Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In particular, embodiments of the present invention provide for the use of a combination of reagents for preparing a product for purification of a GST-tag-free protein of interest from a product after cleavage of a GST fusion protein, the combination of reagents comprising: anti-GST antibodies, antithrombin antibodies, and solid supports for conjugated antibodies; the mass ratio of the anti-GST antibody to the antithrombin antibody is (10-150): 1, depending on the amount of GST protein to be removed and the amount of thrombin remaining.
According to the method, through specific nano magnetic beads coupled with the anti-GST antibody and the antithrombin antibody, GST tag protein, non-digested GST fusion protein and residual thrombin which need to be removed after enzyme digestion are subjected to affinity adsorption in a 1 single buffer reaction system, and the required pure target protein is obtained at one time. The method for obtaining target proteins by enzyme digestion of different GST fusion proteins is unified into a single convenient mode, and the mixed nano magnetic beads coupled with the anti-GST antibody and the antithrombin antibody can be used as a general tool reagent for purifying the GST fusion proteins after enzyme digestion of thrombin, so that a general technical platform and a general kit for efficiently and conveniently cleaving tag proteins of GST fusion proteins by thrombin are established.
In some embodiments, the mass ratio of the anti-GST antibody to the antithrombin antibody is (range between any one or any two of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150): 1.
In some embodiments, the anti-GST antibody and the antithrombin antibody are each conjugated to a solid support.
In some embodiments, the anti-GST antibody and the antithrombin antibody may independently be antibodies or antigen-binding fragments thereof.
In some embodiments, the antibody comprises at least one of a monoclonal antibody, a polyclonal antibody, and a recombinant antibody.
In some embodiments, the source of the antibody (titer over 1:8000 detected by ELISA) is selected from at least one of rabbit, rat, mouse, and goat.
In some embodiments, the anti-GST antibody comprises a mouse anti-GST monoclonal antibody.
In some embodiments, the antithrombin antibody comprises a rabbit antithrombin polyclonal antibody.
In some embodiments, the solid support comprises at least one of nitrocellulose membrane, nylon membrane, ion exchange resin, polystyrene microwell plates, and magnetic beads.
In some embodiments, the solid support comprises magnetic beads.
In some embodiments, the magnetic beads comprise nanomagnetic beads.
In some embodiments, the amount of the anti-GST antibody coupled to the magnetic nanoparticle is 20-100 μg/mg, and specifically may be any one or any range between any two of 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg/mg.
In some embodiments, the amount of the antibody of the nano magnetic bead coupled antithrombin antibody is 20-100 mug/mg, and specifically can be any one or any two or any range between any two of 20, 30, 40, 50, 60, 70, 80, 90 and 100 mug/mg.
In some embodiments, the particle size of the nanomagnetic beads is 200 to 2000nm, and may specifically be in a range between any one or any two of 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, and 2000 nm.
In some embodiments, the active group of the surface of the nanomagnetic beads for coupling with an antibody comprises one or more of a carboxyl group, an amino group, and an epoxy group; preferably, the active group of the surface of the nano magnetic bead for coupling the antibody comprises carboxyl.
The present invention also provides a product for purifying a GST-tag-free target protein from a product after cleavage of GST fusion protein, which comprises the reagent combination as described in the previous examples.
In some embodiments, the product comprises reagents and/or kits.
In some embodiments, the product further comprises reagents for coupling antibodies to the surface of the magnetic beads, including but not limited to PBS, activating solutions, buffers, blocking solutions, and the like.
In addition, the embodiment of the invention also provides a method for purifying a target protein without GST tag from a product after GST fusion protease cleavage, which comprises the following steps: the combination of reagents described in any of the preceding examples or the product described in any of the preceding examples is mixed with the product of the GST fusion protease cleavage.
In some embodiments, when the solid support is a magnetic bead, the mixing ratio of the magnetic bead to the product after cleavage of the GST fusion protein is (1 mL or 50 mg): (500-2000) μg.
In some embodiments, the mixing ratio is (1 mL or 50 mg): (800-1500) μg, which may be (1 mL or 50 mg): (in the range between any one or any several of 800, 900, 1000, 1100, 1200, 1300, 1400 and 1500).
Preferably, the mass ratio of the anti-GST antibody to the antithrombin antibody coupled to the magnetic beads is (10-150): 1, which is described in any of the foregoing examples, and will not be repeated.
In some embodiments, the conditions of the mixing are: room temperature (5-25 ℃) for 1-4 h. The time may specifically be in a range between any one or any two of 1h, 2h, 3h, and 4h.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
A method for purifying a GST-tag-free protein of interest from a product after cleavage of a GST fusion protein, comprising the steps of.
1. Preparation of self-produced mouse anti-GST monoclonal antibody and rabbit antithrombin polyclonal antibody coupled nanometer magnetic beads:
activating: 50mg of magnetic beads (carboxyl functional groups 800nm particle size beads, 1 mL) were washed three times with MES buffer (pH 6.0): 100mM MES and 500mM NaCl), 50mg of EDC and 25mg of sulfa-NHS were added, dissolved in MES buffer, added to the beads, and reacted at room temperature in the dark for 15 minutes.
Coupling: the magnetic beads are recovered by a magnetic frame, washed once by an MES buffer solution, resuspended in the MES buffer solution, 1mg (1 mg/mL) of the pre-prepared antibody is added according to the coupling amount of the monoclonal antibody of the nano magnetic bead target, and the mixture is placed on a mixing instrument for coupling for 4 hours at 37 ℃.
Closing: the product after coupling was added to 2mL of stop buffer (TBS, 5-10 mM hydroxylamine, pH 8.0) for 0.5h at room temperature. The magnetic beads are recovered by the magnetic rack, washed three times by PBS, and the nano magnetic beads are resuspended by 2mL of PBS and stored in a refrigerator at 4 ℃ for standby.
2. Mixing anti-GST monoclonal antibody coupled nano magnetic beads and rabbit antithrombin polyclonal antibody coupled nano magnetic beads: mixing anti-GST monoclonal antibody coupled nano magnetic beads and rabbit antithrombin polyclonal antibody coupled nano magnetic beads according to the mass ratio of 50:1, and preserving at 4 ℃ for standby.
3. Reaction treatment after thrombin cleavage of the GST fusion protein: the thrombin-cleaved reaction was subjected to phosphate buffer (PBS, pH7.4:137mM NaCl, 8.1mM Na)2 HPO4 、1.47m M KH2 PO4 And 2.7mM KCl) was dialyzed overnight at 4℃to adjust the protein concentration to 1-3 mg/ml for use.
4. The mouse anti-GST monoclonal antibody and the rabbit antithrombin polyclonal antibody are coupled with the nano magnetic beads to capture the corresponding antigen protein: the target GST fusion protein thrombin-cleaved reaction was mixed with the mixed mouse anti-GST monoclonal antibody-conjugated magnetic beads and rabbit antithrombin polyclonal antibody-conjugated nano magnetic beads according to 1000. Mu.g protein (about 1 ml) added to 1ml magnetic beads, mixed well, and incubated at room temperature with shaking for 2 hours.
5. Collecting the purified target protein (in this example, STOML2 is a human protein): and collecting magnetic beads by using a magnetic frame, and keeping the solution as the purified target protein.
6. Enzyme-linked immunosorbent assay (ELISA) assay removes residual thrombin effects. Coating the reactant of each step according to the protein quantity of 0.2 mug per hole, and washing for three times; rabbit antithrombin antibody (0.8 mg/mL) was added after gradient dilution, incubated for 30 min at room temperature, and washed three times; adding goat anti-rabbit IgG antibody (1:8000) marked by horseradish peroxidase as secondary antibody, incubating for 30 minutes at room temperature, and washing for three times; the substrate was added to TMB and after 10 minutes development was stopped with 2M sulfuric acid. The OD at a wavelength of 450nm was measured and the results are shown in Table 1.
TABLE 1 enzyme-linked immunosorbent assay (ELISA) detection of thrombin determination of OD values
Remarks: the Cutoff value is 2.5 times of the negative standard; the thrombin titer of the first behavior coating thrombin pre-cleavage reactant is 0K; the thrombin titer of the second behavior coated thrombin post-cleavage reactant was 64K; the third action coats the thrombin titer in the purified protein of interest to 1K; the fourth behavior coat GST fusion protein thrombin titer, negative standard is 0.1475.
7. After Gel Electrophoresis (PAGE) detects the thrombin cleavage of the GST fusion protein, the purity of the protein of interest is determined. The reaction was subjected to 10. Mu.g protein amount per step, and subjected to 10% PAGE electrophoresis, coomassie brilliant blue staining, and the result was shown in FIG. 1, and a single 63kD band was seen, with purity estimated to be 90% or higher.
Example 2
A method for purifying a GST-tag-free target protein from a GST fusion protein cleaved product was substantially the same as in example 1, except that:
in the step 2, the mixing ratio of the anti-GST monoclonal antibody coupled nano magnetic beads and the rabbit antithrombin polyclonal antibody coupled nano magnetic beads is 80:1.
As a result, the target protein having a purity of 90% or more was obtained, and the residual thrombin was detected at 2 to 4K.
Example 3
A method for purifying a GST-tag-free target protein from a GST fusion protein cleaved product was substantially the same as in example 1, except that:
in the step 2, the mixing ratio of the anti-GST monoclonal antibody coupled nano magnetic beads and the rabbit antithrombin polyclonal antibody coupled nano magnetic beads is 100:1.
As a result, the target protein having a purity of 90% or more was obtained, and the residual thrombin was detected at 4K.
Example 4
A method for purifying a GST-tag-free target protein from a GST fusion protein cleaved product was substantially the same as in example 1, except that:
in the step 2, the mixing ratio of the anti-GST monoclonal antibody coupled nano magnetic beads and the rabbit antithrombin polyclonal antibody coupled nano magnetic beads is 150:1.
As a result, the target protein having a purity of 90% or more was obtained, and the residual thrombin was detected at 16K.
Example 5
A method for purifying a GST-tag-free target protein from a GST fusion protein cleaved product was substantially the same as in example 1, except that:
in the step 1, the rabbit anti-GST polyclonal antibody is coupled with the nano magnetic beads, and in the step 2, the mixing ratio of the rabbit anti-GST polyclonal antibody coupled with the nano magnetic beads and the rabbit antithrombin polyclonal antibody coupled with the nano magnetic beads is 60:1.
As a result, the target protein having a purity of 90% or more was obtained, and the residual detection of thrombin was 1K or less, which was comparable to that obtained by using an anti-GST monoclonal antibody.
Example 6
A method for purifying a GST-tag-free target protein from a GST fusion protein cleaved product was substantially the same as in example 1, except that:
in step 3, GST fusion proteins of different molecular weight sizes were used:
GST fusion protein 1: GST tagged fusion protein NPY (molecular weight 36 kD);
GST fusion protein 2: TEAD3 (molecular weight 48 kD) with GST tag fusion protein.
Both GST fusion proteins can obtain the target protein with 90% purity.
Comparative example 1
A method for purifying a GST-tag-free target protein from a GST fusion protein cleaved product was substantially the same as in example 1, except that:
in the step 2, the mixing ratio of the anti-GST monoclonal antibody coupled nano magnetic beads and the rabbit antithrombin polyclonal antibody coupled nano magnetic beads is 20:1.
As a result, a target protein was obtained, but a GST-tagged protein band was found which was not cleared, and the residual detection of thrombin was 1K or less.
Comparative example 2
A method for purifying a GST-tag-free target protein from a GST fusion protein cleaved product was substantially the same as in example 1, except that:
in the step 2, the mixing ratio of the anti-GST monoclonal antibody coupled nano magnetic beads and the rabbit antithrombin polyclonal antibody coupled nano magnetic beads is 40:1.
As a result, a target protein was obtained, but a weak, uncleaned GST-tagged protein band was observed, and the residual detection of thrombin was 1K or less.
Comparative example 3
A method for purifying a GST-tag-free target protein from a GST fusion protein cleaved product was substantially the same as in example 1, except that:
in the step 2, the mixing ratio of the anti-GST monoclonal antibody coupled nano magnetic beads and the rabbit antithrombin polyclonal antibody coupled nano magnetic beads is 180:1.
As a result, the target protein having a purity of 90% or more was obtained, and the residual thrombin was detected at 16K.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.