Application of sericin to improvement of protein stability under various environmental pressures

Abstract

The invention relates to application of sericin to improvement of protein stability under various environmental pressures. Specifically, the sericin can promote denatured and inactivated protein renaturation / refolding so as to improve activity and stability, and can effectively stabilize various proteins including proteins, cell factors, enzymes, antibodies and the like under various environmental pressures, and the environmental pressures include high temperature, mechanical stress, acidic conditions, organic solvents, freeze-drying and multiple freeze-thawing. The sericin can also enhance the anticancer activity of specific glucose oxidase. The natural macromolecular sericin provided by the invention has the advantages of good biocompatibility, easiness in obtaining, low price and the like, and can be used as a wide protein stabilizer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the application of sericin in improving protein stability in harsh environments. Background Art

[0002] Proteins possess remarkable specificity, high activity, and excellent biocompatibility, making them an important therapeutic agent widely used in the treatment of various major diseases, including cancer. Furthermore, protein enzyme preparations, due to their high selectivity and catalytic activity, are also widely used in industrial production. However, proteins have poor stability and are easily subjected to various stresses during production, manufacturing, storage, transportation, and use, causing conformational changes that can reduce or even eliminate their biological activity, ultimately severely impacting their stability and effectiveness. Therefore, effective measures are often required to prevent protein denaturation and inactivation.

[0003] In organisms, natural molecular chaperones are produced in large quantities under harsh conditions to prevent protein aggregation and promote their refolding after stress, thereby helping the body resist external stimuli. However, natural chaperones are difficult to extract, expensive, and not readily available, which greatly limits their application. Small molecule chaperones such as trehalose and sucrose can effectively enhance protein folding and stability. However, the ability of low molecular weight chaperones to stabilize proteins is limited. Compared with small molecules, polymers have better protein stability. However, they usually require chemical synthesis to obtain, which is cumbersome and costly, and there is still much room for improvement.

[0004] Therefore, there is an urgent need to develop a preparation that is widely available, inexpensive, easily obtainable, and has good protein stabilization effects under various environmental pressures. This preparation also has low immunogenicity and good biocompatibility, and can achieve a wide and efficient protein stabilization effect. Summary of the Invention

[0005] The present invention provides a protein stabilizer which has good stabilizing effect on various proteins under various environmental pressures and has chaperone activity.

[0006] In a first aspect of the present invention, a method for promoting protein renaturation / refolding is provided, the method comprising: adding sericin to a denatured and inactivated protein, thereby obtaining a renaturated / refolded protein.

[0007] In another preferred embodiment, the protein is denatured and inactivated due to application of environmental pressure to the protein.

[0008] In another preferred embodiment, the ambient pressure is high temperature.

[0009] In another preferred embodiment, the environmental pressure is heating the protein at 45-100° C. for 5-60 min.

[0010] In another preferred embodiment, the protein is selected from the group consisting of horseradish peroxidase, glucose oxidase, and β-galactosidase.

[0011] In another preferred embodiment, the protein is horseradish peroxidase.

[0012] In another preferred embodiment, the added amount of the sericin is 0.1 to 200 equiv of the protein.

[0013] In another preferred embodiment, the added amount of the sericin is 10 to 100 equiv of the protein, preferably 20 to 60 equiv.

[0014] In another preferred embodiment, the method comprises the step of contacting the denatured and inactivated protein with sericin at 0° C.-50° C. for 8-24 hours.

[0015] In another preferred embodiment, the method comprises the step of contacting the denatured and inactivated protein with sericin at 4° C.-37° C. for 8-24 hours.

[0016] In another preferred embodiment, the sericin is naturally occurring sericin extracted from silk.

[0017] In another preferred embodiment, the molecular weight of the sericin is 1 kDa-200 kDa.

[0018] In another preferred embodiment, the amino acid sequence of the sericin protein includes the following repeating motif: SSTGSSSNTDSNSNSVGSSTSGGSSTYGYSSNSRDGSV (SEQ ID No. 1).

[0019] In another preferred embodiment, the present invention further provides a protein renaturation / refolding promoter, wherein the promoter comprises sericin.

[0020] In another preferred embodiment, the promoter further includes a buffer, a buffer, and / or other protein refolding agents.

[0021] In another preferred embodiment, the buffer or buffer is a conventional buffer or buffer, including but not limited to phosphate buffer.

[0022] In another preferred embodiment, the sericin is used to prepare a protein renaturation / refolding promoter.

[0023] In another aspect of the present invention, a method for improving protein stability under specific environmental pressure is provided, the method comprising: adding sericin to protein under specific environmental pressure, thereby improving protein stability;

[0024] The specific environmental pressure is selected from the following group: acidic environment, organic solvent, mechanical force, freeze-drying, multiple freeze-thaw cycles, and high temperature.

[0025] In another preferred embodiment, the added amount of the sericin is 0.1 to 200 equiv of the protein.

[0026] In another preferred embodiment, the added amount of the sericin is 10 to 100 equiv of the protein, preferably 20 to 60 equiv.

[0027] In another preferred embodiment, the specific environmental pressure is selected from the following group: freeze-drying, acidic environment, organic solvent, mechanical force, and repeated freezing and thawing.

[0028] In another preferred embodiment, the specific environmental pressure treatment procedure comprises: incubating the protein under environmental pressure for 15 minutes to 96 hours.

[0029] In another preferred embodiment, the acidic condition is a hydrogen ion concentration in the system of 0.0001-1M hydrogen ion concentration.

[0030] In another preferred embodiment, the organic solvent is selected from the following group: N,N-dimethylformamide, methanol, and acetonitrile.

[0031] In another preferred embodiment, the multiple freeze-thaw cycles refer to: freezing and thawing at -196°C to 37°C, repeated for 1 to 20 times.

[0032] In another preferred embodiment, the mechanical force can be achieved by conventional equipment such as a stirrer, etc., the time can be determined according to the specific situation, such as 4h, 12h, 24h, 48h, 96h, etc., and the speed can be determined according to the specific situation, such as 100-500rpm.

[0033] In another preferred embodiment, the freeze-drying can be completed by conventional equipment such as a vacuum high-pressure freeze dryer, and the freeze-drying time can be determined according to the specific situation, such as 4h, 12h, 24h, 48h, 96h, etc.

[0034] In another preferred embodiment, the high temperature refers to heating the protein at 30-100° C. for 15 min to 96 h. The heating can be accomplished by conventional methods such as water bath heating.

[0035] In another preferred embodiment, the protein is selected from the group consisting of horseradish peroxidase, glucose oxidase, β-galactosidase, lysozyme, laccase, pancreatic enzyme, alcohol dehydrogenase, acetaldehyde dehydrogenase, formate dehydrogenase, lyticase, uricase, pectinase, tannase, phytase, ribonuclease, xylanase, catalase, lactate dehydrogenase, elastase, transglutaminase, esterase, lipase, α-chymotrypsin, β-glucanase, thrombin, asparaginase, trypsin, protease, proteinase K, papain, pepsin, superoxide dismutase, cytochrome c, xanthine oxidase, Carbonic anhydrase, organophosphorus hydrolase, matrix metalloproteinase, bovine serum albumin, human serum albumin, fibrinogen, fibronectin, collagen, bone morphogenetic protein, amyloid protein, myoglobin, actin, lactoferrin, green fluorescent protein, phycocyanin, insulin, glucagon, interferon, interleukin, β-defensin, growth hormone, avidin (also known as avidin or antibiotin), streptavidin, fibroblast growth factor, colony-stimulating factor, tumor necrosis factor, vascular endothelial growth factor, epidermal growth factor, IgM antibody, IgG antibody, IgA antibody, IgE antibody, IgD antibody.

[0036] In another preferred embodiment, the protein is selected from the group consisting of horseradish peroxidase, glucose oxidase, β-galactosidase, and goat anti-bovine serum albumin IgG polyclonal antibody.

[0037] In another preferred embodiment, the protein is glucose oxidase.

[0038] In a second aspect of the present invention, a protein composition is provided, comprising: an effective amount of active protein, and sericin for improving the stability of the protein.

[0039] In another preferred embodiment, the improving protein stability includes: increasing the stability of the protein under environmental stress conditions, or improving the activity of the protein under environmental stress conditions.

[0040] In another preferred embodiment, the specific environmental pressure is selected from the following group: freeze-drying, acidic environment, organic solvent, mechanical force, and repeated freezing and thawing.

[0041] In another preferred embodiment, in the composition, the molar ratio of active protein to sericin is 1:0.1-200.

[0042] In another preferred embodiment, the sericin is naturally occurring sericin extracted from silk.

[0043] In another preferred embodiment, the amino acid sequence of the sericin protein includes the following repeating motif: SSTGSSSNTDSNSNSVGSSTSGGSSTYGYSSNSRDGSV (SEQ ID No. 1).

[0044] In another preferred embodiment, the protein is selected from the group consisting of horseradish peroxidase, glucose oxidase, β-galactosidase, lysozyme, laccase, pancreatic enzyme, alcohol dehydrogenase, acetaldehyde dehydrogenase, formate dehydrogenase, lyticase, uricase, pectinase, tannase, phytase, ribonuclease, xylanase, catalase, lactate dehydrogenase, elastase, transglutaminase, esterase, lipase, α-chymotrypsin, β-glucanase, thrombin, asparaginase, trypsin, protease, proteinase K, papain, pepsin, superoxide dismutase, cytochrome c, xanthine oxidase, Carbonic anhydrase, organophosphorus hydrolase, matrix metalloproteinase, bovine serum albumin, human serum albumin, fibrinogen, fibronectin, collagen, bone morphogenetic protein, amyloid protein, myoglobin, actin, lactoferrin, green fluorescent protein, phycocyanin, insulin, glucagon, interferon, interleukin, β-defensin, growth hormone, avidin (also known as avidin or antibiotin), streptavidin, fibroblast growth factor, colony-stimulating factor, tumor necrosis factor, vascular endothelial growth factor, epidermal growth factor, IgM antibody, IgG antibody, IgA antibody, IgE antibody, IgD antibody.

[0045] In another preferred embodiment, the protein is selected from the group consisting of horseradish peroxidase, glucose oxidase, β-galactosidase, and goat anti-bovine serum albumin IgG polyclonal antibody.

[0046] In another preferred embodiment, the protein is glucose oxidase.

[0047] In a third aspect of the present invention, a method for improving protein stability is provided, the method comprising: mixing sericin and protein to obtain a protein with improved stability;

[0048] The protein is selected from the group consisting of horseradish peroxidase, D-lactate dehydrogenase, pectinase, elastase, lipase, and α-chymotrypsin.

[0049] In another preferred embodiment, the protein with improved stability refers to the temperature at which the protein begins to change its conformation (T onset-ss ) and melting temperature (T m-ss ) were higher than the T values of proteins without adding sericin. onset and T m .

[0050] In another preferred embodiment, the T onset-ss and T onset , and / or T m-ss and T m The difference is ≥5, preferably, the difference is ≥8.

[0051] In another preferred embodiment, the sericin is naturally occurring sericin extracted from silk.

[0052] In another preferred embodiment, the amino acid sequence of the sericin protein includes the following repeating motif: SSTGSSSNTDSNSNSVGSSTSGGSSTYGYSSNSRDGSV (SEQ ID No. 1).

[0053] In a fourth aspect of the present invention, a pharmaceutical composition is provided, comprising: glucose oxidase and 0.1 to 200 equiv of sericin.

[0054] In another preferred embodiment, the sericin is naturally occurring sericin extracted from silk.

[0055] In another preferred embodiment, the amino acid sequence of the sericin protein includes the following repeating motif: SSTGSSSNTDSNSNSVGSSTSGGSSTYGYSSNSRDGSV (SEQ ID No. 1).

[0056] In the fifth aspect of the present invention, there is provided a use of the pharmaceutical composition according to the fourth aspect of the present invention for preparing anticancer drugs.

[0057] In another preferred embodiment, the anticancer drug is an anti-melanoma drug.

[0058] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The invention relates to the protection of sericin to horseradish peroxidase, β-galactosidase, glucose oxidase and goat anti-bovine serum albumin IgG polyclonal antibody at high temperature.

[0060] Figure 2 The invention relates to the protection of sericin to horseradish peroxidase, β-galactosidase, glucose oxidase and goat anti-bovine serum albumin IgG polyclonal antibody under freeze-drying.

[0061] Figure 3The invention relates to the protection of sericin to horseradish peroxidase, β-galactosidase, glucose oxidase and goat anti-bovine serum albumin IgG polyclonal antibody under stirring conditions.

[0062] Figure 4 The invention relates to the protection of sericin to horseradish peroxidase, β-galactosidase, glucose oxidase and goat anti-bovine serum albumin IgG polyclonal antibody under acidic conditions.

[0063] Figure 5 The invention relates to the protection of sericin to horseradish peroxidase, β-galactosidase and glucose oxidase in the presence of N,N-dimethylformamide organic solvent.

[0064] Figure 6 The invention relates to the protection of glucose oxidase by sericin in the presence of three organic solvents: methanol, acetonitrile and dimethyl sulfoxide.

[0065] Figure 7 The protection of sericin against goat anti-bovine serum albumin IgG polyclonal antibody under repeated freeze-thaw conditions.

[0066] Figure 8 Horseradish peroxidase-assisted refolding / renaturation of sericin.

[0067] Figure 9 Sericin binds glucose oxidase to enhance anticancer activity. DETAILED DESCRIPTION

[0068] After extensive and in-depth research, and through extensive screening and testing, the inventors have provided a method for improving protein stability under various environmental pressures using a natural macromolecular sericin protein. The sericin protein can promote the refolding / renaturation of denatured and inactivated proteins, such as horseradish peroxidase. The sericin protein has a wide range of stabilizing effects on proteins under various environmental pressures, including acidic environments, organic solvents, mechanical forces, freeze-drying, multiple freeze-thaw cycles, and high temperatures. It can also improve the thermal stability of specific proteins, such as horseradish peroxidase, D-lactate dehydrogenase, pectinase, elastase, lipase, and α-chymotrypsin, significantly increasing T onset-ss and melting temperature T m The sericin can also enhance the anti-tumor activity of glucose oxidase. This is the basis for the completion of the present invention.

[0069] Active ingredient

[0070] Sericin is a water-soluble, colloidal protein that encapsulates the fibroin protein found in silk, accounting for approximately 20-30% of the total weight of silk. It acts as an adhesive within the cocoon, binding the fibroin fibers together. Sericin is rich in hydrophilic amino acids (such as serine, aspartic acid, and glycine), which impart high hygroscopicity, emulsification, and biocompatibility. Sericin is widely available, primarily from the cocoons of the domesticated silkworm (Bombyx mori), but can also be obtained from wild silkworm species (such as ricin and tussah). Sericin can be separated by removing the fibroin protein using hot water, alkaline solutions, or enzymatic hydrolysis.

[0071] The active ingredient of the present invention is natural macromolecular sericin, and the average molecular weight of the natural macromolecular sericin is in the range of 1 kDa-200 kDa, preferably, 3000-10000 kDa, such as about 5000 kDa or 10000 kDa.

[0072] The natural sericin mentioned in this invention is derived from the outer layer of silk and includes Ser1, Ser2, and Ser3. Sericin is composed of various amino acids, including glycine, alanine (also known as alanine or α-aminopropionic acid), valine, leucine, isoleucine, phenylalanine, methionine (also known as methionine), proline, serine, tyrosine (also known as tyrosine), cysteine, threonine, aspartic acid (also known as aspartic acid), glutamic acid, lysine, arginine, and histidine. Among them, the components with an amino acid composition ratio of more than 10% are believed to play a very important role in stabilizing proteins, including serine (31.0%), glycine (19.1%), and aspartic acid (17.8%). The components with an amino acid composition ratio of 2%-10% are believed to play an important role in stabilizing proteins, including threonine (8.0%), glutamic acid (4.4%), arginine (3.9%), alanine (3.8%), tyrosine (3.3%), valine (3.1%), and lysine (2.7%). Other amino acids are believed to play a moderately important role in stabilizing proteins.

[0073] Sericin includes but is not limited to the following repeating motif "SSTGSSSNTDSNSNSVGSSTSGGSSTYGYSSNSRDGSV". Polypeptides having this sequence or components or sequences similar thereto also have the function of stabilizing proteins.

[0074] use

[0075] The present invention provides a use of the natural macromolecular sericin as described above. The natural macromolecular sericin has the function of improving protein stability under different environmental pressures, and can thus be used to prevent and / or stop protein denaturation and inactivation.

[0076] Preferably, the protein includes (but is not limited to): horseradish peroxidase, glucose oxidase, β-galactosidase, lysozyme, laccase, pancreatin, alcohol dehydrogenase, acetaldehyde dehydrogenase, formate dehydrogenase, lyticase, uricase, pectinase, tannase, phytase, ribonuclease, xylanase, catalase, lactate dehydrogenase, elastase, transglutaminase, esterase, lipase, α-chymotrypsin, β-glucanase, thrombin, asparaginase, trypsin, protease, proteinase K, papain, pepsin, superoxide dismutase, cytochrome c, xanthine oxidase, carbonic anhydrase, organophosphorus hydrolase, matrix metalloproteinase , bovine serum albumin, human serum albumin, fibrinogen, fibronectin, collagen, bone morphogenetic protein, amyloid protein, myoglobin, actin, lactoferrin, green fluorescent protein, phycocyanin, insulin, glucagon, interferon, interleukin, β-defensin, growth hormone, avidin (also known as avidin or anti-biotin), streptavidin, fibroblast growth factor, colony stimulating factor, tumor necrosis factor, vascular endothelial growth factor, transforming growth factor, epithelial growth factor, insulin growth factor, platelet-derived growth factor, nerve growth factor, hepatocyte growth factor, IgM antibody, IgG antibody, IgA antibody, IgE antibody, IgD antibody.

[0077] Preferably, the proteins include hydrophilic proteins (such as serum albumin), hydrophobic proteins (such as fibrinogen), positively charged proteins (such as lysozyme) and negatively charged proteins (such as glucose oxidase).

[0078] Preferably, the protein includes cytokines (such as fibroblast growth factor), enzymes (such as horseradish peroxidase), antibodies (such as IgG antibodies), and proteins (such as fibronectin).

[0079] Preferably, the method of stabilizing the protein with sericin in the present invention includes (but is not limited to): contacting the protein to be protected with sericin to obtain the protein protected by sericin. The sericin includes (but is not limited to) the following forms: hydrogel, scaffold, self-assembly, vesicle, fiber, microsphere, etc.

[0080] Preferably, the protein to be protected is contacted with sericin in a phosphate buffer solution at 25° C. or 4° C. for 3 min-2 h.

[0081] Preferably, the amount of the sericin added is 0.1 wt equiv-200 wt equiv relative to the amount of the protein to be protected.

[0082] Preferably, the protein alone and the protein protected by sericin are subjected to environmental pressure respectively, and then the activity or melting temperature of the protein is detected to evaluate the stabilizing effect of the compound on the protein.

[0083] Preferably, the environmental pressure includes (but is not limited to) high temperature, acidic conditions, organic solvents, mechanical force, freeze drying, repeated freeze-thaw, including:

[0084] High temperature: Heat in a water bath at 30-100°C for 15 minutes to 96 hours;

[0085] Acidic conditions: incubate at 0.0001-0.1 M hydrogen ion concentration at 4-37°C for 15 minutes to 96 hours, preferably at 0-10°C for 15 minutes to 1 hour;

[0086] Organic solvent: incubate in an organic solvent containing 20% to 99% of methanol, acetonitrile, N,N-dimethylformamide, etc. at 0-50°C for 15 minutes to 96 hours, preferably at 2-10°C for 15 minutes to 1 hour;

[0087] Mechanical force: stirring at 100-500 rpm at 0-50°C for 1-96 hours, preferably incubating at 25-40°C for 12 to 36 hours;

[0088] Freeze drying: Freeze drying under vacuum and high pressure for 4-96 hours;

[0089] Repeated freeze-thaw: Freeze and thaw at -196℃-37℃, and repeat the cycle 1-20 times.

[0090] The sericin can be used to bind proteins; and / or to protect protein activity.

[0091] The superiority of sericin in stabilizing proteins

[0092] Sericin exhibits excellent protein stabilization under a variety of environmental stresses, including high temperature, freeze-drying, acidic conditions, organic solvents, mechanical force, and multiple freeze-thaw cycles. However, some other common stabilizers can only stabilize proteins under a single or specific environmental stress and cannot achieve the same stability under multiple environmental stresses.

[0093] Common stabilizers include bovine serum albumin (BSA) or polyethylene glycol (PEG).

[0094] BSA can have a relatively good protective effect on proteins under acidic conditions and freeze-drying conditions. Therefore, in practical applications, an appropriate amount of BSA can be added when diluting or storing low-concentration proteins, or used in combination with sugars (such as trehalose) to reduce protein denaturation during the freeze-drying process. However, the protective effect on proteins under high temperature and stirring conditions is extremely weak. In addition, BSA may contain trace amounts of proteases, fatty acids or IgG, which may interfere with the experiment or require the selection of higher-purity BSA, increasing costs. Moreover, some proteins such as coagulation factors may be inactivated by BSA binding, and BSA cannot be used as a stabilizer.

[0095] Similarly, polyethylene glycol has a better protective effect on proteins under stirring and freeze-drying conditions. Therefore, in practical applications, adding 5-20% PEG (usually PEG 2000-8000) can extend the shelf life of liquid or freeze-dried proteins (such as antibodies and enzyme preparations), or be used in combination with sugars (such as sucrose) to reduce ice crystal damage (better than using glycerol alone). However, it has almost no protective effect on proteins under high temperature, acidic conditions, and in the presence of organic solvents. Moreover, the addition of PEG may also interfere with ultraviolet absorbance (UV 280nm) or chromatographic analysis (such as SEC-HPLC). In some immunoassays, the addition of PEG may also mask antigen epitopes and affect antibody binding. At the same time, high concentrations of PEG have low biocompatibility and are likely to affect cell activity.

[0096] In summary, sericin stabilizes proteins under a wide range of environmental stresses and can be used in a variety of harsh environments. It is suitable for all aspects of protein production, manufacturing, storage, transportation, and use. It has a broad and universal stabilizing effect on a wide range of proteins, does not affect subsequent assays, and exhibits high biocompatibility.

[0097] Sericin's chaperone-like activity aids protein refolding

[0098] The present invention provides a use of the sericin as described above. After the protein is denatured by applying environmental pressure, sericin is added to contact the protein. The sericin can help the protein renature and / or refold, thereby restoring the activity of the protein.

[0099] Preferably, after the individual proteins are denatured and inactivated by applying environmental pressure, sericin is added to interact with the proteins, and then the sericin partially or completely restores the activity of the proteins. The method comprises the steps of:

[0100] (a) Applying environmental stress to individual proteins;

[0101] (b) The ambient pressure is removed and sericin is added to help the protein renaturation / refolding.

[0102] Preferably, the applied environmental pressure is high temperature, such as heating in a water bath at 30-100° C. for 15 minutes to 48 hours; and adding sericin to assist protein refolding is incubating at 0-50° C. for 8-24 hours, preferably at 0-10° C. for 8-24 hours.

[0103] Protein-binding drugs or pharmaceutical compositions

[0104] In the present invention, a "protein-binding drug or pharmaceutical composition" refers to a pharmaceutical agent comprising the sericin protein described above, which is a protein-binding agent. The protein has therapeutic functions, including (but not limited to) tumor treatment (e.g., glucose oxidase, interferon), ischemic disease treatment (e.g., vascular endothelial growth factor), diabetes treatment (e.g., insulin), lactose intolerance (e.g., β-galactosidase), oral disease treatment (e.g., lysozyme), alcoholism treatment or hangover relief (e.g., alcohol dehydrogenase), hyperuricemia treatment (e.g., uricase), tissue repair (e.g., fibroblast growth factor), hemostasis (e.g., thrombin), ocular disease treatment (e.g., vascular endothelial growth factor), neurodegenerative disease treatment (e.g., amyloid), growth hormone deficiency (e.g., growth hormone), wound repair (e.g., fibronectin), and anti-infective and antiviral treatments (e.g., IgG antibodies).

[0105] In addition, the sericin of the present invention can be used alone or together with other stabilizers (such as formulated in the same pharmaceutical composition). For example, the stabilizer can be a protein stabilizer, such as trehalose.

[0106] The present invention provides a use of the sericin as described above. After the protein drug contacts the sericin, the sericin can bind to the protein, thereby enhancing the stability of the protein drug and thus improving the therapeutic effect of the protein drug.

[0107] Preferably, the therapeutic effect of the individual proteins can be enhanced after interacting with the added sericin.

[0108] Preferably, the protein is glucose oxidase, and the treatment refers to enhancing anti-tumor activity.

[0109] The main advantages of the present invention are:

[0110] 1. The sericin of the present invention stabilizes proteins under various environmental pressures, helps them maintain their conformation, inhibits protein aggregation, and prevents protein denaturation and inactivation. It also exhibits no cytotoxicity or hemolytic toxicity to normal cells and exhibits excellent biocompatibility.

[0111] 2. The sericin of the present invention has a broad stabilizing effect on a variety of proteins, and is suitable for hydrophilic proteins (such as serum albumin), hydrophobic proteins (such as fibrinogen), positively charged proteins (such as lysozyme), and negatively charged proteins (such as glucose oxidase).

[0112] 3. The sericin of the present invention has a broad stabilizing effect on a variety of proteins, including cytokines (such as fibroblast growth factor), enzymes (such as horseradish peroxidase), antibodies (such as IgG antibodies), and proteins (such as fibronectin).

[0113] 4. The sericin of the present invention has a broad stabilizing effect on various proteins and is suitable for disease treatment (such as IgG antibodies), biosensors (such as glucose oxidase), and industrial applications (such as lipase), so it has wide application value.

[0114] 5. The sericin of the present invention stabilizes proteins through intermolecular interactions with proteins and the volume exclusion effect, thereby having a stabilizing effect on various proteins.

[0115] 6. The sericin of the present invention can be combined with proteins to form protein-combined drugs or pharmaceutical compositions, and the combined proteins can be used in actual clinical treatments for clinical disease treatments.

[0116] 7. The sericin of the present invention has good biocompatibility and is substantially free of cytotoxicity and hemolytic toxicity.

[0117] 8. The sericin of the present invention can be added after protein denaturation to refold it and restore its activity.

[0118] 9. The natural macromolecular sericin of the present invention is derived from the outer layer of silk, which is often discarded in the textile industry. It has the advantages of being inexpensive, widely available, and easily obtainable.

[0119] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise stated.

[0120] In the present invention, the sericin used is commercially available natural sericin (CAS: 60650-88-6) with a molecular weight of approximately 10 kDa (SS2).

[0121] Testing the ability of sericin to improve protein stability

[0122] Example 1 Protein thermal stability test

[0123] The proteins selected include horseradish peroxidase (HRP), glucose oxidase (GOX), β-galactosidase (β-Gal), lysozyme, alcohol dehydrogenase (ADH), pectinase, tannase, D-lactate dehydrogenase (D-LDH), elastase, lipase, α-chymotrypsin (α-CMT), proteinase K, cytochrome C (Cyt C), bovine serum albumin (BSA), fibrinogen protein (Fg), fibronectin (Fn), streptavidin, fibroblast growth factor (GF), mitochondrial isothiocyanate (MS / MS), cytochrome c (Cyt C), thiazolidinone (TMA), pyrin (P-Y), thiazolidinone (P-Y), thiazolidinone (P-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), mitochondrial isothiocyanate (M-Y), Factor, bFGF), IgG antibody. The natural macromolecule selected is sericin. The protein was dissolved in phosphate buffer solution at 0.5 mg / mL, and the sericin was dissolved in phosphate buffer solution at 20 mg / mL (40wt equiv). Then, the protein was mixed with phosphate buffer solution and sericin solution in equal volumes at a ratio of 1:1 to obtain a protein protected by sericin, wherein the final concentration of the protein in the mixed solution was 0.25 mg / mL, and the final concentration of the sericin was 10 mg / mL. 10 uL of the mixed solution was drawn using a capillary tube, and the temperature was raised from 20°C to 95°C at a rate of 2°C / min. The fluorescence value changes at 330 nm and 350 nm of the protein were detected by differential scanning fluorimetry (nano DSF), and the temperature at which the protein conformation began to change (T onset ) and melting temperature (T m ). Among them, sericin itself has no tertiary structure, so there is no T onset and T m , which will not affect the determination of protein.

[0124] The experimental results are shown in Table 1. Sericin has a very obvious and prominent effect on improving the stability of certain specific proteins, and can significantly increase the temperature at which the protein conformation begins to change (T onset ) and / or melting temperature (T m ), the maximum increase can reach about 18℃, among which the most obvious effect is achieved by pectinase. After adding sericin, T onset The melting temperature T m It increased by 11.7℃, indicating that sericin can significantly improve the thermal stability of various proteins.

[0125] Table 1 Experimental results of sericin significantly improving the thermal stability of proteins

[0126]

[0127] In addition to the proteins listed in Table 1, sericin also has an excellent thermal stability improvement effect on other proteins, as shown in Table 2.

[0128] Table 2 Experimental results of improving the thermal stability of proteins with different physicochemical properties using sericin

[0129]

[0130]

[0131] Example 2 Test of Sericin's Protection of Protein at High Temperature

[0132] The proteins used were horseradish peroxidase (HRP), glucose oxidase (Gox), β-galactosidase (β-Gal), goat anti-bovine serum albumin IgG polyclonal antibody (G-IgG), and laccase (Lacs). The natural macromolecule used was sericin (SS). The proteins were dissolved in phosphate-buffered saline (PBS) to prepare a 0.15 mg / mL or 0.1 mg / mL solution. Sericin (SS) was dissolved in PBS to a 40 wt equiv (6 mg / mL or 4 mg / mL) solution. The protein and PBS were mixed in a 1:1 ratio to create a total volume of 40 μL for a control group. The protein and sericin (SS) were mixed in a 1:1 ratio to create a total volume of 40 μL. The mixed solutions were heated in a glass bottle in a water bath at 70°C or 50°C for 1 hour or 30 minutes, cooled to room temperature, and centrifuged. The solutions were then collected and assayed for protein activity.

[0133] HRP activity assay: Take 10 μL of the heated mixed solution and dilute it 500 times. Then take 5 μL of the diluted solution and add it to a 48-well plate. Add 500 μL of the color developer catechol solution and incubate in the dark for 15 minutes. Add 500 μL of 2M sulfuric acid solution to quench the solution. Read the absorbance (OD value) at 492 nm and use the formula Calculate the relative activity of HRP;

[0134] β-Gal activity assay: Add 20 μL of β-galactosidase solution and 50 μL of 4 mg / mL ONPG solution to a 96-well plate and incubate for 5 minutes in the dark. Subsequently, add 50 μL of 1 mol / L Na2CO3 to quench the reaction and read the OD value at 405 nm using a microplate reader. The protein activity value of each well is calculated using the formula

[0135] Gox activity assay: Take 0.1mL of 10mg / mL o-diazetidine (ODA) methanol solution and dissolve it in 12mL of pH 6 phosphate buffer. Prepare 180g / L glucose aqueous solution and 90U / mL HRP solution respectively. Subsequently, the ODA solution, glucose solution and HRP solution were mixed in a ratio of 25:3:1 to obtain a working solution. Add 290μL of color development working solution to a 48-well plate, and then add 10μL of glucose oxidase solution for analysis. Then add 200μL of 2N H2SO4 to quench, and use a microplate reader to read the OD value at 460nm. The protein activity value of each well is calculated using the formula

[0136] G-IgG activity is measured by its antigen binding capacity: 100 μL of bovine serum albumin (BSA) solution at a concentration of 10 μg / mL was added to a 96-well plate and incubated overnight. After removing the BSA solution, 200 μL of 5% skim milk solution was added and incubated at room temperature for 2 hours for blocking. Subsequently, the plate solution was replaced with 200 μL of 0.05% PBST solution, incubated for 30 seconds, and then the solution in each well was removed. This process was repeated five times to ensure complete removal of residual skim milk solution. 50 μL of the diluted G-IgG solution was added to each well and incubated for 2 hours. Each well was then washed five times with 0.05% PBST. Subsequently, 50 μL of a rabbit anti-goat antibody solution diluted 1:2000 in PBS was added. After incubation for 45 minutes, the wells were washed five times with 0.05% PBST. 100 μL of a 1 mg / mL OPD solution was added for color development. The reaction was shielded from light for 15 minutes and then quenched by adding 100 μL of 2N H2SO4. The absorbance at 492 nm was read. The G-IgG binding activity value for each well was calculated using the formula

[0137] The experimental results are as follows Figure 1 As shown in Table 3, the addition of sericin significantly increased the activity retention of various proteins after high temperature incubation. Taking HRP as an example, the activity of the protein retained up to 92.5% after the addition of sericin, while only 13.4% was retained without the addition of sericin. This represents a seven-fold increase in activity compared to the absence of sericin. This indicates that sericin can improve the stability of various proteins, thereby retaining higher protein activity.

[0138] Table 3 Experimental results of sericin improving protein activity at high temperature

[0139]

[0140] Example 3 Test of protein protection by sericin under freeze-drying conditions

[0141] The proteins used were horseradish peroxidase (HRP), glucose oxidase (Gox), β-galactosidase (β-Gal), and goat anti-bovine serum albumin IgG polyclonal antibody (G-IgG). The natural macromolecule used was sericin (SS). The proteins were dissolved in phosphate-buffered saline (PBS) to prepare solutions at 0.15 mg / mL or 0.1 mg / mL. Sericin (SS) was dissolved in PBS at 40 wt equiv (6 mg / mL or 4 mg / mL). The protein and PBS were mixed at a ratio of 1:3, resulting in a total volume of 100 μL, serving as controls. The protein and sericin (SS) were mixed at a ratio of 1:3, resulting in a total volume of 100 μL. The mixed solutions were placed in centrifuge tubes, lyophilized overnight, and then reconstituted in 100 μL of ultrapure water. Protein activity was determined using the same method as described in Example 2.

[0142] The experimental results are as follows Figure 2 As shown in Table 4, the addition of sericin ensures that proteins retain high activity after lyophilization, mitigating protein inactivation caused by water loss and vacuum during the lyophilization process. For example, HRP, β-Gal, and Gox retain over 90% of their activity after lyophilization when protected by sericin. This demonstrates that sericin improves protein stability during lyophilization, resulting in higher protein activity retention, and this protective effect is not limited to the type of protein.

[0143] Table 4 Experimental results of improving the activity of freeze-dried protein by sericin

[0144]

[0145] Example 4: Test of Sericin's Protection of Protein under Stirring Conditions

[0146] The proteins used were horseradish peroxidase (HRP), glucose oxidase (Gox), β-galactosidase (β-Gal), and goat anti-bovine serum albumin IgG polyclonal antibody (G-IgG). The natural macromolecule used was sericin (SS). The proteins were dissolved in phosphate-buffered saline (PBS) to prepare a 0.15 mg / mL or 0.1 mg / mL solution. Sericin (SS) was dissolved in PBS to a 40 wt equiv (6 mg / mL or 4 mg / mL) solution. The protein and PBS were mixed in a 1:1 ratio to create a total volume of 50 μL for a control group. The protein and sericin (SS) were mixed in a 1:1 ratio to create a total volume of 50 μL. The mixed solution was placed in a glass bottle and stirred at 250 rpm for 24 hours. The protein activity was then determined using the same method as described in Example 2.

[0147] The experimental results are as follows Figure 3 As shown in Table 5, stirring, i.e., mechanical stress exposure, significantly disrupts protein activity, with GOX activity remaining at only 1.46%. However, the addition of sericin significantly reduced protein inactivation under mechanical stress, increasing activity to 90%. This suggests that sericin can enhance protein stability under stirring conditions, thereby maintaining higher protein activity.

[0148] Table 5 Experimental results of sericin improving protein activity under stirring conditions

[0149]

[0150] Example 5: Test of Sericin's Protection of Proteins under Acidic Conditions

[0151] The proteins used were horseradish peroxidase (HRP), glucose oxidase (Gox), β-galactosidase (β-Gal), and goat anti-bovine serum albumin IgG polyclonal antibody (G-IgG). The natural macromolecule used was sericin (SS). The proteins were dissolved in phosphate-buffered saline (PBS) to prepare solutions at 1.5 mg / mL or 1 mg / mL. Sericin (SS) was dissolved in PBS at 40 wt equiv, i.e., 60 mg / mL or 40 mg / mL. The protein and PBS were mixed in a 1:1 ratio to create a total volume of 10 μL, serving as a control. The protein and sericin (SS) were mixed in a 1:1 ratio to create a total volume of 10 μL. The mixed solutions were then placed in hydrochloric acid solutions of varying concentrations and incubated at 4°C for 30 minutes. The protein activity was then determined using the same method as described in Example 2.

[0152] The experimental results are as follows Figure 4 As shown in Table 6, an acidic environment significantly impairs protein activity, with GOX activity remaining at only 0.3%. However, the addition of sericin significantly reduces protein inactivation in acidic conditions, increasing it to 95%. This suggests that sericin can enhance protein stability in acidic conditions, thereby maintaining higher protein activity.

[0153] Table 6 Experimental results of sericin improving protein activity under acidic conditions

[0154]

[0155] Example 6: Test of Sericin Protection on Protein in Organic Solvents

[0156] The proteins used were horseradish peroxidase (HRP), glucose oxidase (Gox), and β-galactosidase (β-Gal), and the natural macromolecule used was sericin (SS). The proteins were dissolved in phosphate-buffered saline (PBS) to prepare solutions at 1.5 mg / mL or 1 mg / mL. Sericin (SS) was dissolved in PBS at 40 wt equiv, i.e., 60 mg / mL or 40 mg / mL. The proteins were mixed with PBS at a ratio of 1:1, resulting in a total volume of 10 μL, serving as a control. The proteins were then mixed with sericin (SS) at a ratio of 1:1, resulting in a total volume of 10 μL. The mixed solutions were then placed in N,N-dimethylformamide (DMF) solutions of varying concentrations and incubated at 4°C for 30 minutes. The solutions were then sampled and assayed for protein activity. The protein activity assay was performed using the same method as described in Example 2.

[0157] The experimental results are as follows Figure 5As shown in Table 7, sericin can improve the stability of various proteins such as horseradish peroxidase (HRP), glucose oxidase (Gox), and β-galactosidase (β-Gal) in N,N-dimethylformamide (DMF), thereby increasing their activity.

[0158] Table 7 Experimental results of sericin improving the activity of various proteins in N,N-dimethylformamide

[0159]

[0160] In addition, the protective ability of sericin against glucose oxidase (GOX) in methanol (MeOH) and acetonitrile (CH3CN) organic solvents was tested using the same testing method as above.

[0161] The experimental results are as follows Figure 6 As shown in Table 8, it was shown that sericin could improve the stability of GOX in the presence of methanol and acetonitrile, thereby increasing its activity.

[0162] Table 8 Experimental results of improving the activity of GOX in various organic solvents by sericin

[0163]

[0164] Example 7: Test of protein protection by sericin under multiple freeze-thaw cycles

[0165] The protein used was a goat anti-bovine serum albumin IgG polyclonal antibody (G-IgG), and the natural macromolecule used was sericin (SS). The protein was dissolved in phosphate-buffered saline (PBS) to a 1 mg / mL solution, while the sericin (SS) was dissolved in PBS at a 40 wt equiv (40 mg / mL) concentration. The protein and PBS were mixed in a 1:1 ratio, resulting in a total volume of 100 μL. This served as a control. The protein and sericin (SS) were mixed in a 1:1 ratio, resulting in a total volume of 100 μL, with a final protein concentration of 0.05 mg / mL and a sericin concentration of 2 mg / mL. The mixed solution was then flash-frozen at -196°C (liquid nitrogen), thawed in a 37°C water bath, and then frozen again. This process involved 10 freeze-thaw-freeze cycles, and the solutions were then sampled for protein activity. The protein activity assay was performed using the same method as described in Example 2.

[0166] The experimental results are as follows Figure 7 As shown in Table 9, after adding sericin, the activity of goat anti-bovine serum albumin IgG polyclonal antibody can be increased to about twice that of the case without sericin, indicating that sericin can improve the stability of protein under repeated freezing and thawing conditions and thus improve its activity.

[0167] Table 9 Results of experiments on improving protein activity of sericin under repeated freezing and thawing conditions

[0168]

[0169] Example 8 Comparative protection test of different protective agents on proteins under various environmental pressures

[0170] Examples 1-7 demonstrate that sericin exhibits excellent protein stabilization under various environmental stresses, including high temperature, freeze-drying, acidic conditions, the presence of organic solvents, mechanical force, and multiple freeze-thaw cycles. To demonstrate that this unique property of sericin is protein stabilization under these diverse environmental stresses, bovine serum albumin (BSA) and polyethylene glycol (PEG, Mw = 4000) were used as controls. HRP (pI = 8.9) and Gox (pI = 4.6) proteins were used for evaluation, following the same experimental procedures as above.

[0171] Table 10 Experimental results of different protective agents to improve HRP stability under various environmental pressures

[0172]

[0173] The results showed that sericin significantly outperformed bovine serum albumin (BSA) and polyethylene glycol (PEG) in protecting HRP under various environmental stresses. BSA had little protective effect on HRP under high temperature and stirring conditions, but had some protective effect in the presence of organic solvents and was more effective under acidic and lyophilized conditions. PEG had little protective effect under high temperature, acidic conditions, and the presence of organic solvents, but was more effective under stirring and lyophilized conditions. Sericin, on the other hand, exhibited excellent protective effects on HRP under various environmental stresses, including high temperature, acidic conditions, organic solvents, stirring, and lyophilization.

[0174] Table 11 Experimental results of different protective agents to improve the stability of Gox under various environmental pressures

[0175]

[0176] The effect of stabilizing Gox is similar to that of HRP. BSA has little protective effect on Gox under high temperature and stirring conditions, but has some protective effect under lyophilization and in the presence of organic solvents, and has a stronger protective effect under acidic conditions. PEG has little protective effect under high temperature, acidic conditions, and in the presence of organic solvents, but has some protective effect under lyophilization and stirring conditions. Sericin has an excellent protective effect on Gox under various environmental stresses, including high temperature, acidic conditions, organic solvents, stirring, and lyophilization.

[0177] The results show that sericin has excellent protein stabilization effects under various environmental pressures such as high temperature, freeze-drying, acidic conditions, the presence of organic solvents, mechanical force, and multiple freeze-thaw cycles, which is not available with conventional protective agents.

[0178] Example 9 Testing of Sericin-Assisted Protein Refolding / Refolding

[0179] The proteins used were horseradish peroxidase (HRP), glucose oxidase (Gox), and β-galactosidase (β-Gal), and the natural macromolecule used was sericin (SS). HRP was dissolved in phosphate-buffered saline (PBS) to a 0.15 mg / mL solution, and sericin (SS) was dissolved in PBS at a 40 wt equiv (6 mg / mL) concentration. 50 μL of each protein solution was placed in a glass vial and heated in a 70°C water bath for 30 minutes to unfold / denature the protein. Subsequently, 50 μL of sericin was added to facilitate protein refolding / renaturation. A control solution was added with 50 μL of PBS. The protein solutions were incubated at 4°C overnight. Protein activity was then measured using the same method as described in Example 2. Heat denaturation conditions for Gox and β-Gal were 0.1 mg / mL at 65°C for 15 minutes and 0.1 mg / mL at 50°C for 30 minutes, respectively.

[0180] The experimental results are as follows Figure 8 As shown in Table 12, the addition of sericin can significantly help the renaturation / refolding of proteins after denaturation and inactivation, and the activity is significantly improved, especially HRP, whose activity increases by about 2 times, indicating that sericin helps promote the renaturation / refolding of proteins after high-temperature denaturation.

[0181] Table 12 Experimental results of sericin promoting protein renaturation / refolding after high temperature denaturation

[0182]

[0183] Sericin-binding proteins enhance therapeutic effects

[0184] Example 10 Anticancer Activity Test

[0185] The cancer cell line used was the B16 mouse melanoma cell line, the protein used was glucose oxidase (GOX), and the natural macromolecule used was sericin (SS). Tumor cells in the logarithmic growth phase were selected and trypsinized. These cells were diluted to a cell suspension of 30,000 cells per mL and seeded into 96-well plates at 100 μL per well. Three replicates were plated per group and incubated overnight in a 37°C, 5% CO2 cell incubator to allow the cells to adhere.

[0186] Experimental Groups: Sericin (SS) was dissolved in phosphate buffer to prepare a 4 mg / mL stock solution. The protein was then prepared in phosphate buffer to a 1.5 mg / mL solution and diluted with culture medium to 156 ng / mL. The sericin was then diluted with culture medium to a protein concentration of 40 wt equiv, or 6240 ng / mL. Protein (GOX) and sericin (SS) were mixed in a 1:1 ratio, and the original culture medium was replaced with the protein-containing culture medium (the GOX group). The original culture medium was replaced with the culture medium containing the protein-sericin (SS) mixture (the GOX+SS group).

[0187] Control group: Compound-free culture medium served as the negative control group, and pure DMSO served as the blank control group. After treating cells with either protein alone or a mixture of protein and sericin (SS) for 24 hours, the original culture medium was discarded, and 100 μL of serum-free culture medium containing 10% μL of the cell viability assay reagent MTT solution (5 mg / mL) was added to each well. The cells were incubated in the dark at 37°C for 4 hours, and the absorbance at 570 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability = (OD value of experimental group - OD value of blank control group) / (OD value of negative control group - OD value of blank control group) × 100%.

[0188] The experimental results are as follows Figure 9 As shown, the combination of sericin and GOX enhanced the anticancer activity of GOX against cancer cells. The cell viability of the GOX group was 36.0%, and the cell viability of the GOX+SS group was 2.6%, indicating that the anticancer activity of the combined use of sericin and GOX was about 14 times that of GOX alone.

[0189] Sericin biocompatibility testing

[0190] Example 11: Hemolytic Toxicity Test

[0191] Fresh blood collected from mice was stored at 4°C until use. For testing, sufficient mouse blood was collected, diluted with an appropriate amount of TBS, and centrifuged at 4000 rpm for 3 minutes. The supernatant was discarded, and TBS was added to mix the red blood cells at the bottom. Centrifugation was repeated three times, and then TBS was added to dilute the red blood cells to 5% before use. The sericin (SS) stock solution of the present invention was dissolved in TBS to 4 mg / mL. 100 μL of the sericin (SS) solution to be tested was added to the first row of a 96-well plate. Serial dilutions were performed starting from the second row, using 0.1% TX100 as a positive control and TBS as a negative control. 50 μL of the TBS-diluted red blood cells was added to each well and incubated at 37°C for 1 hour. The 96-well plate was transferred to a centrifuge and centrifuged at 3700 rpm for 5 minutes. 80 μL of the sample was transferred from each well to a fresh 96-well plate and read on a microplate reader at a wavelength of 405 nm. Finally, the hemolysis rate was calculated according to the formula (% hemolysis rate = (OD polymer - OD blank) / (OD control - OD blank) × 100%). In the hemolytic activity test, each sample had two replicates, and the experiment was repeated three times.

[0192] The experimental results showed that the hemolytic activity of sericin (HC 50 ) were all greater than 2000 μg / mL, and they had no serious hemolytic toxicity to red blood cells, indicating that sericin had good biocompatibility.

[0193] Example 12: Cytotoxicity test of sericin on mammalian cells

[0194] Mammalian cells in the logarithmic growth phase, human umbilical vein endothelial cells, were selected. After trypsin digestion, the cells were diluted to 8,000 cells per mL of cell suspension and inoculated into 96-well plates at 100 μL per well, with 3 replicates per group. The cells were cultured overnight in a cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere.

[0195] Experimental Group: Sericin was dissolved in ultrapure water to prepare a 4 mg / mL stock solution, which was then diluted with culture medium to 2 mg / mL. A two-fold concentration gradient dilution was then performed with culture medium to obtain 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, and 31.3 μg / mL. The original culture medium was replaced with culture medium containing the aforementioned sericin concentrations.

[0196] Control group: medium without compound was used as negative control group, and pure DMSO was used as blank control group; after treating cells with different concentrations of compound for 24 hours, the original medium was discarded, and 100 μL of serum-free medium containing 10% μL of cell activity detection reagent MTT solution (5 mg / mL) was added to each well, and the cells were incubated in the dark at 37°C for 4 hours. The absorbance at 570 nm was detected by a microplate reader; the cell viability was calculated using the following formula: cell viability = (OD value of experimental group - OD value of blank control group) / (OD value of negative control group - OD value of blank control group) × 100%.

[0197] The experimental results showed that the half-lethal concentration (IC 50 ) were all higher than 2000 μg / mL, indicating that sericin had no obvious cytotoxicity to mammalian cells and had good biocompatibility.

[0198] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for promoting protein renaturation / refolding, characterized in that: The method comprises: adding sericin to a denatured and inactivated protein, thereby obtaining a renatured / refolded protein.

2. The method according to claim 1, wherein The added amount of the sericin is 0.1 to 200 equiv of the protein.

3. The method according to claim 1, wherein The method comprises the steps of: contacting the denatured and inactivated protein with sericin at 0° C.-50° C. for 8-24 hours.

4. The method according to claim 1, wherein The sericin is naturally occurring sericin extracted from silk.

5. A protein composition, characterized in that The protein composition comprises an effective amount of active protein and sericin for improving the stability of the protein.

6. The protein composition according to claim 5, wherein The protein is selected from the group consisting of horseradish peroxidase, glucose oxidase, β-galactosidase, lysozyme, laccase, pancreatin, alcohol dehydrogenase, acetaldehyde dehydrogenase, formate dehydrogenase, lyticase, uricase, pectinase, tannase, phytase, ribonuclease, xylanase, catalase, lactate dehydrogenase, elastase, transglutaminase, esterase, lipase, α-chymotrypsin, β-glucanase, thrombin, asparaginase, trypsin, protease, proteinase K, papain, pepsin, superoxide dismutase, cytochrome c, xanthine oxidase, carbonic anhydrase , organophosphorus hydrolases, matrix metalloproteinases, bovine serum albumin, human serum albumin, fibrinogen, fibronectin, collagen, bone morphogenetic protein, amyloid protein, myoglobin, actin, lactoferrin, green fluorescent protein, phycocyanin, insulin, glucagon, interferon, interleukin, β-defensin, growth hormone, avidin (also known as avidin or anti-biotin), streptavidin, fibroblast growth factor, colony stimulating factor, tumor necrosis factor, vascular endothelial growth factor, epithelial growth factor, IgM antibody, IgG antibody, IgA antibody, IgE antibody, IgD antibody.

7. A method for improving protein stability, characterized in that: The method comprises: mixing sericin and protein to obtain protein with improved stability; The protein is selected from the group consisting of horseradish peroxidase, D-lactate dehydrogenase, pectinase, elastase, lipase, and α-chymotrypsin.

8. The method according to claim 7, wherein The protein with improved stability refers to the protein at a temperature at which the conformation of the protein begins to change (T onset-ss ) and melting temperature (T m-ss ) were higher than the T values of proteins without adding sericin. onset and T m .

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: glucose oxidase and 0.1 to 200 equiv of sericin.

10. Use of the pharmaceutical composition according to claim 9, characterized in that: It is used to prepare anticancer drugs.

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