Recombinant BMP4 protein renaturation solution and preparation method of recombinant BMP4 protein

Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant BMP4 protein renaturation solution and a preparation method of a recombinant BMP4 protein. On one hand, the invention provides a recombinant BMP4 protein renaturation solution, and the recombinant BMP4 protein renaturation solution is characterized in that the renaturation solution is prepared from the following components in concentration: Tris-HCl with the concentration of 46 to 52 mM, EDTA (Ethylene Diamine Tetraacetic Acid) with the concentration of 4 to 6 mM, Arg with the concentration of 0.48 to 0.54 M, CHAPS with the concentration of 30 to 35 mM, GSSG with the concentration of 1.5 to 2.5 mM, GSH with the concentration of 0.8 to 1.2 mM, and TritonX-114 with the concentration of 0.3 to 0.6 percent by volume. On the other hand, the invention also provides a renaturation method of the recombinant BMP4 protein by adopting the renaturation liquid and a preparation method of the recombinant BMP4 protein. The composition proportion of the recombinant BMP4 protein renaturation liquid is designed, and a specific proportion design is adopted, so that the renaturation rate of the denatured recombinant BMP4 protein can be greatly improved, and the activity of the recombinant BMP4 protein can be effectively maintained.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a recombinant BMP4 protein renaturing solution and a method for preparing the recombinant BMP4 protein. Background Art

[0002] BMP4 protein is an important growth factor belonging to the TGF-β superfamily. It plays a key role in biological processes such as embryonic development, bone formation and tissue repair. It participates in the regulation of cell proliferation, differentiation and apoptosis by activating Smad protein and MAPK pathway. It has extensive medical research and regenerative medicine application potential and is closely related to the development of many human diseases. Therefore, BMP4 protein has important research and application value in the biomedical field.

[0003] At present, commercially available recombinant BMP4 usually comes from NS0 and HEK293 cell lines, but there are problems of low yield and high cost. Due to its cystine knot structure, BMP4 is produced in the form of inactive aggregates in prokaryotic expression systems such as Escherichia coli. The refolding process must obtain correctly folded monomers and successful protein dimers to form a cystine knot, which makes renaturation more difficult. Therefore, most BMP4 proteins expressed in E. coli contain a His tag and are obtained by denaturation purification without renaturation. At the same time, the renaturation time of the BMP4 recombinant protein expressed in E. coli is relatively long, and the final renaturation protein yield is less than 15%.

[0004] Therefore, there is an urgent need to provide a method for improving the renaturation rate of BMP4 recombinant protein. Summary of the invention

[0005] In order to overcome the above technical problems, the present application provides a preparation process for a recombinant BMP4 protein with a high renaturation rate and a high activity of the prepared recombinant BMP4 protein.

[0006] On the one hand, the present application provides a recombinant BMP4 protein refolding solution, which is composed of the following components in concentration: 46-52mM Tris-HCl, 4-6mM EDTA, 0.48-0.54M L-arginine (Arg), 30-35mM CHAPS, 1.5-2.5mM oxidized glutathione (GSSG), 0.8-1.2mM reduced glutathione (GSH), and 0.3-0.6% volume percentage concentration of TritonX-114.

[0007] By adopting the above technical solution, the present application designs the component ratio of the recombinant BMP4 protein renaturation solution. The specific ratio design can greatly improve the renaturation rate of the denatured recombinant BMP4 protein and effectively maintain the activity of the recombinant BMP4 protein.

[0008] Furthermore, the concentrations of the components are: 50-52 mM Tris-HCl, 4-6 mM EDTA, 0.48-0.54 M L-arginine (Arg), 33-35 mM CHAPS, 1.5-2.5 mM oxidized glutathione (GSSG), 0.8-1.2 mM reduced glutathione (GSH), and 0.3-0.6% volume percent concentration of TritonX-114.

[0009] Furthermore, the concentrations of the components are: 50 mM Tris-HCl, 5 mM EDTA, 0.5 M L-arginine (Arg), 33 mM CHAPS, 2 mM oxidized glutathione (GSSG), 1 mM reduced glutathione (GSH), and 0.4% volume percent concentration of TritonX-114.

[0010] Furthermore, the pH value of the Tris-HCl is 8.5.

[0011] On the other hand, the present application also provides a method for renaturing a recombinant BMP4 protein, using the recombinant BMP4 protein renaturing solution.

[0012] Furthermore, the method comprises the following steps: S1. In an ice water bath, adding the denatured recombinant BMP4 protein to the recombinant BMP4 protein renaturation solution to obtain a renaturation reaction solution; S2, gradually heating the renaturation reaction solution obtained in step S1 to 10° C. under stirring, and then continuing to keep the temperature under stirring to react, thereby obtaining renatured recombinant BMP4 protein; Furthermore, in step S2, the total duration of the gradual heating and heat preservation reaction is controlled to be more than 72 hours.

[0013] By adopting the above technical scheme, the present application designs a method for renaturing recombinant BMP4 protein. The use of the above recombinant BMP4 protein renaturing liquid not only effectively ensures that the renaturation treatment has a high renaturation rate, but also ensures that the recombinant BMP4 protein obtained by the renaturation treatment has a high activity.

[0014] Furthermore, in the step S1, the added concentration of the denatured recombinant BMP4 protein is not greater than 0.1 μg / μl.

[0015] Furthermore, in the step S1, the temperature of the renaturation reaction solution is controlled at 4°C.

[0016] Further, in step S2, the gradual heating is carried out in a uniform heating manner, and the duration of the gradual heating is controlled within 20-32 hours. Preferably, the duration of the gradual heating is controlled within 24-32 hours or 24-28 hours. More preferably, the duration of the gradual heating is controlled within 24 hours, 28 hours or 32 hours.

[0017] On the other hand, the present application also provides a method for preparing a recombinant BMP4 protein, comprising the following steps: a. Synthesize the recombinant BMP4 protein with optimized base sequence into a vector of E. coli expression system; b. Transforming the vector containing the recombinant BMP4 protein synthesized in step a into competent cells of Escherichia coli, culturing and inducing expression, collecting the cells, lysing and purifying them to obtain denatured recombinant BMP4 protein; c. renaturing the denatured recombinant BMP4 protein obtained in step b by the renaturation method to obtain a renaturation mixed solution; d. The renaturation mixture obtained in step c is centrifuged and purified to obtain recombinant BMP4 protein.

[0018] Furthermore, in the step d, the purification method comprises the following steps: d1. The crude recombinant BMP4 protein obtained by centrifugation was dialyzed into 20 mM Tris-HCl, pH=8.0, 4 M Urea buffer in an ice water bath, and enriched by an sp column to obtain enriched protein; d2, dialyzing the enriched protein obtained in step d1 again into 20 mM Tris-HCl, pH=8.0, 4 M Urea buffer to obtain a secondary dialyzed protein; d3. The secondary dialyzed protein obtained in step d2 is separated and purified using a heparin column to obtain a pure recombinant BMP4 protein.

[0019] By adopting the above technical scheme, the present application has designed a method for preparing recombinant BMP4 protein using the above renaturation treatment scheme of the present application. Through specific renaturation treatment steps and purification steps, in addition to effectively improving the purity of the prepared recombinant BMP4 protein, the preparation yield is also high.

[0020] On the other hand, the present application also provides the use of the recombinant BMP4 protein renaturation solution in the renaturation of recombinant BMP4 protein or in the preparation of recombinant BMP4 protein.

[0021] Compared with the existing technology, 1) The present application designs a component ratio of a recombinant BMP4 protein refolding solution. The specific ratio design can significantly increase the refolding rate of denatured recombinant BMP4 protein to more than 29%, and can effectively maintain the activity of the recombinant BMP4 protein.

[0022] 2) The present application designs a method for renaturing recombinant BMP4 protein. The use of the above-mentioned recombinant BMP4 protein renaturing solution not only effectively ensures that the renaturation treatment has a high renaturation rate, but also ensures that the recombinant BMP4 protein obtained by the renaturation treatment has a high activity.

[0023] 3) The present application has designed a method for preparing recombinant BMP4 protein using the above-mentioned renaturation treatment scheme of the present application. Through specific renaturation treatment steps and purification steps, in addition to effectively improving the purity of the prepared recombinant BMP4 protein, the preparation yield is also high. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the SDS-PAGE detection band diagram of the denatured recombinant BMP4 protein prepared in the examples of this application.

[0025] Figure 2 It is a bar graph showing the test results of the renaturation rate (%) of the recombinant BMP4 protein in Comparative Examples 1-4.

[0026] Figure 3 It is a line graph of the detection results of the renaturation rate (%) of the recombinant BMP4 protein in Examples 4-7 and Comparative Examples 5-7.

[0027] Figure 4 The results of the activity test of the recombinant BMP4 protein of Example 9 and a commercially available competitor are shown. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, conditions described in laboratory manuals or conditions recommended by manufacturers, and all experimental raw materials are sourced from commercially available sources.

[0029] Preparation Example Preparation of denatured recombinant BMP4 protein a. Synthesize the recombinant BMP4 protein with optimized base sequence into a vector of E. coli expression system; b. The vector containing the recombinant BMP4 protein synthesized in step a is transformed into competent cells of Escherichia coli, cultured, induced for expression, and the cells are collected, lysed, and purified to obtain denatured recombinant BMP4 protein, the SDS-PAGE detection band of which is as follows: Figure 1 shown.

[0030] Examples 1-3 Examples 1 to 3 of the present application are examples of the recombinant BMP4 protein renaturation solution of the present application, and the raw materials used are all commercially available.

[0031] Example 1 The recombinant BMP4 protein refolding solution of this embodiment has a concentration ratio of each component of the refolding solution: 46mM Tris-HCl (pH=8.5), 4mM EDTA, 0.48M L-arginine (Arg), 30mM CHAPS, 1.5mM oxidized glutathione (GSSG), 0.8mM reduced glutathione (GSH), and 0.3% volume percentage concentration of TritonX-114.

[0032] Example 2 The recombinant BMP4 protein refolding solution of this embodiment has a concentration ratio of each component of the refolding solution of 52mM Tris-HCl (pH=8.5), 6mM EDTA, 0.54M L-arginine (Arg), 35mM CHAPS, 2.5mM oxidized glutathione (GSSG), 1.2mM reduced glutathione (GSH), and 0.6% volume percentage concentration of TritonX-114.

[0033] Example 3 The recombinant BMP4 protein refolding solution of this embodiment has a concentration ratio of each component of the refolding solution, 50mM Tris-HCl (pH=8.5), 5mM EDTA, 0.5M L-arginine (Arg), 33mM CHAPS, 2mM oxidized glutathione (GSSG), 1mM reduced glutathione (GSH), and 0.4% volume percentage concentration of TritonX-114.

[0034] Comparative Examples 1-3 The sources of the raw materials and recombinant BMP4 protein used in the comparative examples of the present application are the same as those in the examples.

[0035] Comparative Example 1 The difference between the recombinant BMP4 protein refolding solution in this comparative example and Example 3 is that the refolding solution in this comparative example does not contain 33 mM CHAPS, which is replaced by a combination of guanidine hydrochloride (GuHCl) and 1 M NaCl at different concentrations: 0.1 M GuHCl+1 M NaCl, 0.2 M GuHCl+1 M NaCl, 0.3 M GuHCl+1 M NaCl, 0.4 M GuHCl+1 M NaCl and 0.5 M GuHCl+1 M NaCl.

[0036] Comparative Example 2 The difference between the recombinant BMP4 protein refolding solution in this comparative example and Example 3 is that the refolding solution in this comparative example does not contain 33 mM CHAPS, which is replaced by Tween-20 of different concentrations (v / v): 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%.

[0037] Comparative Example 3 The difference between the recombinant BMP4 protein refolding solution in this comparative example and Example 3 is that the refolding solution in this comparative example does not contain 33 mM CHAPS, which is replaced by NP-40 of different concentrations (v / v): 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%.

[0038] Comparative Example 4 The difference between the recombinant BMP4 protein refolding solution in this comparative example and Example 3 is that the refolding solution in this comparative example does not contain 33 mM CHAPS, which is replaced by Triton X-100 of different concentrations (v / v): 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%.

[0039] Performance test 1 In this experiment, the following renaturation method was used to detect the renaturation effect of the recombinant BMP4 protein renaturation solution in Examples 1-3 and Comparative Examples 1-4. The specific steps are: in an ice water bath, the temperature of the recombinant BMP4 protein renaturation solution was controlled at 4°C, and the denatured recombinant BMP4 protein was added to the recombinant BMP4 protein renaturation solution to a protein concentration of 0.1 μg / μl to obtain a renaturation reaction solution, and the reaction was stirred at 4°C for 72 hours. The test results are shown in Table 1 and Figure 2 as shown in .

[0040] Table 1 Test results of Examples 1-3 Example Recombinant BMP4 protein renaturation rate (%) Example 1 19.1 Example 2 19.3 Example 3 19.8 It can be seen from Table 1 that after the recombinant BMP4 protein renaturation solution of Examples 1-3 of the present application was stirred and reacted at 4°C for 72 hours, the renaturation rate of the recombinant BMP4 protein can reach more than 19%, which is higher than the current renaturation rate of the protein in the industry.

[0041] At the same time, from Figure 2 It can be seen that under the same renaturation conditions, adding CHAPS to the renaturation solution has a significant advantage in improving the renaturation rate of BMP4 protein compared to adding other components (such as GuHCl+NaCl, Tween-20, NP-40 and Triton X-100).

[0042] Embodiment 4-7 Examples 4-6 of the present application are examples of the renaturation method of the recombinant BMP4 protein of the present application, and all use the renaturation solution of Example 3.

[0043] Example 4 The renaturation method of the recombinant BMP4 protein of this embodiment specifically comprises the following steps: S1. In an ice water bath, control the temperature of the recombinant BMP4 protein renaturation solution at 4°C, and add the denatured recombinant BMP4 protein to the recombinant BMP4 protein renaturation solution to a protein concentration of 0.1 μg / μl to obtain a renaturation reaction solution; S2. The renaturation reaction solution obtained in step S1 is gradually heated to 10° C. under stirring conditions, and the heating time is controlled to be 20 h. Then, the reaction is continued at 10° C. under stirring conditions for 52 h to obtain the renatured recombinant BMP4 protein.

[0044] Example 5 The renaturation method of the recombinant BMP4 protein of this embodiment specifically comprises the following steps: S1. In an ice water bath, control the temperature of the recombinant BMP4 protein renaturation solution at 4°C, and add the denatured recombinant BMP4 protein to the recombinant BMP4 protein renaturation solution to a protein concentration of 0.1 μg / μl to obtain a renaturation reaction solution; S2. The renatured reaction solution obtained in step S1 is gradually heated to 10° C. under stirring conditions, and the heating time is controlled to be 24 hours. Then, the reaction is continued at 10° C. under stirring conditions for 48 hours to obtain the renatured recombinant BMP4 protein.

[0045] Example 6 The renaturation method of the recombinant BMP4 protein of this embodiment specifically comprises the following steps: S1. In an ice water bath, control the temperature of the recombinant BMP4 protein renaturation solution at 4°C, and add the denatured recombinant BMP4 protein to the recombinant BMP4 protein renaturation solution to a protein concentration of 0.1 μg / μl to obtain a renaturation reaction solution; S2. The renatured reaction solution obtained in step S1 is gradually heated to 10° C. under stirring conditions, and the heating time is controlled to be 28 hours. Then, the reaction is continued at 10° C. under stirring conditions for 44 hours to obtain the renatured recombinant BMP4 protein.

[0046] Example 7 The renaturation method of the recombinant BMP4 protein of this embodiment specifically comprises the following steps: S1. In an ice water bath, control the temperature of the recombinant BMP4 protein renaturation solution at 4°C, and add the denatured recombinant BMP4 protein to the recombinant BMP4 protein renaturation solution to a protein concentration of 0.1 μg / μl to obtain a renaturation reaction solution; S2. The renatured reaction solution obtained in step S1 is gradually heated to 10° C. under stirring conditions, and the heating time is controlled to be 32 hours. Then, the reaction is continued at 10° C. under stirring conditions for 40 hours to obtain the renatured recombinant BMP4 protein.

[0047] Comparative Examples 5-7 Comparative Example 5 The renaturation method of the recombinant BMP4 protein of this comparative example specifically comprises the following steps: S1. In an ice water bath, control the temperature of the recombinant BMP4 protein renaturation solution at 4°C, and add the denatured recombinant BMP4 protein to the recombinant BMP4 protein renaturation solution to a protein concentration of 0.1 μg / μl to obtain a renaturation reaction solution; S2. The renatured reaction solution obtained in step S1 is gradually heated to 10° C. under stirring conditions, and the heating time is controlled to be 12 h. Then, the reaction is continued at 10° C. under stirring conditions for 60 h to obtain the renatured recombinant BMP4 protein.

[0048] Comparative Example 6 The renaturation method of the recombinant BMP4 protein of this comparative example specifically comprises the following steps: S1. In an ice water bath, control the temperature of the recombinant BMP4 protein renaturation solution at 4°C, and add the denatured recombinant BMP4 protein to the recombinant BMP4 protein renaturation solution to a protein concentration of 0.1 μg / μl to obtain a renaturation reaction solution; S2. The renaturation reaction solution obtained in step S1 is gradually heated to 10° C. under stirring conditions, and the heating time is controlled to be 16 hours. Then, the reaction is continued at 10° C. under stirring conditions for 56 hours to obtain the renatured recombinant BMP4 protein.

[0049] Comparative Example 7 The renaturation method of the recombinant BMP4 protein of this comparative example specifically comprises the following steps: S1. In an ice water bath, control the temperature of the recombinant BMP4 protein renaturation solution at 4°C, and add the denatured recombinant BMP4 protein to the recombinant BMP4 protein renaturation solution to a protein concentration of 0.1 μg / μl to obtain a renaturation reaction solution; S2. The renatured reaction solution obtained in step S1 is gradually heated to 10° C. under stirring conditions, and the heating time is controlled to be 36 hours. Then, the reaction is continued at 10° C. under stirring conditions for 36 hours to obtain the renatured recombinant BMP4 protein.

[0050] Performance test 2 The renaturation rates of the renatured recombinant BMP4 proteins prepared in Examples 4-7 and Comparative Examples 5-7 were tested. The test results are shown in Figure 3 .

[0051] from Figure 3 It can be seen that the present application adopts the recombinant BMP4 protein renaturation liquid of Example 3, and the optimal renaturation temperature parameter is an initial temperature of 4°C, which is gradually heated to 10°C; the optimal reaction time parameter is gradually heated to 10°C, the heating time is controlled at 20-32h, and the total reaction time is controlled at more than 72h. Using this renaturation temperature and reaction time parameters, the renaturation rate of the obtained recombinant BMP4 protein can reach more than 29%.

[0052] Example 8 The method for preparing the recombinant BMP4 protein of this embodiment comprises the following steps: a. Synthesize the recombinant BMP4 protein with optimized base sequence into a vector of E. coli expression system; b. Transforming the vector containing the recombinant BMP4 protein synthesized in step a into competent cells of Escherichia coli, culturing and inducing expression, collecting the cells, lysing and purifying them to obtain denatured recombinant BMP4 protein; c. renaturing the denatured recombinant BMP4 protein obtained in step b by the renaturation method to obtain a renaturation mixed solution; d. The renaturation mixture obtained in step c is centrifuged and purified to obtain recombinant BMP4 protein.

[0053] Example 9 The difference between this embodiment and embodiment 8 is that in step d of this embodiment, the purification method comprises the following steps: d1. The crude recombinant BMP4 protein obtained by centrifugation was dialyzed into 20 mM Tris-HCl, pH=8.0, 4 M Urea buffer in an ice water bath, and enriched by an sp column to obtain enriched protein; d2, dialyzing the enriched protein obtained in step d1 again into 20 mM Tris-HCl, pH=8.0, 4 M Urea buffer to obtain a secondary dialyzed protein; d3. The secondary dialyzed protein obtained in step d2 is separated and purified using a heparin column to obtain a pure recombinant BMP4 protein.

[0054] Comparative Example 8 The commercially available competitor used is the recombinant human BMP4 protein of the original Abbkine.

[0055] Performance test 3 This experiment tests the preparation method of the recombinant BMP4 protein and the activity of the recombinant BMP4 protein obtained. In this experiment, the recombinant BMP4 protein obtained in Examples 8-9 of the present application and the original Abbkine recombinant human BMP4 protein were tested for activity.

[0056] Test results such as Figure 4As shown, by using the recombinant BMP4 protein renaturation solution and the preparation method of recombinant BMP4 protein provided in the present application, the recombinant BMP4 protein can finally be renatured within 72 hours, and the activity is equivalent to that of commercialized BMP4, which greatly reduces the production cost of BMP4 protein, improves production efficiency, and further promotes the research of BMP4 protein.

[0057] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. A recombinant BMP4 protein renaturation solution, characterized in that: The refolding solution is composed of the following components: 46-52 mM Tris-HCl, 4-6 mM EDTA, 0.48-0.54 M Arg, 30-35 mM CHAPS, 1.5-2.5 mM GSSG, 0.8-1.2 mM GSH, and 0.3-0.6% volume percent concentration of TritonX-114.

2. The recombinant BMP4 protein renaturation solution according to claim 1, characterized in that: The concentrations of the components are: 50-52 mM Tris-HCl, 4-6 mM EDTA, 0.48-0.54 M Arg, 33-35 mM CHAPS, 1.5-2.5 mM GSSG, 0.8-1.2 mM GSH, and 0.3-0.6% volume percent concentration of TritonX-114.

3. The recombinant BMP4 protein renaturation solution according to claim 1, characterized in that: The pH value of the Tris-HCl is 8.

5.

4. A method for renaturing a recombinant BMP4 protein, characterized in that: The recombinant BMP4 protein renaturing solution as described in any one of claims 1 to 3 is used.

5. The renaturation method according to claim 4, characterized in that: The method comprises the following steps, S1. In an ice water bath, adding the denatured recombinant BMP4 protein to the recombinant BMP4 protein renaturation solution according to any one of claims 1 to 3 to obtain a renaturation reaction solution; S2, gradually heating the renaturation reaction solution obtained in step S1 to 10° C. under stirring, and then continuing to keep the temperature under stirring to react, thereby obtaining renatured recombinant BMP4 protein; In step S2, the total duration of the gradual heating and heat preservation reaction is controlled to be more than 72 hours.

6. The renaturation method according to claim 5, characterized in that: In the step S1, the added concentration of the denatured recombinant BMP4 protein is not greater than 0.1 μg / μl.

7. The renaturation method according to claim 5, characterized in that: In the step S1, the temperature of the renaturation reaction solution is controlled at 4°C.

8. The renaturation method according to claim 7, characterized in that: In step S2, the gradual heating is carried out in a uniform heating manner, and the duration of the gradual heating is controlled within 20 to 32 hours.

9. A method for preparing a recombinant BMP4 protein, characterized in that: The following steps are involved: a. Synthesize the recombinant BMP4 protein with optimized base sequence into a vector of E. coli expression system; b. Transforming the vector containing the recombinant BMP4 protein synthesized in step a into competent cells of Escherichia coli, culturing and inducing expression, collecting the cells, lysing and purifying them to obtain denatured recombinant BMP4 protein; c. renaturing the denatured recombinant BMP4 protein obtained in step b by the renaturation method described in any one of claims 4 to 8 to obtain a renaturation mixed solution; d. The renaturation mixture obtained in step c is centrifuged and purified to obtain recombinant BMP4 protein.

10. Use of the recombinant BMP4 protein renaturation solution according to any one of claims 1 to 3 in the renaturation of recombinant BMP4 protein or in the preparation of recombinant BMP4 protein.

Images