Gp96 extraction and purification lysis buffer and extraction and purification method

Abstract

The invention provides an extraction and purification lysate of Gp96 and an extraction and purification method, and belongs to the technical field of protein extraction. The invention provides an improved cell lysis solution which is a mixed solution containing NaHCO3 and EDTA (Ethylene Diamine Tetraacetic Acid), and a toxic protease inhibitor PMSF is replaced by nontoxic EDTA. An extraction and purification method of Gp96 is established based on the cell lysis solution, and meanwhile, a harmful substance Tritonx-100 with protein modification and the like is changed into repeated freezing and thawing operation, so that cell membranes are damaged, and the yield is increased. By utilizing the extraction and purification method disclosed by the invention, the yield of the Gp96 protein can reach 68mu g / g tissue, the submitted purity of the protein can reach 88%, the protein is almost non-toxic to 3T3 cell proliferation, and the clinical application range of the Gp96 can be obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of protein extraction, and particularly relates to a Gp96 extraction and purification lysate and an extraction and purification method. Background Art

[0002] Heat shock protein (HSP) is a class of proteins that are highly conserved in biological evolution and widely present in prokaryotes and eukaryotes. Its main biological functions include: molecular chaperones, participating in the folding and assembly of newly synthesized proteins; binding to other peptide proteins in the cell, especially denatured proteins, participating in the cell's anti-damage, repair and heat tolerance processes; participating in the protein hydrolysis process; binding to antigenic peptides, processing and presenting tumor antigens and maintaining the stability of the intracellular environment; and having a certain regulatory effect on cell growth, development, differentiation and death.

[0003] HSP can be divided into multiple subfamilies such as HSP110, HSP90, HSP70, HSP60, HSP40, small molecule HSP and ubiquitin according to the degree of homology and molecular weight. Heat shock protein gp96 belongs to the HSP90 subfamily. This protein is the most abundant heat shock protein in the endoplasmic reticulum of cells and also has significant biological activity. At present, the source of Gp96-peptide complex is mainly extracted and purified from tumor tissue removed from patients. For example, Chinese patent CN112111016A has improved the extraction and purification method of Gp96-peptide complex (CTL-OT), but there is still a problem of small extraction and purification amount; Zhang Tianyi et al. used Buffer A composed of toxic Triton X-100 for lysis in "Extraction and Purification of Heat Shock Protein gp96-Peptide Complex". Although the extraction and purification amount was increased, there were safety hazards, which also limited clinical application.

[0004] Before Gp96 is extracted and purified, it is usually necessary to lyse it, and the lysate often contains PMSF (phenylmethylsulfonyl fluoride), which is an irreversible serine / cysteine ​​protease inhibitor. Serine proteases are a family of proteases that break peptide bonds in macromolecular proteins to make them small molecular proteins. In mammals, serine proteases play an important role, especially in digestion, coagulation and complement systems. Then the use of PMSF may cause problems in digestion, coagulation and complement systems. Human adenosine homocysteinease is a tetramer present in the cytoplasm, and each monomer subunit is tightly bound to the cofactor nicotinamide adenine dinucleotide (NAD). Cysteine ​​proteases include cathepsin B, H, L, etc., which are generally present in lysosomes and are mainly involved in cell phagocytosis and the removal and digestion of excess intracellular substances. In humans and other animals, they are responsible for aging and apoptosis (programmed cell death), MHC class II immune response, prohormone processing and extracellular matrix remodeling that is important for bone development. If PMSF is used, it may cause cell dysfunction. In addition, PMSF has various toxic manifestations on biological systems. At the cellular level, it can induce cytotoxic reactions through multiple pathways. For example, Huang et al. (2011) used p-Nitroanisole (a compound similar to PMSF) to induce cell cycle arrest, activated the ATM / CHK2 pathway and induced autophagy in colon cancer cells. In addition, studies have pointed out that PMSF may cause structural damage to cell membranes. For example, Xu et al. (2015) found that pH-responsive nanocomplexes can cause mitochondria-mediated apoptosis by producing ROS (reactive oxygen species), which is similar to the mechanism of action of PMSF. For example, PMSF may induce autophagic death (Zhou et al., 2019), which is a process of cell self-elimination. If it is out of control, it may promote cell death. PMSF may inactivate membrane proteins and ultimately cause cell death by changing the acid-base balance (pH) of the internal cell environment. Therefore, the improvement of cell lysate is also an important part of Gp96 extraction and purification. Summary of the invention

[0005] The present invention provides a Gp96 extraction and purification lysate and an extraction and purification method, which can efficiently obtain a natural extract containing Gp96, and the protein extraction amount and purity are significantly improved.

[0006] The invention provides a cell lysis solution, which is a mixed solution containing NaHCO3 and EDTA.

[0007] Preferably, the final concentration of NaHCO3 in the lysate is 30 mM, and the mass percentage of EDTA is 1%.

[0008] The present invention also provides a method for extracting and purifying Gp96, comprising the following steps: mixing human in vitro placental tissue or tumor tissue with the above-mentioned cell lysate to prepare a homogenate, and extracting a supernatant by centrifugation after repeated freezing and thawing; extracting and purifying the supernatant by Gp96 monoclonal antibody-Sepharose 4B affinity chromatography and HiTrap-Q Sepharose ion exchange chromatography in sequence to obtain an extract containing Gp96.

[0009] Preferably, the number of repeated freeze-thaw cycles is 3 to 5 times.

[0010] Preferably, centrifugation is performed after the repeated freezing and thawing, and the centrifugation is performed 1 to 2 times, the rotation speed of each centrifugation is 10000 rpm, the time of the first centrifugation is 4 hours, and the time of the second centrifugation is 0.5 hours.

[0011] Preferably, extracting the supernatant after the centrifugation further comprises salting out the supernatant using ammonium sulfate, and redissolving the salted-out precipitate and then dialyzing;

[0012] The solvent for reconstitution and the dialysate for dialysis both include a PBS solution containing NaCl, with a pH value of 7.2.

[0013] Preferably, the concentration of the PBS solution containing NaCl is 0.01 M, wherein the final concentration of NaCl is 150 mM.

[0014] Preferably, the eluent used in the Gp96 monoclonal antibody-Sepharose 4B affinity chromatography is a 0.01 M glycine-hydrochloric acid buffer solution with a pH value of 2.8.

[0015] Preferably, the pH value of the elution solution of the Gp96 monoclonal antibody-Sepharose 4B affinity chromatography is adjusted to neutral.

[0016] Preferably, the eluent of the HiTrap-Q Sepharose ion exchange chromatography comprises a Tris-HCl buffer solution containing NaCl.

[0017] Preferably, after the extraction and purification, the collected eluate is further concentrated by ultrafiltration using a 30KD membrane package.

[0018] Beneficial effects: The present invention provides a cell lysis solution, which is a mixed solution containing NaHCO3 and EDTA. The conventionally used toxic PMSF is replaced with non-toxic EDTA to ensure product safety. The present invention also provides a method for extracting and purifying Gp96. By using an improved cell lysis solution and replacing Tritonx-100, a harmful substance such as protein modification, with a repeated freeze-thaw operation, the cell membrane is destroyed and the yield is increased. Using the extraction and purification method of the present invention, the amount of Gp96 protein can reach 68μg / g tissue, the purity of the protein submitted for inspection can reach 88%, and it is almost non-toxic to 3T3 cell proliferation, which can significantly improve the clinical application range of Gp96. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a comparison result of the protein yield of Example 1 (after improvement) of the present invention and Comparative Example 1 (before improvement);

[0020] Figure 2 The total ion current chromatogram of the protein in Comparative Example 1;

[0021] Figure 3 is the total ion current chromatogram of the protein in Example 1;

[0022] Figure 4 This is the proliferation curve of HaCat cells at different Tritonx-100 concentrations;

[0023] Figure 5 The effects of Gp96 prepared by different methods on the proliferation of 3T3 cells are shown in the figure. The conventional method represents comparative example 1, and the improved method represents example 1.

[0024] Figure 6 This is a verification diagram of Gp96 extracted and purified in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The invention provides a cell lysis solution, which is a mixed solution containing NaHCO3 and EDTA.

[0026] The final concentration of NaHCO3 in the lysate of the present invention is 30 mM, and the mass percentage of EDTA is 1%. In the present invention, the lysate is prepared by mixing EDTA (pH 8.0) and NaHCO3 (pH 7.2) of corresponding concentrations before use.

[0027] The present invention also provides a method for extracting and purifying Gp96, comprising the following steps: mixing human in vitro placental tissue or tumor tissue with the above-mentioned cell lysate to prepare a homogenate, and extracting a supernatant by centrifugation after repeated freezing and thawing; extracting and purifying the supernatant by Gp96 monoclonal antibody-Sepharose 4B affinity chromatography and HiTrap-Q Sepharose ion exchange chromatography in sequence to obtain an extract containing Gp96.

[0028] The present invention uses human ex vivo placental tissue or tumor tissue as raw material. When ex vivo placental tissue is used as raw material, the capsule and connective tissue are preferably removed. When cryopreserved ex vivo placenta is used as raw material, it is preferably thawed first, and then the capsule and connective tissue are removed, and the tissue is repeatedly washed until it is nearly white. When tumor tissue is used as raw material, the tumor tissue preferably includes liver cancer or kidney cancer.

[0029] The present invention preferably cuts the raw material into pieces and mixes it with a lysis solution to prepare a homogenate. The present invention directly grinds the lysis solution and the cut raw material after mixing. The present invention preferably prepares a homogenate by grinding until there are no obvious block particles. The present invention repeatedly freezes and thaws the homogenate to destroy the cell membrane and increase the yield. The number of repeated freeze-thaws is preferably 3 to 5 times. When freezing, the homogenate is directly placed in a -20°C environment, and when thawing, it is placed in a 4°C environment.

[0030] The present invention preferably performs centrifugation after the repeated freezing and thawing, and the centrifugation is preferably performed 1 to 2 times, the speed of each centrifugation is preferably 10000 rpm, the time of the first centrifugation is preferably 4 hours, and the time of the second centrifugation is preferably 0.5 hours. The centrifugation of the present invention is preferably performed at 4°C, and the supernatant is collected after the centrifugation for the next step.

[0031] The present invention extracts the supernatant after the centrifugation, and preferably also includes salting out with ammonium sulfate, and the salting out precipitate is redissolved and dialyzed. The present invention preferably performs the ammonium sulfate salting out twice, and the operation of the ammonium sulfate salting out is not particularly limited, and the salting out can be performed using conventional methods in the art. The present invention preferably redissolves the salting out precipitate, and the redissolved solvent preferably includes a PBS buffer containing NaCl, the pH value is 7.2, and the concentration of the PBS buffer is preferably 100mM, wherein the final concentration of NaCl is preferably 150mM. The present invention preferably also includes dialysis after the redissolution, and the dialysate for dialysis preferably includes a PBS solution containing NaCl, the pH value is 7.2, and the concentration of the PBS buffer is preferably 0.01M, wherein the final concentration of NaCl is preferably 150mM. The composition ratio of the reconstituted solvent and the dialysate of the present invention can ensure that the Gp96 protein is in a physiological environment close to the natural one, thereby ensuring the integrity of the protein; a 30KD dialysis bag is used during the dialysis, which can dialyze away the added EDTA and improve the safety of products with a molecular weight of about 90KD.

[0032] The present invention loads the above-mentioned dialyzed clear solution onto a Gp96 monoclonal antibody-Sepharose 4B affinity chromatography column, and the loading flow rate of the supernatant is preferably 0.5 mL / min. When the present invention utilizes the Gp96 monoclonal antibody-Sepharose 4B affinity chromatography column for elution, it is preferred that a Tris-HCl buffer solution containing NaCl is first used to elute the chromatography column, and then a glycine-hydrochloric acid buffer solution is used as an eluent to elute the chromatography column, and the concentration of the glycine-hydrochloric acid buffer solution is preferably 0.01 M, and the pH value is 2.8. The present invention preferably adjusts the pH value of the finally collected eluate to neutral, and in the embodiment, preferably uses a 0.5 mol / L TrisHCl buffer containing NaCl to adjust to pH=7.0.

[0033] In the present invention, the eluent adjusted to neutrality is loaded onto a HiTrap-Q Sepharose ion exchange chromatography column, and the loading speed is preferably 1.5 mL / min, and then the chromatography column is eluted with a 400 mM Tris-HCl buffer solution containing NaCl.

[0034] The present invention preferably further comprises ultrafiltration and concentration of the collected eluate to obtain an extract containing Gp96. In the embodiment, a 30KD membrane package is preferably used to ultrafiltration and concentration of the collected eluate.

[0035] In order to further illustrate the present invention, a method for extracting and purifying Gp96 provided by the present invention is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] The preparation of all buffer solutions used in the embodiments of the present invention is as follows:

[0037] Lysis buffer: 30 mM NaHCO3 pH 7.2, 1% EDTA (pH 8.0);

[0038] Dialysate: 100 mM PBS pH 7.2, 150 mM NaCl;

[0039] Affinity chromatography eluent: 100 mM glutamate-hydrochloric acid pH 7.2;

[0040] Ion exchange chromatography eluent: 150 mM Tris-HCl pH 7.2, 600 mM NaCl.

[0041] Example 1

[0042] (1) Pretreatment of human ex vivo placental tissue: remove the capsule and connective tissue of the ex vivo placental tissue or use tumor tissue as raw material, then cut the placental tissue or tumor tissue into pieces, add lysis solution (30mMNaHCO3, 1% EDTA), grind it into a homogenate, and repeat freezing and thawing 3 to 5 times; centrifuge the homogenate twice (the first centrifugation speed is 10000rpm, the time is 1h, and the temperature is 4°C; the second centrifugation speed is 10000rpm, the time is 0.5h, and the temperature is 4°C), and the supernatant is collected after the end.

[0043] After two ammonium sulfate salt precipitations, the salt precipitation precipitate was redissolved in 150 mM NaCl 0.01 M, pH 7.2 PBS, and dialyzed using 150 mM NaCl 0.01 M, pH 7.2 PBS for later use;

[0044] (2) Gp96 monoclonal antibody-Sepharose 4B affinity chromatography: The supernatant obtained in step (1) was dialyzed against 0.01 M PBS containing 150 mM NaCl, pH 7.2, and then loaded onto a Gp96 monoclonal antibody-Sepharose 4B affinity chromatography column at a rate of 0.5 mL / min. The column was repeatedly washed with 0.01 M PBS containing 150 mM NaCl, pH 7.2, for about 5 column volumes until the flow-through was free of protein. The column was then eluted with a pH 2.8, 0.01 M glycine-HCl buffer solution, and the collected eluate was immediately adjusted to pH = 7.0 with 0.5 M TrisHCl containing NaCl.

[0045] (3) HiTrap-Q Sepharose ion exchange chromatography: The eluate obtained in step (2) was loaded onto a HiTrap-Q Sepharose ion exchange chromatography column at a rate of 1.5 mL / min, and then the ion exchange chromatography column was eluted with a Tris-HCl buffer solution containing NaCl. The collected eluate was ultrafiltered and concentrated using a 30 KD membrane package to obtain an extract containing Gp96 ( Figure 6 ).

[0046] Comparative Example 1

[0047] Gp96 was extracted and purified with reference to the method of Chinese patent CN112111016A (a method for extracting and purifying a Gp96-polypeptide complex (CTL-OT), Hehong Shangyi (Chengdu) Biotechnology Co., Ltd., applied for on September 24, 2020).

[0048] The protein concentration relative to the tissue was determined by BCA protein concentration assay, and the protein purity was determined by protein total ion current chromatography. Figure 1 , Figure 2 and Figure 3 As shown, the protein yield of Comparative Example 1 is 9 μg / g tissue, and the purity is 48%; while the protein yield of Example 1 is 67 μg / g tissue, and the purity is 88%.

[0049] Experimental Example 1

[0050] Determination of Gp96 cytotoxicity using CCK-8 assay

[0051] 1. Effect of Tritonx-100 on HaCat cell proliferation:

[0052] 1) Preparation: Resuscitate HaCat (human immortalized keratinocytes, purchased from Changsha Baiyiyuan Biotechnology Co., Ltd.), and expand and culture them in DMEM medium containing 10% FBS. Collect cells in the logarithmic growth phase, digest them with 0.25% trypsin containing 1 mg / ml EDTA, collect the cell suspension by pipetting with DMEM medium containing 10% FBS, wash by centrifugation at 1000g, discard the supernatant, suspend the cell pellet with DMEM medium containing 10% FBS, and adjust the cell concentration to 5×10 4 Pieces / ml.

[0053] 2) Inoculate into a 96-well cell culture plate, 100 μL per well, and culture in a carbon dioxide incubator at 37° C., 5% CO 2 , and saturated humidity for 24 hours.

[0054] 3) Methods:

[0055] (1) Under sterile conditions, Tritonx-100 (purchased from China National Pharmaceutical Group) was diluted in DMEM medium containing 10% FBS to different gradients: 1‰, 0.25‰, 0.0625‰, 0.015625‰, 0.0090625‰, 0.0009765625‰, 0.00024414062‰, 0.00006103515‰, 0.00001525878‰, 0.00000381469‰, 0.000000956367‰ and 0 (negative control);

[0056] (2) Transfer the serially diluted Tritonx-100 to the 96-well cell culture plate in the "Preparation" process, 100 μL per well, 3 replicates per concentration, and gently shake to mix;

[0057] (3) Place in an incubator at 37°C, 5% CO2, and saturated humidity for 20 hours;

[0058] (4) 4 hours before the end of culture, 10 μL of CCK8 detection reagent was added to each well;

[0059] (5) After the culture is completed, measure the OD using an ELISA reader. 450 value.

[0060] 4) Results: With Tritonx-100 concentration as the horizontal axis, the OD values ​​of the triplicate wells of HaCat cells at different Tritonx-100 concentrations are shown. 450 The mean is the vertical axis, and the proliferation curve is produced. The result is as follows Figure 4 As shown, Tritonx-100, a component in the lysis buffer, has significant toxicity to the proliferation of HaCat cells.

[0061] 2. Effects of Gp96 obtained in Example 1 and Gp96 obtained in the comparative example on the proliferation of 3T3 cells (mouse fibrosarcoma cells, purchased from Changsha Baiyiyuan Biotechnology Co., Ltd.):

[0062] 1) Preparation: Resuscitate 3T3 (mouse fibrosarcoma cell line) and culture with DMEM medium containing 10% FBS. Collect cells in the logarithmic growth phase, digest with 0.25% trypsin containing 1 mg / ml EDTA, collect the cell suspension by pipetting with DMEM medium containing 10% FBS, wash by centrifugation at 1000 g, discard the supernatant, suspend the cell pellet with DMEM medium containing 10% FBS, and adjust the cell concentration to 5×10 4 Pieces / ml.

[0063] The cells were inoculated into a 96-well cell culture plate, with 100 μL per well, and cultured for 24 hours in a carbon dioxide incubator at 37°C, 5% CO2, and saturated humidity.

[0064] 2) Methods:

[0065] (1) Under sterile conditions, GP96 prepared by conventional and improved methods were diluted to 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0 (cell control) μg / ml;

[0066] (2) Transfer the GP96 samples prepared by the conventional and modified methods after gradient dilution to the 96-well cell culture plate in the "Preparation" process, 100 μL per well, 3 replicates per concentration, and gently shake to mix;

[0067] (3) Place in an incubator at 37°C, 5% CO2, and saturated humidity for 20 hours;

[0068] (4) 4 hours before the end of culture, 10 μL of CCK8 detection reagent was added to each well;

[0069] (5) After the culture is completed, measure the OD using an ELISA reader. 450 value.

[0070] 3) Results: With protein concentration as the horizontal axis, the OD values ​​of the three replicate wells corresponding to the GP96 samples prepared in Example 1 and Comparative Example 1 450 The mean is the vertical axis, and the proliferation curve is made as follows Figure 5 As shown, the Gp96 prepared in Example 1 has almost no toxicity to the proliferation of 3T3 cells, while the GP96 prepared in Comparative Example 1 has significant toxicity to 3T3 cells at a concentration of 2.5 μg / ml and above.

[0071] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A cell lysis solution, characterized in that The lysate is a mixed solution containing NaHCO3 and EDTA.

2. The cell lysate according to claim 1, characterized in that The final concentration of NaHCO3 in the lysate is 30 mM, and the mass percentage of EDTA is 1%.

3. A method for extracting and purifying Gp96, characterized in that: The following steps are involved: The human ex vivo placental tissue or tumor tissue is mixed with the cell lysate according to claim 1 or 2 to prepare a homogenate, and the supernatant is extracted by centrifugation after repeated freezing and thawing; the supernatant is extracted and purified by Gp96 monoclonal antibody-Sepharose4B affinity chromatography and HiTrap-Q Sepharose ion exchange chromatography in sequence to obtain an extract containing Gp96.

4. The extraction and purification method according to claim 3, characterized in that: The number of repeated freezing and thawing is 3 to 5 times.

5. The extraction and purification method according to claim 3 or 4, characterized in that: The centrifugation was performed 1 to 2 times, the rotation speed of each centrifugation was 10000 rpm, the time of the first centrifugation was 4 hours, and the time of the second centrifugation was 0.5 hours.

6. The extraction and purification method according to claim 3, characterized in that: After extracting the supernatant, the method further comprises salting out the supernatant with ammonium sulfate, redissolving the salting-out precipitate and then dialyzing; The solvent for reconstitution and the dialysate for dialysis both include a PBS solution containing NaCl, with a pH value of 7.

2.

7. The extraction and purification method according to claim 3, characterized in that: The eluent used in the Gp96 monoclonal antibody-Sepharose 4B affinity chromatography is a 0.01 M glycine-hydrochloric acid buffer solution with a pH value of 2.

8.

8. The extraction and purification method according to claim 3 or 7, characterized in that: The method comprises adjusting the pH value of the elution solution of the Gp96 monoclonal antibody-Sepharose 4B affinity chromatography to neutral.

9. The extraction and purification method according to claim 3, characterized in that: The eluent of the HiTrap-Q Sepharose ion exchange chromatography includes a Tris-HCl buffer solution containing NaCl.

10. The extraction and purification method according to claim 3, characterized in that: After the extraction and purification, the collected eluate is ultrafiltered and concentrated using a 30KD membrane package.

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