Universal protein renaturation method and application thereof

Abstract

The present invention provides a universal protein renaturation method and an application thereof, and relates to the technical field of protein renaturation. The method includes the following steps: (1) add 2mercaptoethanol to urea solution to obtain denaturing solution; (2) dissolve a sample including to-be-renatured protein by using the denaturing solution and then freeze the sample, to obtain a frozen body; (3) thaw the frozen body at room temperature, and extract the thawed liquid as soon as the liquid is thawed; (4) dilute the extracted liquid with water, to terminate renaturation and obtain supernatant and precipitate through centrifugation, where the supernatant includes the protein that has been successfully renatured. According to the present invention, protein renaturation recovery efficiency can be improved, and a production batch is stable and reproducible. Operations in the present invention are simple, almost no waste liquid is produced, and no protein is lost. According to the present invention, nine allergen proteins are renatured, to significantly improve the protein renaturation yield. In addition, compared with that in the conventional technology, the protein activity according to the present invention is increased by about 3 times or more.

Description

TECHNICAL FIELD The present invention relates to the technical field of protein renaturation, in particular to a universal protein renaturation method and an application thereof. BACKGROUND A prokaryotic expression system with Escherichia coli as a host cell is currently the most widely used method for gene cloning, offering advantages such as simple operation, rapid growth, low cost, and high yield. However, the biggest problem with the expression system is that expression products are often insoluble and biologically inactive inclusion bodies, and additional steps of denaturation-renaturation and further purification need to be performed to obtain high-purity active proteins. The inclusion body is solid aggregates of amorphous proteins with membrane structure formed by intracellular aggregation, including some DNA, RNA and other bacterial proteins in addition to inactive recombinant proteins. The formation of the inclusion body can avoid proteases in the cell from degrading the protein, to protect host cells from some toxic protein products, and facilitate protein separation. The primary structure (that is, amino acid sequence) of the protein in the inclusion body is correct, but the three-dimensional structure is wrong, so the inclusion body is not biologically active. To obtain a target protein product that is active in a native state, it is necessary to dissolve the inclusion body and restore the native conformation and activity of the target protein after isolating and recovering the inclusion body. However, the spontaneous protein renaturation efficiency is extremely low, and due to the large differences in physicochemical properties among different proteins, different proteins have different renaturation methods. In the conventional technology, most research is on the study of protein renaturation of a specific kind, while few studies addressing the renaturation of various allergen proteins. It is shown in the conventional technology that protein folding is a physical process that can be performed in test tubes. Anfisen has observed that fully reduced ribonuclease A can spontaneously fold into the native conformation with full activity in vitro. The primary structure of a protein includes all information that guides formation 1 of the native conformation, and the native structure may be at the lowest point of total free energy. The method of protein folding mechanisms is based on the classical "folding pathway" model, in which denatured protein reaches a native state in an order of folding intermediates through a determined pathway. In the past, due to little understanding of the free energy of conformational stability, it was difficult to give a conformational energy factor to determine the activity of proteins. In addition, the possible conformational space was very large and complex, so it was difficult in both theory and practice to predict the folding conformation of protein molecules directly from the amino acid sequence. Recently, with the large-scale application of Al technology represented by AlphaFold 3, folding structure of nearly 200,000 proteins are successfully predicted and resolved, so the field now faces a significant bottleneck in transitioning from calculation to production. Therefore, there is an urgent need to obtain a renaturation method simultaneously applicable to various proteins, to facilitate the further application of proteins. In addition, in the patent application (CN 106243186 B) of "cyclic operation method for independent use in protein renaturation or as precursor step for protein renaturation", a protein renaturation method is provided. According to the method, a denaturant is gradually crystallized and precipitated from the solution at a low- temperature environment, and the solution with a decreasing concentration gradient of the denaturant creates a smooth denaturation salt concentration gradient, to further promote protein renaturation. Although the protein with successful renaturation can be successfully obtained, the applicant aims to simplify the renaturation method and further improve the protein renaturation efficiency. SUMMARY The present invention aims to provide a universal renaturation method applicable to a variety of proteins, to improve protein renaturation efficiency, and improve a protein recovery rate. To achieve the foregoing invention objectives, the following technical solutions are provided in the present invention. The present invention provides a universal protein renaturation method, including the following steps: (1) add 2mercaptoethanol to urea solution to obtain denaturing solution; 2 (2) dissolve a sample including to-be-renatured protein by using the denaturing solution and then freeze the sample, to obtain a frozen body; (3) thaw the frozen body at room temperature, and extract the thawed liquid as soon as the liquid is thawed; (4) dilute the extracted liquid with water to urea concentration of less than 2M, to terminate renaturation and obtain supernatant and precipitate through centrifugation, where the supernatant includes the protein that has been successfully renatured. Preferably, the urea concentration of the urea solution in step (1) is greater than 7M. Preferably, a volume ratio of the urea solution and the 2-mercaptoethanol is: 1 mL: 0.8~4 pL. Preferably, in configuring the urea solution in step (1 ), 10~ 30% volume of glycerol is first added in water, and then urea is added, to obtain the urea solution. Preferably, the sample including the to-be-renatured protein in step (2) is a protein inclusion body; and a mass-volume ratio of the protein inclusion body (wet weight) and the denaturing solution is: 0.5~4 g: 50 mL. Preferably, the freezing temperature in step (2) is -80~-8 °C. Preferably, the room temperature in step (3) is 18~26 °C. Preferably, when diluting with the water to terminate renaturation in step (4), a volume ratio of the water and the extracted liquid is 2~4:1, and the urea concentration in the diluted liquid is not more than 2M. Preferably, the speed of the centrifugation is 8,000~22,000 rpm, and the time is 8~15 min. Preferably, after step (4) is performed, the precipitate obtained in step (4) is used as the sample including the to-be-renatured protein, and step (2) to step (4) are repeated to achieve a renaturation cycle. The present invention provides the application of the universal protein renaturation method in protein renaturation. Preferably, the protein is an allergen. Preferably, the allergen protein includes Bos d 4, Bos d 5, Pen a 1, Der p 1, Amb a 1, Phi p 12, Chea 1, Betv 1, and Plaa 1. According to the present invention, matters undergo complete changes in a frozen state, the solution becomes solid, and there is no concentration concept at all. 3 The concept is replaced by "abundance". In this case, the concentration of urea (denaturant) drops to zero, resulting in the complete loss of protein denaturation ability. Consequently, polypeptide chains will either naturally fold into the protein of natural formation, or misfold into the structurally defective protein. In other words, the state represents an end. During the heating process, the frozen body begins to thaw and the urea redissolves, and denaturation is performed again. This is understood as a reverse process of freezing, and the sooner the solution is extracted, the better the performance. However, because the method for extracting the solution by inserting the needle along the wall is earlier and faster than the method for directly dumping the solution after the solution is thawed, urea crystals are not easy to redissolve into the liquid. This significantly enhances protein renaturation recovery efficiency while improving batch stability and reproducibility in production. Operations in the present invention are simple, almost no waste liquid is produced, and no protein is lost. According to the present invention, proteins are renatured, to significantly improve the protein renaturation recovery efficiency. In addition, compared with that in Comparative Example, the protein activity according to the present invention is increased by about 3 times or more. DETAILED DESCRIPTION OF EMBODIMENTS The technical solutions according to the present invention are described in detail below with reference to embodiments, but the technical solutions constitute no limitation on the protection scope of the present invention. Example 1 A universal protein renaturation method is provided, including the following steps. (1) Prepare saturated urea solution (with urea solution concentration of 8M) with water including 10% volume of glycerol, and then add 2-mercaptoethanol at a ratio of 1 mL of urea solution: 1 pL of dithioethanol, to obtain denaturing solution. (2) Dissolve an inclusion body of an allergen protein by using the denaturing solution in step (1 ), with 2 g of the inclusion body dissolved by using per 50 mL of the denaturing solution, and then freeze the mixed solution in the refrigerator at -35 °C until a frozen body becomes solid. (3) Take out the frozen body from the refrigerator, put the frozen body in a centrifuge tube, and thaw the frozen body at room temperature (20 °C) in the 4 laboratory. Extract the thawed liquid by using a syringe as soon as the liquid begins to thaw, where the needle should be inserted as deep as possible against the wall of the centrifuge tube when the liquid is extracted. (4) Rapidly add twice the volume of distilled water after the liquid is extracted, dilute the liquid to terminate renaturation (in this case, the urea concentration in the solution drops to about 1M), with centrifugation at 20,000 rpm for 10 min, to obtain supernatant and precipitate, where the supernatant includes allergen proteins that have successfully renatured and can be used for activity assay. The centrifuged precipitate can be redissolved with the denaturing solution and mixed into a new inclusion body solution to achieve a renaturation cycle. Comparative Example 1 Different from Example 1, step (3) is: take out the frozen body from the refrigerator, put the frozen body in a centrifuge tube, and thaw the frozen body at room temperature (22 °C). After the frozen body is thawed, the liquid obtained by thawing is directly dumped into 4 times the volume of water for dilution, to terminate renaturation, and then the liquid is centrifuged at 20,000 rpm for 10 min, to obtain supernatant, where the supernatant includes allergen proteins that have been successfully renatured and can be used for activity assay. Test Example: Nine allergen proteins, namely, Bos d 4, Bos d 5, Pen a 1, Der p 1, Amb a 1, Phi p 12, Che a 1, Bet v 1, and Pla a 1 are separately renatured in the method of Example 1 and Comparative Example 1. Each protein is tested twice in the group, the supernatant obtained through centrifugation is used as the raw protein for activity assays, and the Blank group is a blank control group with single addition of serum. The activity assay results are shown in Table 1. 1. Activity Assay Method: 1. Elisa plate coating 1.1. Dilution: Dilute the raw protein by 50 or 100 times. 1.2. Coating: Add 100 pL per well in a 96-well reaction plate (elisa plate) and refrigerate at 6 °C for 20H. 1.3. Washing: Take out the elisa plate and wash 3 times by using the washing solution. 1.4. Blocking: Add 200 pL of blocking solution to each well and put it in a 37 °C oven at a constant temperature for 2H. 5 1.5. Washing: Take out the elisa plate, wash the elisa plate 3 times, and set aside. 2. Reagent preparation 2.1. Washing solution Dilute the concentrated washing buffer (20X) 20 times, that is, take one part (one volume of concentrated washing solution (20X)) and add 19 parts of distilled water, mix the solution and the water well and set aside. 2.2. HRP-Linked Secondary Antibody (ready-to-use) Prepare HRP-linked secondary antibody solution A and HRP-linked secondary antibody solution B at a ratio of 1:9, mix the solution A and the solution B well and set aside. 2.3. Sample serum preparation Dilute the sample serum 100-fold with sample diluent. The reagents used in Test Sample are all commercially available products of Hangzhou ZheDa Dixun Biological Gene Engineering Co., Ltd. 3. Assay 3.1. Pipette 100 L of diluted sample and add the sample to the corresponding elisa plate wells and mix gently for 103. Incubate the sample for 30 min at room temperature. 3.2. Wash the plate: Pour all the liquid in the plate, add 250 L of diluted washing solution to wash the elisa plate, and then pour out the liquid. Wash 5 times repeatedly, and finally turn the elisa plate over and pat the elisa plate dry on a thick stack of absorbent paper. 3. Add 100 L of prepared HRP-linked secondary antibody solution to each well and incubate the solution at room temperature for 30 min. 3.4. Washing: Same as 3.2 operation. 3.5. Color development: Add 100 L of TMB substrate chromogenic solution to each well, and carry out the reaction at room temperature in the dark for 10 min. 3.6. Termination: Add 100 L of terminator solution to each well. 3.7. Assay: Measure the absorbance (A450 nm) of each well at a wavelength of 450 nm by using a standard microplate reader. 2. Activity Assay Results 6 Table 1 Comparison of OD values of Different Proteins Recovered by Different Methods Protein Coating Comparative I-W 5 Amb a 1 / 50 II-III-I Phl p 1 / 50 ÍI-IIII- n---- Comparing the present invention with Comparative Example 1, the activity of the allergen protein obtained by the refoIding method according to the present application is more than 3 times the activity of the allergen protein obtained by the direct dumping method according to Comparative Example 1. The above descriptions are merely the preferred embodiments of the present invention. It should be noted that, for a person of ordinary skill in the art, various modifications and refinements may be made without departing from the principles of the present invention, and the modifications and refinements shall fall within the protection scope of the present invention. 7

Claims

1. A method for universal protein renaturation, characterized in the sense that it the includes steps of: (1) Adding 2-mercaptoethanol to a urea solution to a to obtain a denaturing solution; (2) The use of the denaturing solution to dissolve a sample that the protein to be renatured contains and subsequently to freeze it to a to obtain a frozen body; (3) Allow the said frozen body to melt at room temperature and the extract molten liquid as soon as the liquid melts out; (4) Rapidly diluting the extracted liquid with water, the ending the renaturation, centrifuging, obtaining a supernatant and a precipitate, in which the said supernatant the successful contains renatured protein.

2. The method for universal protein renaturation as asserted in claim 1, characterized in the sense that the urea concentration of the mentioned urea solution in step (1) is greater than 7M. The volume ratio of the said urea solution and 2-mercapto- ethanol is: 1 ml: 0.8-4 µL.

3. The method for universal protein renaturation as asserted in claim 2, characterized in the sense that in the preparation of the said urea solution in step (1), 10-30% volume glycerol is first added to water, and Subsequently, urea is added to obtain a urea solution.

4. The method for universal protein renaturation as asserted in claim 3, characterized in the sense that the said sample that the sample that the in step (2) contains protein to be renatured, is a protein inclusion body; the amount protein inclusion body is measured by wet weight, and the mass-volume ratio of the said protein inclusion body to the said denaturing solution is: 05-49: 50 ml; The freezing temperature mentioned in step (2) is -80-8 °C.

5. The method for universal protein renaturation as asserted in claim 4, characterized in the sense that the room temperature in step (3) is 18-26°C.

6. The method for universal protein renaturation as asserted in claim 5, 8 characterized in the sense that in step (4), when diluting with water to the to terminate renaturation, the volume ratio of water to the mentioned extracted liquid is 2-4:1, and the urea concentration in the liquid after dilution is not greater than 2M.

7. The method for universal protein renaturation as asserted in claim 6, characterized in the sense that the centrifugation speed is 8,000-22,000 rpm and time 8-15 min.

8. The method for universal protein renaturation as asserted in claim 7, characterized in the sense that after completing step (4), the precipitate obtained in step (4) is used as a sample that contains the protein to be renatured, and steps (2) to (4) are repeated to obtain a renaturation cycle.

9. The application of the method for universal protein renaturation as claimed in claims 1-8 in protein renaturation.

10. The application as asserted in claim 9, characterized in the sense that the said protein is an allergen protein; The mentioned allergen protein comprises Bos d 4, Bos d 5, Pen a 1, Der p 1, Amba 1, Phl p 12, Chea 1, Betv1 and Plaa 1. 9 PATENT APPLICATION NO.: NO 200058 RESEARCH REPORT CONCERNING THE RESULT OF THE STATE OF THE ART RESEARCH RELEVANT LITERATURE 1 Literature with, where necessary, indication of of particular importance for Classification (IPC) Category text sections or figures. conclusion(s) no: A CN 106 243 186 B ​​(ZHANG PENG) 1-10 INV. December 25, 2020 (2020-12-25) C07K1 / 113 * the entire document * C07K1 / 14 ----- Investigated areas of technology C07K If amended conclusions have been submitted, this report relates to the conclusions submitted on: Place of investigation: Date on which the investigation was conducted Competent official: completed: The Hague March 17, 2026 Schleifenbaum, A 1 CATEGORY OF THE SIGNED LITERATURE X: the conclusion is deemed not new or not inventive T: after the filing date or the priority date considered in relation to this literature published literature that is not detrimental to the patent application, but is mentioned for clarification of Y: the conclusion is considered non-inventive at 1 the theory or principle that underlies the in relation to the combination of this literature with other cited literature of the same category, invention where the combination is obvious to the skilled person E: earlier patent (application), published on or after the is deemed to be the filing date on which the same invention is described A: literature not belonging to category X or Y that the D: stated in the patent application describes the state of the art O: non-written state of the art L: literature mentioned for other reasons P: between the priority date and the filing date &: member of the same patent family or corresponding published literature patent publication EOB FORM 02.83 (P0414B) APPENDIX TO THE REPORT CONCERNING THE RESEARCH INTO THE STATE OF THE ART, NO 200058 CARRIED OUT IN PATENT APPLICATION NO. The appendix contains a list of patent applications or patents published elsewhere (so-called members of the same patent family), that correspond to patent specifications mentioned in the report. The statement has been compiled based on data from the European Patent Office's computer file as of The accuracy and completeness of this statement is guaranteed neither by the European Patent Office nor by the Industrial Office. Property guaranteed; the data is provided for informational purposes. 17-03-2026 In the report Date of Corresponding Date of mentioned patent document publication document(s) publication CN 106243186 B ​​25-12-2020 NONE ----------------------------------------------------------------------- General information regarding this appendix has been published in the 'Official Journal' of the European Patent Office No. 12 / 82, pp. 448 et seq. WRITTEN OPINION FILE NUMBER SUBMISSION DATE PRIORITY DATE APPLICATION NUMBER NO200058 09.07.2025 06.12.2024 CLASSIFICATION INV. C07K1 / 113 C07K1 / 14 APPLICANT Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd. This written opinion contains an explanation of the following sections: Part I Basis of the written opinion Part II Priority Part III Determination of novelty, inventiveness and industrial applicability not possible Part IV The application relates to more than one invention Part V Reasoned statement regarding novelty, inventiveness and industrial applicability Part VI Other cited documents Part VII Other defects Part VIII Other remarks THE COMPETENT OFFICIAL Schleifenbaum, A Application no.: WRITTEN OPINION Part I Basis of the Written Opinion 1. This written opinion has been prepared on the basis of the most recent conclusions submitted prior to the commencement of the research.

2. This justification has been drawn up with respect to nucleotide and / or amino acid sequences mentioned be in the application, based on a sequence list that: a. is included in the application as originally submitted b. has been submitted after the submission date for the purpose of the research and was accompanied by a statement that the sequence list contains no more information than the application as originally submitted.

3. This justification has been drawn up with respect to nucleotide and / or amino acid sequences mentioned be included in the application, insofar as a meaningful justification could be formed without a sequence list that complied with WIPO standard ST.

26.

4. Other remarks: Part V Reasoned statement regarding novelty, inventiveness and industrial applicability 1. Explanation Novelty Yes: Conclusions 1-10 No: Conclusions Inventiveness Yes: Conclusions 1-10 No: Conclusions Industrial applicability Yes: Conclusions 1-10 No: Conclusions 2. Citations and comments: See separate page