EST protease activity detection method

Abstract

The invention discloses an EST protease activity detection method which comprises the following steps: a, setting an EST experimental group, a negative control group and a blank control group; b, carrying out incubation reaction on the EST experimental group, the negative control group and the blank control group to obtain respective reaction mixtures; c, detecting the influence of the reaction mixture on breast cancer cell proliferation to obtain the proliferation rate of each group; and d, calculating the enzymatic activity of the EST protein. The method disclosed by the invention is simple and easy to implement, good in repeatability and capable of effectively detecting the activity of the EST protease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a method for detecting EST protease activity. Background Technology

[0002] The EST (Estrogen Sulfotransferase) gene, also known as SULT1E1, encodes estrogen sulfotransferase, belonging to the sulfotransferase gene family (SULTs). The gene products of this family are key enzymes in the body's sulfation metabolism, catalyzing the formation of sulfate (SO4). 3- The process of transferring a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) group from the universal sulfate donor PAPS to the hydroxyl or amino residue of other acceptor substrates is called sulfonation. Within the SULT1 subfamily, SULT1E1 primarily acts on steroid hormones. The SULT1E1 protein has an affinity for steroid hormones such as estrone (E1) and estradiol (E2), and exerts its greatest effect when these substances undergo sulfation; therefore, SULT1E1 is also known as EST (estrone sulfotransferase).

[0003] Currently, research on EST protease activity focuses on the human species. Human EST proteins can inactivate estrogen through sulfation, playing a crucial role in regulating estrogen levels and gonadal maturation. However, research on human EST protease activity primarily relies on isotope labeling methods, which are not only costly, complex, and technically demanding, but also pose health risks. Furthermore, numerous influencing factors, such as the selection of sampling time points, tracer dosage selection, and the purity and chemical form of the label, can affect the accuracy of enzyme activity assays. In addition, some companies also use sulfate transfer activity detection methods belonging to the SULT family. While these methods can indirectly reflect the activity of SULT family-related enzymes, they lack the ability to specifically identify EST enzymes themselves.

[0004] Therefore, developing a simple, lower-cost, safe and reliable method for detecting EST-specific enzyme activity has become an urgent research need. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a simple, easy-to-use, and specific method for detecting EST protease activity.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for detecting EST protease activity includes the following steps:

[0008] a. Set up the EST experimental group, negative control group, and blank control group:

[0009] Add the same volume of buffer system to each group, then add the EST protein to be tested and E2 (17-β estradiol) to the EST experimental group, add E2 to the negative control group, and add the same solvent as the first two groups to the blank control group, so that the solvent concentration and final volume are the same as the first two groups.

[0010] The buffer system includes Tris-HCl buffer, MgCl2, DTT, BSA, and PAPS.

[0011] b. The EST experimental group, negative control group, and blank control group were incubated to obtain their respective reaction mixtures;

[0012] c. Detect the effect of the reaction mixture on the proliferation of breast cancer cells and obtain the proliferation rate of each group;

[0013] d. Calculate the enzyme activity of the EST protein using the following formula:

[0014] EST protein enzyme activity = {[(negative control group proliferation rate - experimental group proliferation rate) / (negative control group proliferation rate - blank control group proliferation rate)]} Estradiol content / incubation time.

[0015] In step a, the buffer system contains Tris-HCl buffer with a pH of 7.4 and a concentration of 50 mM; MgCl2 with a concentration of 1 mM; DTT with a concentration of 1 mM; BSA with a concentration of 625 μg / mL; and PAPS with a concentration of 10 μM.

[0016] In step a, the concentrations of the EST protein and E2 in the EST experimental group were 0.3 μg / 50 μl and 1 μM, respectively; the concentration of E2 in the negative control group was 1 μM.

[0017] In step a, E2 is dissolved in DMSO; in the blank control group, the solvent is DMSO and water.

[0018] In step b, the incubation reaction involves incubating each group at 37°C, followed by an ice bath to terminate the reaction.

[0019] Furthermore, the incubation time is 10 minutes; the ice bath time is 30 minutes.

[0020] In step c, the detection method is as follows: MCF-7 cells are cultured in phenol red-free DMEM containing 10% estrogen-free serum for 6-8 days, and the cell density is adjusted to 2×10⁶ cells / day. 4Cells were seeded at a density of 1 / ml in a 96-well plate, incubated for 24 hours, and then the drug was added. After another 24 hours of incubation, the cell proliferation rate was measured.

[0021] The method of adding the medicine is as follows:

[0022] Take 50 μl of the reaction mixture from step b and mix it with 950 μl of culture medium. Then add 100 μl to each well of a 96-well plate.

[0023] The culture medium is: 89% phenol red-free DEME medium + 10% estrogen-free serum + 1% penicillin-streptomycin bispecific antibody.

[0024] Among them, the CCK-8 assay was used to detect cell proliferation rate.

[0025] This invention presents a method for detecting EST protease activity based on MCF-7 cell proliferation experiments. This method is simple to operate, highly reproducible, free from radioactive contamination, safer, and inexpensive. The detection method can be widely used for EST protease activity detection, capable of detecting not only the widely studied human EST protein but also the EST protein expressed in the prokaryotic cells of the less common mollusc, the scallop, demonstrating significant application value.

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the embodiments do not limit the present invention in any way. For those skilled in the art, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. Attached Figure Description

[0027] Figure 1 Bar chart showing the proliferation rate of MCF-7 cells in different treatment groups of commercial human EST.

[0028] Figure 2 Bar graph showing the proliferation rate of MCF-7 cells in different treatment groups of EST protein expressed in the prokaryotic cells of Scallop scallop. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited thereto.

[0030] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0031] Example 1: Establishment of the EST protease activity detection method of the present invention

[0032] Experimental materials:

[0033] 1. Cells: Human breast cancer cells (MCF-7 cells).

[0034] 2. Reagents: Phenol red-free DMEM medium, dextran-activated carbon hormone-free fetal bovine serum, 17-β estradiol (E2), 3'-phosphoadenosine-5'-phosphosulfate (PAPS), dimethyl sulfoxide (DMSO), dithiothreitol (DTT), MgCl2, BSA (bovine serum albumin), recombinant human EST protein (i.e., commercially available human EST protein, MCE product code HY-P76099A), EST expressed in prokaryotic cells of *Ctenophora indica*, CCK-8, sterile water, 1×PBS, etc.

[0035] 3. Instruments and equipment: cell culture incubator, inverted microscope, enzyme-linked immunosorbent assay (ELISA) reader, clean bench, etc.

[0036] II. Experimental Methods:

[0037] 1. Commercially available human EST protein first reacts with E2:

[0038] This invention establishes an incubation system for the reaction of EST and E2, ensuring a sufficient reaction and preventing toxicity or other damage to MCF-7 cells in subsequent cell experiments.

[0039] Set up three groups:

[0040] 1) Experimental group: An incubation system was established: reaction buffer (50 mM Tris-HCl buffer (pH 7.4), 1 mM MgCl2, 1 mM DTT (final concentration in cell slurry 0.05 mM), BSA (625 μg / mL)), 1 μM E2 (dissolved in DMSO, stock concentration 0.02 mM), 10 μM PAPS (dissolved in DMSO, stock concentration 0.6 mM), and 0.3 μg EST, with a final volume of 50 μl (final DMSO concentration in cell slurry 0.33%). The incubation system was incubated at 37°C for 10 minutes, and the centrifuge tubes were placed in an ice bath for 30 minutes to stop the reaction.

[0041] 2) Negative control group: An incubation system was established: reaction buffer (50 mM Tris-HCl buffer (pH 7.4), 1 mM MgCl2, 1 mM DTT, BSA (625 μg / mL)), 1 μM E2 (dissolved in DMSO, stock solution concentration 0.02 mM), and 10 μM PAPS (dissolved in DMSO, stock solution concentration 0.6 mM)), with a final volume of 50 μl. The incubation system was incubated at 37°C for 10 minutes, and the centrifuge tubes were placed in an ice bath for 30 minutes to stop the reaction.

[0042] 3) Blank control group: Establish an incubation system: reaction buffer (50 mM Tris-HCl buffer (pH 7.4), 1 mM MgCl2, 1 mM DTT, BSA (625 μg / mL)) and 10 μM PAPS (dissolved in DMSO, stock solution concentration 0.6 mM)), with a final volume of 50 μl. Incubate the system at 37°C for 10 minutes, then place the centrifuge tubes in an ice bath for 30 minutes to stop the reaction.

[0043] 2. Assay for the activity identification of commercial human EST protease (referencing the E-SCREEN assay, with modifications to parameters and detection methods: based on the fact that E2 can promote the proliferation of MCF-7 cells, the assay detects whether E2 still has activity after reacting with EST, thereby verifying the enzyme activity of commercial human EST protein):

[0044] 1) MCF-7 cells were cultured for 6-8 days in phenol red-free DMEM medium containing 10% estrogen-free serum. (The phenol red in DMEM medium acts as an indicator to observe and monitor cell growth and proliferation during culture. When the medium turns orange-red, it indicates that the medium is slightly alkaline, requiring close monitoring of cell density and timely passage. Phenol red-free medium is used because phenol red can mimic the effects of steroid hormones, especially estrogen, to avoid interference with experimental results. Estrogen-free serum is used to remove interference from endogenous estrogen.)

[0045] MCF-7 cells were cultured in phenol red-free DMEM containing 10% de-estrogen serum for 6-8 days. The culture medium used in subsequent experiments consisted of 89% phenol red-free DMEM medium + 10% de-estrogen serum + 1% penicillin-streptomycin.

[0046] 2) Cell plating:

[0047] ① Set up a blank control group, a control group, a negative control group, and an experimental group.

[0048] ②The other three groups, except the control group: Logarithmic growth phase cells were taken and analyzed at a concentration of 2 × 10⁻⁶ cells / mL. 4Cells were seeded at a density of 100 μL / ml in 96-well plates. 100 μL of culture medium was added to each well for the control group. After seeding, the cell species, seeding time, and patient name were labeled, and the plates were incubated for 24 hours.

[0049] 3) Drug addition: Remove the 96-well plate and gently aspirate the culture medium from the drug-treated group. Care should be taken to avoid scratching or aspirating the cells during this process. After washing the cells twice with 1×PBS, add 100 μl of the drug solution to each well in all drug-treated groups except the control group.

[0050] ① Blank control group: Mix 950 μl of culture medium and 50 μl of the incubation system after the reaction in section II, 1, and 3 above, and add 100 μl of the mixed drug solution to each well.

[0051] ② Negative control group: Mix 950 μl of culture medium and 50 μl of the incubation system after the reaction in step 2, 1, and 2 above, and add 100 μl of the mixed drug solution to each well.

[0052] ③ Experimental group: Mix 950 μl of culture medium and 50 μl of the incubation system after the reaction in section II.1.1) above, and add 100 μl of the mixed drug solution to each well.

[0053] After the medication is added, place the 96-well plate in an incubator and continue culturing for 24 hours.

[0054] 4) CCK-8 experiment (completely protected from light):

[0055] ① After culturing for 24 hours with the added drug, remove the cells from the incubator and observe their survival status and number under a microscope to ensure that there is a difference in number and that the cell density does not exceed 90%. (If the difference is too small to be observed, further steps are required. However, if the cell condition is very poor and there are too many dead cells, it is unnecessary to waste time on further steps. Because the 96-well plate has been treated with TC, the cells can only grow at the bottom, so the number of cells should not be too high. Too many cells will float in the culture medium, and discarding the culture medium later will result in cell loss and affect the results.)

[0056] ② Prepare the CCK-8 medium solution (10%) in the dark and mix thoroughly by pipetting.

[0057] ③ Discard the culture medium in each well of each group (because DTT is a reducing agent and will affect the OD value, so the culture medium containing DTT needs to be removed before adding CCK8 reagent). After discarding the culture medium, wash the cells in each well of each group twice with 1×PBS.

[0058] ④ Add 100 μl of CCK-8 culture medium mixture to each group of wells. Before adding, ensure that the solution is mixed evenly and uniformly. Add the solution one group at a time to reduce the difference between replicates. Add the solution as quickly as possible to prevent the cells from drying for too long and changing their state.

[0059] ⑤ Place the 96-well plate in a 37℃ CO2 incubator for about 1.5 hours. If the color is still light (consider increasing the number of cells per well for the next seeding), the time can be extended appropriately. If the color is uneven at this time, you can gently shake it.

[0060] ⑥ Measure the OD value at 450nm using an ELISA reader.

[0061] Results are expressed as proliferation rate (PR%), where PR% = (OD experimental group - OD blank) / (OD control group - OD blank) × 100%.

[0062] The enzyme activity of EST protein can be calculated based on the proliferation rate. The formula set in this invention is: Enzyme activity = {[(Proliferation rate of negative control group - Proliferation rate of experimental group) / (Proliferation rate of negative control group - Proliferation rate of blank control group)] Estradiol content / incubation time, enzyme activity is expressed in U (the amount of enzyme that catalyzes the conversion of 1 micromolar substrate per minute).

[0063] The experiment was independently replicated three times. Based on the comparison of the proliferation rate of the commercial human EST experimental group and the negative control group, it was determined that the enzyme activity of the experimental group EST inactivating estradiol within 10 min of incubation was 0.049813±0.002863U.

[0064] 3. To increase the rigor of the experiment and verify the general applicability of the experimental method, this study also performed the above experiment on the EST protein expressed in the prokaryotic cells of *Scallop spp.* to verify the feasibility of the experiment. The experimental steps were the same as the method for verifying the activity of commercially available human EST protein. The enzyme activity of the EST protein expressed in the prokaryotic cells of *Scallop spp.* was 0.05679 ± 0.00497 U.

[0065] III. Data Analysis

[0066] SPSS 27.0 software was used to process the CCK-8 experimental data of commercial human EST protein and EST protein expressed in the prokaryote of scallop, and to compare whether there were significant differences between the experimental group, the negative control group and the control group (P < 0.05 was statistically significant).

[0067] IV. Test Results

[0068] See Figure 1 and Figure 2 .

[0069] The results showed that, regardless of whether commercially available human EST protein or EST protein expressed in the prokaryote of *Scallop spp.* was used, the proliferation rate of the negative control group was greater than that of the experimental group. Furthermore, there were significant differences in proliferation rates between the experimental group and the negative control group, and between the negative control group and the control group (P < 0.05). This indicates that the detection method of this invention can detect both the widely studied and commercially available human EST enzyme activity, and the previously unstudied EST protease activity expressed in the prokaryote of the lesser-known mollusc *Scallop spp.*. This demonstrates the universal applicability of the method for detecting EST enzyme activity and its potential for widespread application in EST enzyme activity detection research across various organisms.

[0070] In summary, the detection method of the present invention is simple to operate, has good repeatability, and provides accurate and reliable results, and can effectively detect EST protease activity.

Claims

1. A method for detecting EST protease activity, characterized in that, Includes the following steps: a. Set up the EST experimental group, negative control group, and blank control group: Add the same volume of buffer system to each group, then add the EST protein to be tested and E2 to the EST experimental group, add E2 to the negative control group, and add the same solvent as the first two groups to the blank control group, so that the solvent concentration and final volume are the same as the first two groups. The buffer system includes Tris-HCl buffer, MgCl2, DTT, BSA, and PAPS. b. The EST experimental group, negative control group, and blank control group were incubated to obtain their respective reaction mixtures; c. Detect the effect of the reaction mixture on the proliferation of breast cancer cells and obtain the proliferation rate of each group; d. Calculate the enzyme activity of the EST protein using the following formula: EST protein enzyme activity = {[(negative control group proliferation rate - experimental group proliferation rate) / (negative control group proliferation rate - blank control group proliferation rate)]} Estradiol content / incubation time.

2. The detection method according to claim 1, characterized in that, In step a, the buffer system contains Tris-HCl buffer with a pH of 7.4 and a concentration of 50 mM; MgCl2 with a concentration of 1 mM; DTT with a concentration of 1 mM; BSA with a concentration of 625 μg / mL; and PAPS with a concentration of 10 μM.

3. The detection method according to claim 1, characterized in that, In step a, the concentrations of the EST protein and E2 in the EST experimental group were 0.3 μg / 50 μl and 1 μM, respectively; the concentration of E2 in the negative control group was 1 μM.

4. The detection method according to claim 3, characterized in that, In step a, E2 is dissolved in DMSO; in the blank control group, the solvent is DMSO and water.

5. The detection method according to any one of claims 1-4, characterized in that, In step b, the incubation reaction involves incubating each group at 37°C, followed by an ice bath to terminate the reaction.

6. The detection method according to claim 5, characterized in that, The incubation time is 10 minutes; the ice bath time is 30 minutes.

7. The detection method according to claim 1, characterized in that, In step c, the detection method is as follows: MCF-7 cells are taken and cultured in phenol red-free DMEM containing 10% estrogen-free serum for 6-8 days, and the cell density is adjusted to 2×10⁶ cells / day. 4 Cells were seeded at a density of 1 / ml in a 96-well plate, incubated for 24 hours, and then the drug was added. After another 24 hours of incubation, the cell proliferation rate was measured.

8. The detection method according to claim 7, characterized in that, The method of administering the medicine is as follows: Take 50 μl of the reaction mixture from step b and mix it with 950 μl of culture medium. Then add 100 μl to each well of a 96-well plate.

9. The detection method according to claim 7 or 8, characterized in that, The culture medium was: 89% phenol red-free DEME medium + 10% estrogen-free serum + 1% penicillin-streptomycin bispecific antibody.

10. The detection method according to claim 7, characterized in that, Cell proliferation rate was detected using the CCK-8 assay.

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