Novel reduction method of spherical protein

Abstract

The invention provides a novel reduction method of spherical protein. The spherical protein is of a single-chain structure, and disulfide bonds in molecules of the spherical protein are wrapped in spheres; the novel reduction method of the spherical protein comprises the following steps: (1) simulating and calculating the theoretical isoelectric point pH of the spherical protein according to the gene sequence of the spherical protein; (2) preparing a PBS (Phosphate Buffer Solution) of which the pH value is greater than the pH value of the theoretical isoelectric point; (3) preparing a DTT-containing reduced test sample buffer solution; and (4) diluting the spherical protein with a PBS buffer solution, then mixing the diluted spherical protein with a DTT-containing reduced test sample buffer solution, and then incubating. When the spherical protein is reduced by using the novel method disclosed by the invention, non-reduced target protein can be thoroughly changed into reduced protein under a mild condition, and meanwhile, the specific spherical protein is prevented from being damaged and degraded under a high-temperature condition, so that the detection interference on the drug protein is eliminated; and high stability and antigen activity of the protein in the reduction process are ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological protein reduction, in particular to a novel reduction method of spherical protein. BACKGROUND

[0002] Macromolecular proteins can be divided into fibrous proteins and spherical proteins according to shape. Fibrous proteins are mostly assembled into linear or fibrous structures from single or repeated secondary structures, and the exposure degree of disulfide bonds is high. The structure of spherical proteins is compact and complex, and the disulfide bonds are likely to be buried inside. Therefore, spherical proteins are not easy to be reduced compared with fibrous proteins.

[0003] The research and development team of the present application found that there are two obvious characteristics for a certain specific spherical protein structure in the experimental process: 1. The spherical protein is a single chain structure with a molecular weight of 53kD, containing a pair of intrachain disulfide bonds (C29-C214); three free cysteine thiol groups (C108, C249, C353). 2. The proportion of alpha-helix and random coil structure contained in the protein is relatively high (both about 33%), which makes the protein molecule tightly packed and increases the complexity of the spatial structure, and the protein conformation changes obviously with the increase of temperature. Because the intramolecular disulfide bond is tightly wrapped inside the spherical structure, it is difficult for the reducing agent molecules to contact the disulfide bond, and general reduction methods cannot completely reduce the protein.

[0004] Protein reduction technology is of great significance for scientific research, and it is widely used in protein purification, protein structure analysis and biomedical research in order to detect, identify or study its biological activity. The general reduction method (such as SDS-polyacrylamide gel electrophoresis method in Chinese Pharmacopoeia) cannot completely reduce the specific spherical protein to the target protein; and the protein is sensitive to heat, and severe temperature will cause the target protein to be destroyed and degraded, which will interfere with drug testing and analysis. SUMMARY

[0005] The purpose of the present application is to provide a novel reduction method of spherical protein, so as to solve the problem that the prior art cannot completely reduce the specific spherical protein by using the pharmacopoeia method.

[0006] The present application is implemented as follows:

[0007] A novel reduction method of spherical protein, the spherical protein is a single chain structure, and its intramolecular disulfide bond is wrapped inside the spherical structure;

[0008] The novel reduction method of the spherical protein comprises the following steps:

[0009] (1) According to the gene sequence of the spherical protein, the theoretical isoelectric point pH value is obtained by simulation calculation;

[0010] (2) preparing PBS buffer solution with pH value greater than the theoretical isoelectric point pH value of step (1) ;

[0011] (3) preparing reduced sample buffer solution with DTT concentration of 400-800 mg / mL;

[0012] (4) diluting the protein to be reduced with the PBS buffer solution of step (2) and then mixing with the reduced sample buffer solution of step (3), and then incubating at 27-37℃ for 8-12 min.

[0013] Further, the globular protein has a molecular weight of 53 kD, contains a pair of intrachain disulfide bonds (C29-C214), three free cysteine sulfhydryl groups (C108, C249, C353), and has a proportion of alpha-helix and random coil structure of about 33%.

[0014] Further, the globular protein is derived from the R-67174 strain of the American Type Culture Collection.

[0015] Further, the pH value of the PBS buffer solution of step (2) is 7.1-7.7.

[0016] Further, the PBS buffer solution of step (2) is prepared by dissolving disodium hydrogen phosphate dodecahydrate, sodium chloride, potassium dihydrogen phosphate, and potassium chloride in water, and adjusting the amount of potassium dihydrogen phosphate to be added so that the pH value is 7.1-7.7.

[0017] Further, the incubation condition of step (4) is specifically incubating at 32℃ for 10 min.

[0018] Further, in step (4), the globular protein to be reduced is diluted 10 times with the PBS buffer solution of step (2).

[0019] Further, in step (4), the volume ratio of the diluted protein solution to the reduced sample buffer solution is 3:1.

[0020] Further, the reduced sample buffer solution of step (3) is prepared by dissolving tris-hydroxymethyl aminomethane, bromophenol blue, sodium dodecyl sulfate, and glycerol in water, and adjusting the pH value with hydrochloric acid, and then adding DTT and vortexing to mix.

[0021] The novel reduction method of the application is compared with the following methods: two pharmacopoeia methods, water dilution, boiling after doubling the concentration of mercaptoethanol, water dilution, 32℃ incubation for 10 min after doubling the concentration of mercaptoethanol, PBS dilution at pH 7.4, 32℃ incubation for 10 min with 100 mmol / L DTT, water dilution, 32℃ incubation for 10 min with 600 mg / mL DTT, PBS buffer dilution at pH 6.0-pH 6.8, 32℃ incubation for 10 min with 600 mg / mL DTT, dilution with stock buffer of other components, 32℃ incubation for 10 min with 600 mg / mL DTT, and it has been verified that the novel reduction method can completely reduce the target protein and reduce the generation of degradation products.

[0022] When the spherical protein is reduced by using the novel method of the application, the non-reduced target protein can be completely converted into a reduced protein under mild conditions (27-37℃ incubation for 8-12 min), while avoiding the destruction and degradation of the specific spherical protein under high temperature conditions, eliminating the detection interference on the drug protein, and ensuring the high stability and antigen activity of the protein during the reduction process.

[0023] The application has the following beneficial effects:

[0024] 1) The reduction method is convenient, fast and complete, has wide experimental conditions, high durability and few influencing factors.

[0025] 2) The reduction method has a reaction temperature of 27-37℃, and the reaction conditions are very mild, without degradation products, no interference on drug analysis, and can truly reflect the protein structure under the reduced state; and the reduction reaction time is short, which is suitable for rapid analysis.

[0026] 3) The reduction method ensures that the protein maintains high stability and antigen activity during the reduction process. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the detection result of Example 1 and Comparative Example 1.

[0028] Figure 2 is the detection result of Comparative Example 2.

[0029] Figure 3 is the detection result of Comparative Example 3.

[0030] Figure 4 is the detection result of Comparative Example 4 and Comparative Example 5.

[0031] Figure 5 is the detection result of Comparative Example 6.

[0032] Figure 6 is the detection result of Comparative Example 7.

[0033] Figure 7 are the test results of Comparative Examples 8-10.

[0034] Figure 8 are the test results of Comparative Examples 11-13.

[0035] Figure 9 are the test results of Example 2.

[0036] Figure 10 are the test results of Example 3.

[0037] Figure 11 are the test results of Example 4.

[0038] Figure 12 are the test results of Example 5.

[0039] Figure 13 are the test results of Example 6.

[0040] Figure 14 are the test results of Example 7.

[0041] Figure 15 are the test results of Example 8.

[0042] Figure 16 are the test results of Example 9. DETAILED DESCRIPTION

[0043] The specific spherical protein introduced in the embodiments of the present application is derived from the Methyloceanibacter methanicus R-67174 strain of the American Type Culture Collection (ATCC)

Full Scientific Name (LPSN): Methyloceanibacter methanicus Vekeman et al. 2018

[0044] The amino acid sequence of the specific spherical protein is shown in SEQ ID NO. 1.

[0045] The specific spherical protein is derived from a bacterium of the Hyphomicrobiaceae family and is in a spherical structure. The gene synthesizing the protein is generally expressed in yeast, and the intracellular cysteine concentration is simultaneously increased to promote the production of the protein, thereby supporting the efficient expression of downstream recombinant proteins.

[0046] The present application develops a convenient, efficient, and mild method for reducing the above specific spherical protein. The test sample referred to in each of the following embodiments and comparative examples is the above specific spherical protein.

[0047] Example 1

[0048] 1. The theoretical isoelectric point of the specific globin protein is pH 6.46, calculated by computer simulation according to the gene sequence of the specific globin protein. The pH of the subsequent PBS buffer should be far from this value.

[0049] 2. 200 mmol PBS buffer (pH 7.4): Dissolve 3.58 g of disodium hydrogen phosphate dodecahydrate, 8.00 g of sodium chloride, 0.27 g of potassium dihydrogen phosphate, and 0.20 g of potassium chloride in water, and then add water to 1000 mL, mix well, and filter through a 0.2 μm filter membrane.

[0050] 3. Non-reducing test sample buffer (4X): Weigh 3.03 g of tris-hydroxymethyl aminomethane, 20 mg of bromophenol blue, and 8.0 g of sodium dodecyl sulfate, and measure 40 mL of glycerol. Dissolve and dilute to about 80 mL, adjust the pH to 6.8 with hydrochloric acid, and dilute to 100 mL with water.

[0051] 4. 600 mg / mL DTT reducing test sample buffer (4X): Weigh 600 mg of dithiothreitol (DTT), add 1 mL of non-reducing test sample buffer (4X), and vortex to mix well. Prepare immediately before use.

[0052] 5. Sample processing and loading: Dilute the test sample 10-fold with PBS buffer (pH 7.4), mix with 600 mg / mL DTT reducing test sample buffer (4X) at a volume ratio of 3:1, and incubate at 32°C for 10 minutes. The loading amount is 1.02 μg.

[0053] 6. Use immunoblotting to detect whether the non-reduced bands have been completely converted to reduced bands, and whether there are degradation products during the reduction reaction. The detection steps used in the following examples and comparative examples are the same as this step and will not be repeated.

[0054] Comparative Example 1

[0055] The specific globin protein described above was reduced using the pharmacopoeia method, as follows:

[0056] 1. Reducing test sample buffer (4X): Weigh 3.03 g of tris-hydroxymethyl aminomethane (Tris), 20 mg of bromophenol blue, and 8.0 g of sodium dodecyl sulfate, and measure 40 mL of glycerol. Dissolve and dilute to 80 mL with water, adjust the pH to 6.8 with hydrochloric acid, and dilute to 100 mL with water. Before use, add dithiothreitol (DTT) to 100 mmol / L.

[0057] 2. Test sample processing and loading: Dilute the test sample 10-fold with water to obtain a test sample solution; mix the test sample solution with the reducing test sample buffer (4X) at a volume ratio of 3:1, heat in a water bath or 100°C block heater for 5 minutes, cool to room temperature, and load 1.02 μg.

[0058] The reduction results of Example 1 and Comparative Example 1 are shown in Figure 1 . Figure 1 In the figure, Example 1 corresponds to lane 1 (i.e. the new method reduction), and Comparative Example 1 corresponds to lane 3 (i.e. the pharmacopoeia method (DTT 100 mmol / L)). It can be seen that when the new method of Example 1 is used, only the target band is present; when the pharmacopoeia method (DTT 100 mmol / L) of Comparative Example 1 is used to treat the test sample, there are non-reduced target bands and degradation products.

[0059] The amino acid sequence of the protein after reduction by the method of Example 1 of the present application is the same as before reduction, but the intrachain disulfide bond (C29-C214) of the reduced protein is reduced to (-SH), the spherical structure is destroyed, two (-SH) are added, and the active site inside the spherical structure is exposed to facilitate detection, identification or study of its biological activity.

[0060] Comparative Example 2

[0061] In this comparative example, another pharmacopoeia method is used to reduce the above-mentioned specific spherical protein, as follows:

[0062] 1. Reducing test sample buffer (4X): weigh 3.03 g of tris, 20 mg of bromophenol blue, 8.0 g of sodium dodecyl sulfate, and 40 mL of glycerol, dissolve and dilute to about 80 mL with water, add 20 mL of β-mercaptoethanol, adjust the pH to 6.8 with hydrochloric acid, and dilute to 100 mL with water.

[0063] 2. Test sample treatment and loading: dilute the test sample 10 times with water to obtain a test sample solution; mix the test sample solution with the reducing test sample buffer (4X) at a volume ratio of 3:1, heat in a water bath or 100°C block heater for 5 minutes, cool to room temperature, and load 1.02 μg.

[0064] The detection results of this comparative example are shown in Figure 2 lane 4. Figure 2 In the figure, lane 3 (control) corresponds to the test sample treated by the new reduction method of Example 1. It can be seen from Figure 2 that when the new method is used for reduction, only the target band is present; when the pharmacopoeia method (20% mercaptoethanol) of Comparative Example 2 is used to treat the test sample, there are non-reduced target bands and degradation products.

[0065] Comparative Example 3

[0066] Compared with Comparative Example 2, the concentration of mercaptoethanol in this comparative example is doubled, as follows:

[0067] 1. Reduced sample buffer (4X): weigh Tris 3.03 g, bromophenol blue 20 mg, sodium dodecyl sulfate 8.0 g, take glycerol 40 mL, dissolve and dilute to about 60 mL with water, add β-mercaptoethanol 40 mL, adjust pH to 6.8 with hydrochloric acid, dilute to 100 mL with water.

[0068] 2. Sample treatment and loading: dilute the sample 10 times with water to obtain a sample solution; mix the sample solution with the reduced sample buffer (4X) at a volume ratio of 3:1, heat in a water bath or 100°C block heater for 5 minutes, cool to room temperature, and load 1.02 μg.

[0069] The detection results of the comparative example are shown in Figure 3 lane 4. Figure 3 The control treatment method in this example is to treat the sample using the new reduction method in Example 1. As can be seen from Figure 3 , when the sample is treated using the new method, only the target band is obtained; in Comparative Example 3, when the sample is treated with 40% mercaptoethanol and then boiled, non-reduced target bands and degradation products are generated.

[0070] Comparative Example 4

[0071] Compared with Comparative Example 2, the heating in this comparative example is replaced by incubation. The details are as follows:

[0072] 1. Reduced sample buffer (4X): weigh Tris 3.03 g, bromophenol blue 20 mg, sodium dodecyl sulfate 8.0 g, take glycerol 40 mL, dissolve and dilute to about 80 mL with water, add β-mercaptoethanol 20 mL, adjust pH to 6.8 with hydrochloric acid, dilute to 100 mL with water.

[0073] 2. Sample treatment and loading: dilute the sample 10 times with water to obtain a sample solution; mix the sample solution with the reduced sample buffer (4X) containing 20% mercaptoethanol at a volume ratio of 3:1, incubate at 32°C for 10 min, cool to room temperature, and load 1.02 μg.

[0074] Comparative Example 5

[0075] Compared with Comparative Example 4, the concentration of mercaptoethanol in this comparative example is doubled, i.e. the buffer is reduced sample buffer (4X) containing 40% mercaptoethanol, and the others are the same as in Comparative Example 4.

[0076] The detection results of Comparative Examples 4 and 5 are shown in Figure 4 lanes 4 and 5, respectively. As can be seen from Figure 4It can be seen that only the target band is obtained (the 3rd lane) when the sample is reduced by the new method of Example 1; the non-reduced target band and the aggregate are generated when the sample is treated with 20% mercaptoethanol and incubated at 32°C for 10 min; and the non-reduced target band is generated when the sample is treated with 40% mercaptoethanol and incubated at 32°C for 10 min.

[0077] Comparative Example 6

[0078] In the present comparative example, the concentration of DTT in the reducing sample buffer is 100 mmol / L, and the other conditions are the same as those in Example 1. Specifically, the reducing sample buffer (4X) is prepared as follows:

[0079] 1. Reducing sample buffer (4X): 3.03 g of tris-hydroxymethyl aminomethane, 20 mg of bromophenol blue, 8.0 g of sodium dodecyl sulfate, 40 mL of glycerol, and water are dissolved and diluted to 80 mL, and the pH value is adjusted to 6.8 with hydrochloric acid, and then water is added to dilute to 100 mL. Before use, dithiothreitol is added to 100 mmol / L.

[0080] 2. Sample treatment and loading: the diluent is PBS buffer with pH 7.4; after being diluted by 10 times, the sample solution is mixed with the reducing sample buffer (4X) at a volume ratio of 3:1, incubated at 32°C for 10 min, cooled to room temperature, and loaded with an amount of 1.02 μg.

[0081] The detection results are shown in Figure 5 the 3rd lane. It can be seen that only the target band is obtained (the 2nd lane) when the sample is reduced by the new method of Example 1; the non-reduced target band is generated when the sample is treated with the reducing sample buffer (4X) containing 100 mmol / L DTT and incubated at 32°C for 10 min. Figure 5 Comparative Example 7

[0082] In the present comparative example, water is used to dilute the sample instead of the PBS buffer in Example 1, and the other conditions are the same as those in Example 1. Specifically, the diluent is water; after being diluted by 10 times, the sample is mixed with the reducing sample buffer (4X) containing 600 mg / mL DTT at a volume ratio of 3:1, incubated at 32°C for 10 min, and loaded with an amount of 1.02 μg.

[0083] The results are shown in

[0084] the 3rd and 4th lanes. It can be seen that only the target band is obtained (the 5th lane) when the sample is reduced by the new method of Example 1; the non-reduced target band is generated when the sample is diluted with water and then treated with the reducing sample buffer (4X) containing 600 mg / mL DTT and incubated at 32°C for 10 min. Figure 6 Figure 6 Comparative Example 8

[0085] In the present comparative example, water is used to dilute the sample instead of the PBS buffer in Example 1, and the other conditions are the same as those in Example 1. Specifically, the diluent is water; after being diluted by 10 times, the sample is mixed with the reducing sample buffer (4X) containing 600 mg / mL DTT at a volume ratio of 3:1, incubated at 32°C for 10 min, and loaded with an amount of 1.02 μg. ​

[0086] Compared with Example 1, in the present comparative example, PBS buffer with pH 6.0 is used, and the details are as follows:

[0087] 1. PBS buffer (pH 6.0): 3.58 g of disodium hydrogen phosphate dodecahydrate, 8.00 g of sodium chloride, and 0.20 g of potassium chloride are dissolved in water, and then the amount of potassium dihydrogen phosphate is adjusted to make the pH 6.0, and finally water is added to 1000 mL, mixed, and filtered with a 0.2 μm filter membrane.

[0088] 2. Sample treatment and loading: the sample is diluted 10 times with PBS buffer with pH 6.0, and then mixed with the reduced sample buffer (4X) of 600 mg / mL DTT (the preparation method is the same as that of Example 1) at a volume ratio of 3:1, and incubated at 32°C for 10 min. The loading amount is 1.02 μg.

[0089] Comparative Example 9

[0090] Compared with Comparative Example 8, in the present comparative example, the pH of the PBS buffer is 6.4, and the others are the same as those of Comparative Example 8.

[0091] Comparative Example 10

[0092] Compared with Comparative Example 8, in the present comparative example, the pH of the PBS buffer is 6.8, and the others are the same as those of Comparative Example 8.

[0093] The detection results of Comparative Examples 8-10 are shown in lanes 6, 5, and 4 of FIG. 1, respectively. Figure 7 It can be seen that only the target band (lane 3) is obtained when the sample is reduced by the new method of Example 1; and there is a non-reduced target band when the sample is diluted with PBS buffer with pH 6.0, pH 6.4, or pH 6.8. Figure 7 Comparative Example 11

[0094] Compared with Example 1, in the present comparative example, HEPES buffer is used to replace the PBS buffer in Comparative Example 1, and the details are as follows:

[0095] 1. 0.5 mol HEPES buffer (pH 7.2): 119.15 g of HEPES powder is added to 800 mL of deionized water, stirred to dissolve, and then the pH is adjusted to 7.2 with HCL or NaOH; and finally the volume is adjusted to 1000 mL.

[0096] 2. Sample treatment and loading: the sample is diluted 10 times with HEPES buffer with pH 7.2, and then mixed with the reduced sample buffer (4X) of 600 mg / mL DTT at a volume ratio of 3:1, and incubated at 32°C for 10 min. The loading amount is 1.02 μg.

[0097] 2. Sample treatment and loading: the sample is diluted 10 times with HEPES buffer with pH 7.2, and then mixed with the reduced sample buffer (4X) of 600 mg / mL DTT at a volume ratio of 3:1, and incubated at 32°C for 10 min. The loading amount is 1.02 μg.

[0098] Comparative Example 12

[0099] Comparative Example 12 is the same as Comparative Example 11 except that the pH of the HEPES buffer is adjusted to 7.4.

[0100] Comparative Example 13

[0101] Comparative Example 13 is the same as Comparative Example 11 except that the pH of the HEPES buffer is adjusted to 7.6.

[0102] The detection results of Comparative Examples 11-13 are shown in lanes 1, 2 and 3, respectively, of Figure 1. Figure 8 As can be seen from Figure 1, when the new method of Example 1 is used for reduction, only the desired band (lane 4) is obtained; when the test sample is diluted with HEPES buffer having pH 7.2, pH 7.4 and pH 7.6, respectively, and then incubated, non-reduced desired bands are obtained. Figure 8 Example 2

[0103] Comparative Example 2 is the same as Example 1 except that the incubation temperature is 27°C, the incubation time is 10 min, the PBS buffer has pH 7.4, and the concentration of DTT in the reduced test sample buffer (4X) is 600 mg / mL.

[0104] The detection results are shown in Figure 2.

[0105] As can be seen from Figure 2, under the reaction conditions of this example, only the reduced desired band is obtained, and the reduction is complete; no non-reduced desired band is obtained, and no degradation band is obtained. Figure 9 Figure 9 Example 3

[0106] Comparative Example 3 is the same as Example 1 except that the incubation temperature is 37°C, the incubation time is 10 min, the PBS buffer has pH 7.4, and the concentration of DTT in the reduced test sample buffer (4X) is 600 mg / mL.

[0107] The detection results are shown in Figure 3. As can be seen from Figure 3, under the reaction conditions of this example, only the reduced desired band is obtained, and the reduction is complete; no non-reduced desired band is obtained, and no degradation band is obtained.

[0108] Figure 10 Example 4 Figure 10 Comparative Example 4 is the same as Example 1 except that the incubation temperature is 32°C, the incubation time is 8 min, the PBS buffer has pH 7.4, and the concentration of DTT in the reduced test sample buffer (4X) is 600 mg / mL.

[0109] The detection results are shown in Figure 4.

[0110] As can be seen from Figure 4, under the reaction conditions of this example, only the reduced desired band is obtained, and the reduction is complete; no non-reduced desired band is obtained, and no degradation band is obtained.

[0111] Example 5 Figure 11 Figure 11 ​​It can be seen that under the reaction conditions of this embodiment, only the target band is reduced, and the reduction is complete; there is no non-target band or degradation band.

[0112] Example 5

[0113] Compared with Example 1, the incubation temperature in this example is 32°C, the incubation time is 12 min; the pH of the PBS buffer is 7.4; and the concentration of DTT in the reduced test sample buffer (4X) is 600 mg / mL.

[0114] Test results are shown Figure 12 ,Depend on Figure 12 It can be seen that under the reaction conditions of this embodiment, only the target band is reduced, and the reduction is complete; there is no non-target band or degradation band.

[0115] Example 6

[0116] Compared with Example 1, the incubation temperature in this example is 32°C, the incubation time is 10 min; the pH of the PBS buffer is 7.1; and the concentration of DTT in the reduced test sample buffer (4X) is 600 mg / mL.

[0117] Test results are shown Figure 13 ,Depend on Figure 13 It can be seen that under the reaction conditions of this embodiment, only the target band is reduced, and the reduction is complete; there is no non-target band or degradation band.

[0118] Example 7

[0119] Compared with Example 1, the incubation temperature in this example is 32°C, the incubation time is 10 min; the pH of the PBS buffer is 7.7; and the concentration of DTT in the reduced test sample buffer (4X) is 600 mg / mL.

[0120] Test results are shown Figure 14 ,Depend on Figure 14 It can be seen that under the reaction conditions of this embodiment, only the target band is reduced, and the reduction is complete; there is no non-target band or degradation band.

[0121] Example 8

[0122] Compared with Example 1, the incubation temperature in this example is 32°C, the incubation time is 10 min; the pH of the PBS buffer is 7.4; and the concentration of DTT in the reduced test sample buffer (4X) is 400 mg / mL.

[0123] Test results are shown Figure 15 ,Depend on Figure 15 It can be seen that under the reaction conditions of this embodiment, only the target band is reduced, and the reduction is complete; there is no non-target band or degradation band.

[0124] Example 9

[0125] Compared with Example 1, in the present example, the incubation temperature is 32℃, the incubation time is 10 min; the PBS buffer is pH 7.4; and the concentration of DTT in the reduced sample buffer (4X) is 800 mg / mL.

[0126] The detection results are shown in Table 1. Figure 16 It can be seen that, under the reaction conditions of the present example, only the reduced target band is obtained, and the reduction is complete; no non-reduced target band is obtained, and no degradation band is obtained. Figure 16

[0127] From Examples 1-9, it can be seen that the new reduction method of the present application has very high durability; the pH value range of the PBS buffer can be pH 7.1-pH 7.7; the concentration range of DTT in the reduced sample buffer (4X) can be 400-800 mg / mL; the incubation temperature range can be 27-37℃; and the incubation time range can be 8-12 min; and changes in these parameter ranges can ensure the consistency of the results.

[0128] From the above, it can be seen that, using the pharmacopoeia method, the doubled concentration of mercaptoethanol method (boiling or 32℃ incubation), PBS dilution, 100 mmol / L DTT 32℃ incubation for 10 min, water dilution, 600 mg / mL DTT 32℃ incubation for 10 min, PBS buffer dilution at pH 6.0-6.8, 600 mg / mL DTT 32℃ incubation for 10 min, and dilution with the stock buffer of other components, these methods cannot completely reduce the specific spherical protein to the reduced target protein; and the protein is sensitive to heat, and intense temperature can destroy the target protein, causing degradation and interfering with drug testing and analysis. When the protein is reduced using the new method of the present application, the non-reduced target protein can be completely reduced to the reduced target protein under mild conditions, and the specific protein is avoided from being destroyed and degraded, thereby eliminating the interference with the detection of the drug protein.​

Claims

1. A novel method for reducing a globular protein, characterized by, The spherical protein is a single chain structure, and its intramolecular disulfide bond is wrapped inside the sphere; The novel reduction method of the spherical protein comprises the following steps: (1) According to the gene sequence of the spherical protein, the theoretical isoelectric point pH value is obtained by simulation calculation; (2) Prepare a PBS buffer with a pH value greater than the theoretical isoelectric point pH value of step (1); (3) Prepare a reduced sample buffer with a DTT concentration of 400-800 mg / mL; (4) Dilute the spherical protein to be reduced with the PBS buffer in step (2), then mix with the reduced sample buffer in step (3), and then incubate at 27-37°C for 8-12 min.

2. The novel method of reducing the globular protein according to claim 1, characterized in that, The spherical protein is derived from the R-67174 strain of the American Type Culture Collection.

3. The novel method of reducing the globular protein according to claim 2, characterized in that, The pH value of the PBS buffer in step (2) is 7.1-7.

7.

4. The novel method of reducing the spherical protein according to claim 3, characterized in that, The preparation method of the PBS buffer in step (2) is as follows: dissolve dodecahydrate sodium phosphate dibasic, sodium chloride, potassium phosphate dibasic, and potassium chloride in water, and adjust the amount of potassium phosphate dibasic to make the pH value 7.1-7.

7.

5. The novel method of reducing the spherical protein according to claim 1, characterized in that, The incubation condition in step (4) is specifically: incubate at 32°C for 10 min.

6. The novel method of reducing the spherical protein according to claim 1, characterized in that, In step (4), the spherical protein to be reduced is diluted 10 times with the PBS buffer in step (2).

7. The novel method of reducing the spherical proteins according to claim 6, characterized in that, In step (4), the volume ratio of the diluted protein solution to the reduced sample buffer is 3:

1.

8. The novel method of reducing the spherical protein according to claim 1, characterized in that, The preparation method of the reduced sample buffer in step (3) is as follows: dissolve tris(hydroxymethyl)aminomethane, bromophenol blue, sodium dodecyl sulfate, and glycerol in water, and adjust the pH value with hydrochloric acid, then add DTT and vortex to mix.

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