claim 1, characterized in that: The tissue disruption process includes: rinsing the embryo with pre-cooled physiological saline, adding 2-3 times the volume of cell lysis buffer, the buffer consisting of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100, homogenizing under ice bath conditions, centrifuging at 5000g for 20 minutes at 4°C, and collecting the supernatant.

3. The method for extracting small molecule protein complexes as described in claim 1, characterized in that: The chymotrypsin hydrolysis process includes: determining the protein content of the supernatant using a BCA protein quantification kit, adding chymotrypsin at a protein to chymotrypsin ratio of 250:1, adjusting the temperature to 55℃, pH to 8.0, stirring speed to 150 rpm, reacting for 30 minutes, and terminating the reaction in an ice bath.

4. The method for extracting a small molecule protein complex as described in claim 1, characterized in that: The hydrophobic chromatography enrichment process includes: using phenyl-Sepharose 6FF medium, equilibrating the column with 20mM Tris-HCl pH 7.5 and 0.5M NaCl, loading enzyme digestion solution, washing with equilibration buffer until the absorbance at 280nm is close to the baseline, collecting the target peak by linear gradient elution, concentrating by 3kDa ultrafiltration, and lyophilizing for storage.

5. The method for extracting a small molecule protein complex as described in claim 1, characterized in that: The relative contents of each protein in the protein complex are: Ubiquitin 30%, EFS 24%, ALB 16%, BIN 110%, KPNA 310%, FBLN 16%, and H2AJ 4%.

6. The method for extracting a small molecule protein complex as described in claim 1, characterized in that: The protein complex contains Ubiquitin, which includes an enzymatically cleaved ubiquitin with the sequence AGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDT IENVKAKIQDKEGIPPDQQRLIF, and its content is more than 5 times that of natural ubiquitin.

7. The use of a small molecule protein complex as described in any one of claims 1-6 in the preparation of skin care products.

8. The application of the small molecule protein complex as described in claim 7 in the preparation of skincare products, characterized in that: The skincare product is an emulsion containing 0.1% small molecule protein complex, 0.5% sodium hyaluronate, 5% glycerin, 1% vitamin C, 0.1% retinol, 3% emulsifier, appropriate amount of preservative, and the remainder is deionized water and oil phase raw materials.

9. The application of the small molecule protein complex as described in claim 7 in the preparation of skincare products, characterized in that: The preparation of the skin care product includes: dissolving a small molecule protein complex in deionized water, adding water-soluble ingredients and stirring to dissolve; dissolving oil-soluble ingredients in the oil phase, adding water phase and stirring at high speed to emulsify, adjusting the pH to 5.5-6.5, and then bottling and packaging.

10. The use of the small molecule protein complex as described in claim 7 in the preparation of skincare products, characterized in that: After a 4-week human trial, improvements in skin hydration, elasticity, and wrinkle depth were observed. Cell experiments also revealed increased collagen synthesis and antioxidant enzyme activity.

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