Stabilizing complex composition for maintaining activity of protein and preparation method therefor

Abstract

The present invention relates to: a stabilizing complex composition for maintaining the activity of a protein, which can stably maintain an extracellular superoxide dismutase (EC-SOD) protein even at high temperatures for a long period of time; and a preparation method therefor. The present invention is characterized by mixing the EC-SOD protein with ectoine, human recombinant collagen (collagen-1), hyaluronic acid, or sorbitol alone or a mixture thereof, as an active ingredient.

Description

Stabilizing complex composition for maintaining protein activity and method for producing the same

[0001] The present invention relates to a stabilizing complex composition for maintaining the activity of an extracellular superoxide dismutase (EC-SOD) protein, which can stably maintain the activity of the protein for a long period of time even at high temperatures, and a method for producing the same.

[0002] Superoxide dismutase (SOD) is an antioxidant enzyme found in the human body that removes reactive oxygen species and protects cells. Specifically, SOD converts reactive oxygen species into hydrogen peroxide, enabling other antioxidant enzymes to function. SOD is categorized into Cu / Zn SOD, which contains copper and zinc atoms; Mn SOD, which contains manganese atoms; and EC SOD (extracellular superoxide dismutase), which exists on the cell surface or in the extracellular fluid.

[0003] Among these, EC SOD, like Cu / Zn SOD, contains copper and zinc atoms, but has the unique characteristic of having a heparin binding domain at the C-terminus. Because EC SOD has a heparin binding domain, it is presumed that it binds to heparin in the cell membrane and plays a role in protecting the cell membrane.

[0004] According to the literature, EC SOD is known to be responsible for biological defense functions in plasma and extracellular matrix (Marklund et al., Biochem. J. 266, 213-219, 1990; Su et al., Am J Respir Cell Mol Biol., Feb 16(2), 162-70, 1997; Luoma et al., Thromb. Vasc. Bio. 18, 157-167,1998). Other literature has reported that gene therapy using EC SOD improves aortic restenosis in rabbits and alleviates collagen-induced arthritis in mice (Mikko O et al., Circulation, 106, 1999-2003, 2002; Iyama S et al., Arthritis & Rheumatism, 44,9,2160-67, 2001).

[0005] Recently, it has been reported that EC SOD has a cell regenerative function that inhibits telomere shortening, a cell aging phenomenon, in human fibroblasts (Serra V et al., The journal of Biological Chemistry, 278,9, 6824-30, 2003). In addition, it has been reported that the heparin binding domain of EC SOD acts as a nuclear localization signal to localize in the nucleus of thymus and testis cells, protecting genomic DNA from oxidative stress and regulating transcriptional activity sensitive to redox reactions (Ookawara T et al., BBRC, 296, 54-61, 2002). However, the distribution trend of EC SOD in the skin has not been known until now, and the effect of EC SOD on skin diseases has not been known.

[0006] However, human recombinant extracellular superoxide dismutase (EC-SOD), which has been confirmed to have excellent antioxidant function, is known to have problems with stability and activity on its own. Therefore, it has been reported that when mineral elements such as copper (Cu) or zinc (Zn) are combined to improve this stability, it exhibits stable efficacy on its own (MC Carroll, J Biol Chem, v.281, 28648-28656, 2006; IMAhl, Protein Expr Purif, 37, 311-319, 2004; CSHwang, Microbiology, 148, 3705-3713, 2002; H. Walti, Biol Neonate, 82, 96-102, 2002).

[0007] Despite these efforts to improve stability and activity, the instability of EC-SOD still remains, making it difficult to develop it for therapeutic and cosmetic purposes.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] Korean Patent No. 10-1815317 (December 28, 2017)

[0011] The purpose of the present invention is to provide a stabilizing complex composition for maintaining the activity of an extracellular superoxide dismutase (EC-SOD) protein, which can stably maintain the activity of the protein for a long period of time even at high temperatures, and a method for producing the same.

[0012] The stabilizing complex composition for maintaining the activity of a protein according to the present invention is characterized by mixing Ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol as active ingredients, either singly or in combination, with superoxide dismutase (EC-SOD) protein.

[0013] Preferably, the superoxide dismutase protein is characterized by being any one of EC-SOD1, EC-SOD2, and EC-SOD3.

[0014] Preferably, ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol are used alone or in a mixture in an amount of 0.01 equivalent (W / W) or more relative to superoxide dismutase protein.

[0015] In addition, the method for manufacturing a stabilizing complex composition for maintaining protein activity is characterized by adding ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol as active ingredients, alone or in combination, to superoxide dismutase (EC-SOD) protein.

[0016] Preferably, it is characterized by comprising the steps of (a) dissolving superoxide dismutase (EC-SOD) protein in purified water; and (b) dissolving ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol, alone or in combination.

[0017] Preferably, the method further comprises the step of (c) freeze-drying the superoxide dismutase (EC-SOD) protein solution dissolved in step (b); and (d) preparing the solution into powder after freeze-drying.

[0018] Preferably, the stabilizing complex of the superoxide dismutase (EC-SOD) protein is characterized by maintaining protein stability for more than 4 weeks at a temperature of 45°C.

[0019] The stabilizing complex composition for maintaining the activity of a protein according to the present invention has the effect of stably maintaining the activity of extracellular superoxide dismutase (EC-SOD) protein for a long period of time even at high temperatures by using ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol.

[0020] Figure 1 is a graph showing the activity measurement values ​​of SOD-3 after 4 weeks of storage at 45°C as an experimental example of the present invention.

[0021] Hereinafter, the present invention will be described in detail with reference to preferred embodiments. However, those skilled in the art will recognize that the present invention can be variously modified or altered within the scope of the embodiments described below, and that such modifications or variations are also included within the scope of the present invention.

[0022] A stabilizing complex composition for maintaining the activity of a protein according to the present invention is prepared by mixing Ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol, either alone or in combination, with superoxide dismutase (EC-SOD) protein as active ingredients.

[0023] Here, stabilization of the superoxide dismutase (EC-SOD) protein can be considered as a protein stabilization even when the activity of the protein is lower than in the native state, or when the decrease in protein activity is smaller during preservation.

[0024] The superoxide dismutase (EC-SOD) protein of the present invention refers to a natural or recombinant EC SOD protein or a protein having substantially equivalent physiological activity thereto. Proteins having substantially equivalent physiological activity include chemically synthesized functional equivalents and functional derivatives of natural / recombinant EC SOD proteins. The superoxide dismutase protein of the present invention uses EC-SOD, EC-SOD1, EC-SOD2, and EC-SOD3, and EC-SOD3 is particularly preferred.

[0025] Ectoin of the present invention has the chemical formula C6H 10 It is an organic compound that occurs naturally in bacteria and contains N2O2. In particular, ectoine can be obtained from various bacteria in the Halomonadaceae or Firmicutes genus. In the present invention, ectoine is mixed in an amount of 0.01 equivalents (W / W) or more, preferably 0.5 to 2.5 equivalents (W / W) relative to SOD.

[0026] Furthermore, human recombinant collagen (Collagen-1) is the most abundant type of collagen in humans, and collagen type 1 is the dominant structural protein in bone (more than 90% of the organic mass), tendons, ligaments, cornea, and many interstitial connective tissues. Structurally, collagen type 1 is composed of two identical α-1 type 1 chains and α-2 type 1 chains in a triple helix structure. In the present invention, human recombinant collagen is mixed in an amount of 0.01 equivalent (W / W) or more, preferably 0.5 to 2.5 equivalents relative to SOD.

[0027] Hyaluronic acid (HA) is a biodegradable polymer approved for injection into the body, and is a substance derived from a living organism. The hyaluronic acid contained in the composition of the present invention may be any of hyaluronic acid purified from the body fluid or tissue of a mammal, including a human, hyaluronic acid obtained by culturing a recombinant microorganism or cell, or hyaluronic acid obtained by a chemical synthesis method. The weight average molecular weight of hyaluronic acid may be in the range of about 200,000 to 400,000 Da, and may be used by appropriately changing the concentration. In particular, hyaluronic acid is mixed in an amount of 0.01 equivalent (W / W) or more, preferably 0.5 to 2.5 equivalents relative to SOD.

[0028] Sorbitol is a sugar alcohol with six hydroxyl groups, also called D-sorbitol, D-sorbitol, or D-glucitol. This sorbitol is mixed in an amount of 0.01 equivalent (W / W) or more, preferably 0.5 to 2.5 equivalents relative to SOD.

[0029] Meanwhile, the present invention provides a method for producing a stabilizing complex composition for maintaining the activity of a protein by adding ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol as active ingredients, either singly or in combination, to superoxide dismutase (EC-SOD) protein.

[0030] At this time, it is manufactured by (a) dissolving superoxide dismutase (EC-SOD) protein in purified water; (b) dissolving ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol, alone or in combination. In addition, it may further include (c) a step of lyophilizing the superoxide dismutase (EC-SOD) protein solution dissolved in step (b); and (d) a step of manufacturing the solution into a powder after lyophilization. At this time, ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol, alone or in combination, are mixed in an amount of 0.01 equivalent (W / W) or more, and preferably, 0.5 to 2.5 equivalents, relative to SOD. The stabilized complex of superoxide dismutase (EC-SOD) protein thus produced can maintain protein activity for approximately 4 weeks or more at a temperature of approximately 45°C. Furthermore, since the complex of superoxide dismutase protein can maintain activity even after lyophilization, lyophilization facilitates storage and handling.

[0031]

[0032] Hereinafter, the present invention will be described in detail based on examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0033] <SOD-3 안정화 복합체의 제조>

[0034] 1 wt% of SOD-3 was dissolved transparently in purified water, 1 wt% of Sorbitol was added, and the mixture was freeze-dried to prepare a powder-like EC-SOD / Sorbitol complex of Example 1. In addition, Ectoine was added under the same conditions to prepare an EC-SOD / Ectoine complex of Example 2, hyaluronic acid (HA) was added under the same conditions to prepare an EC-SOD / HA complex of Example 3, and collagen-1 was used under the same conditions to prepare an EC-SOD / Collagen-1 complex of Example 4. In addition, as a comparative example, trehalose used as a stabilizer was added under the same conditions as in the example to prepare an EC-SOD / trehalose complex of Comparative Example 1.

[0035] <SOD-3 의 열적 안정성 실험 >

[0036] In order to comparatively evaluate the thermal stability of the SOD-3 stabilizing complexes manufactured in Examples 1 to 4 and Comparative Example 1, the SOD-3 complexes were transparently dissolved in purified water at a standard concentration of 1% SOD-3 (W / W) and placed in an oven at 45°C for 4 weeks, and the activity of the SOD-3 stabilizing complexes at each week was compared with the activity of the standard solution (Comparative Example). In Comparative Example 2, 1% (W / W) of SOD-3 was transparently dissolved in purified water and placed in an oven at 45°C for 4 weeks, and the activity of SOD-3 at each week was measured.

[0037] To measure the activity of SOD-3 protein, a commercially available SOD activity assay kit (Abcam) was used and the activity was measured using the following method.

[0038] 1) Prepare the WST working solution by diluting it in the buffer solution at a ratio of 1 / 10.

[0039] 2) Prepare xanthine oxidase by diluting it in enzyme dilution solution at a ratio of approximately 1 / 100.

[0040] 3) Prepare each SOD solution sample by diluting it to 1 / 00 ​​in DW.

[0041] 4) In a 96-well plate, react 200 ul / well of WST working solution, 20 ul / well of SOD solution sample dilution or DW, and 20 ul of xanthine oxidase enzyme working solution.

[0042] 5) After reacting for 20 to 30 minutes in an incubator (LabTech) set to 37℃, measure OD 450.

[0043] 6) Measure the percentage of residual SOD activity by comparing the SOD activity in each SOD solution with the activity of the freshly dissolved SOD solution as 100%.

[0044]

[0045] <Activity measurement results>

[0046] The activity results for the SOD-3 stabilization complex were confirmed after 4 weeks in an oven at 45°C for Examples 1 to 4 and Comparative Examples 1 and 2, and are shown in the graphs in Table 1 and Fig. 1.

[0047] Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 SOD-3 / Sorbotol complex SOD-3 / Ectoine complex SOD-3 / HA complex SOD-3 / Collagen-1 complex SOD-3 / Trehalose complex SOD-3 alone Activity 80% 97% 85% 93% 63% 65%

[0048] As shown in Table 1 and Fig. 1, the activity of the SOD-3 stabilizing complex was measured in the order of Ectoine > Collagen-1 > HA > Sorbitol > Trehalose, and it was confirmed that the SOD-3 / Ectoine complex showed the best stability. In particular, in the case of the SOD-3 alone sample, the activity value decreased to 65% of the initial value, but Ectoine and Collagen-1 maintained very high activities of 97% and 93%, respectively, and HA and Sorbitol maintained higher activities of 85% and 80%, respectively, than when SOD-3 was used alone. In this way, it was confirmed that the use of Ectoine, Collagen-1, HA, and Sorbitol could maintain high activity of the EC-SOD protein even for a long period of time, more than 4 weeks, at high temperatures.

Claims

1. A stabilizing complex composition for maintaining the activity of a protein, characterized by mixing ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol as active ingredients, alone or in combination, with superoxide dismutase (EC-SOD) protein.

2. A stabilizing complex composition for maintaining the activity of a protein, characterized in that in claim 1, the superoxide dismutase protein is any one of EC-SOD, EC-SOD1, EC-SOD2, and EC-SOD3.

3. A stabilizing complex composition for maintaining the activity of a protein, characterized in that in claim 1, ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol are used alone or as a mixture in an amount of 0.01 equivalent (W / W) or more relative to superoxide dismutase protein.

4. A stabilizing complex composition for maintaining the activity of a protein, characterized in that in claim 1, ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol are mixed alone or as a mixture in an amount of 0.5 to 2.5 equivalents relative to superoxide dismutase protein.

5. A method for producing a stabilizing complex composition for maintaining the activity of a protein, characterized by adding ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol as active ingredients, alone or in combination, to an EC-SOD protein.

6. A method for producing a stabilizing complex composition for maintaining the activity of a protein, characterized in that the composition comprises the steps of: (a) dissolving superoxide dismutase (EC-SOD) protein in purified water; and (b) dissolving ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol, alone or in combination, in an amount of 0.01 equivalent (W / W) or more relative to the EC-SOD protein.

7. A method for producing a stabilizing complex composition for maintaining the activity of a protein, characterized in that in claim 6, ectoine, human recombinant collagen (Collagen-1), hyaluronic acid, and sorbitol are mixed alone or in a mixture in an amount of 0.5 to 2.5 equivalents relative to superoxide dismutase protein.

8. A method for producing a stabilizing complex composition for maintaining the activity of a protein, characterized in that in claim 5, further comprises the step of (c) freeze-drying the superoxide dismutase (EC-SOD) protein solution dissolved in step (b); and (d) manufacturing the solution into a powder after freeze-drying.

9. A method for producing a stabilizing complex composition for maintaining the activity of a protein according to claim 5, wherein the superoxide dismutase protein is any one of EC-SOD, EC-SOD1, EC-SOD2, and EC-SOD3.

10. A method for producing a stabilizing complex composition for maintaining the activity of a protein, characterized in that the stabilizing complex of the superoxide dismutase (EC-SOD) protein in claim 5 maintains protein stability for 4 weeks or more at a temperature of 45°C.

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