CN119529052A

CN119529052A - A Gp96 extraction and purification lysate and extraction and purification method

Info

Publication number: CN119529052A
Application number: CN202411304427.0A
Authority: CN
Inventors: 王刚
Original assignee: Hehong Shangyi Chengdu Biotechnology Co ltd
Current assignee: Hehong Shangyi Chengdu Biotechnology Co ltd
Priority date: 2024-09-19
Filing date: 2024-09-19
Publication date: 2025-02-28

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 lysate, which is a mixed solution containing NaHCO ₃ and EDTA, and replaces a toxic protease inhibitor PMSF with nontoxic EDTA. The invention establishes an extraction and purification method of Gp96 based on the cell lysate, and simultaneously changes harmful substances Tritonx-100 with protein modification and the like into repeated freezing and thawing operations, damages cell membranes and increases the yield. By using the extraction and purification method disclosed by the invention, the Gp96 protein can reach 68 mug/g tissue, the purity of protein delivery detection can reach 88%, the method is almost nontoxic to 3T3 cell proliferation, and the clinical application range of Gp96 can be obviously improved.

Description

Gp96 extraction and purification lysate and extraction and purification method

Technical Field

The invention belongs to the technical field of protein extraction, and particularly relates to an extraction and purification lysate of Gp96 and an extraction and purification method.

Background

Heat shock proteins (Heat shockprotein, HSP) are a class of proteins which are highly conserved in biological evolution and widely exist in prokaryotes and eukaryotes, and have the main biological functions of molecular chaperones, participating in folding and assembling of newly synthesized proteins, combining with other peptide proteins in cells, especially denatured proteins, participating in the processes of anti-injury, repair and heat tolerance of cells, participating in the proteolytic process, combining antigen peptides, processing and presenting tumor antigens, maintaining the stability of the environment in cells and the like, and having a certain regulation effect on the growth, development, differentiation and death of cells.

HSPs can be divided into subfamilies of HSP110, HSP90, HSP70, HSP60, HSP40, small molecule HSPs, ubiquitin, and the like, depending on the degree of homology and molecular weight. Heat shock protein gp96 belongs to a member of the HSP90 subfamily, which is the most abundant heat shock protein on the cytoplasmic reticulum, which also has significant biological activity. At present, the source of the Gp 96-polypeptide complex is mainly extracted and purified from tumor tissues excised from patients, for example, the extraction and purification method of the Gp 96-polypeptide complex (CTL-OT) is improved as shown in Chinese patent CN112111016A, but the problem of small extraction and purification amount still exists, and Zhang Tianyi et al in heat shock protein Gp 96-peptide complex extraction and purification uses BufferA composed of toxic Triton X-100 for cracking, so that the extraction and purification amount is improved, but potential safety hazards exist, and clinical application is limited.

Cleavage is generally required before Gp96 is extracted and purified, and PMSF (phenylmethylsulfonyl fluoride) is often contained in the lysate, PMSF being an irreversible serine/cysteine protease inhibitor. Serine proteases are a family of proteases whose function is to cleave peptide bonds in macromolecular proteins, making them small molecule proteins. Serine proteases play an important role in mammals, particularly in the digestive, coagulation and complement systems. Then the use of PMSF may cause problems with the digestive, clotting and complement systems. Human adenylhomocysteine is a tetramer that exists in the cytoplasm, each monomeric subunit being tightly bound to the cofactor nicotinamide adenine dinucleotide (Nicotinamide adenine dinucleotide, NAD). Cysteine proteases, including cathepsins B, H, L and the like, are commonly found in lysosomes and are primarily involved in phagocytosis of cells and removal and digestion of intracellular excess material. In humans and other animals, they are responsible for aging and apoptosis (programmed cell death), mhc class ii immune responses, prohormone treatment and extracellular matrix remodeling important for skeletal development. If PMSF is used, it may cause disturbance of cell functions. In addition, PMSF exhibits various toxicity to biological systems. At the cellular level, it can induce a cytotoxic response through multiple pathways, such as Huang et al (2011) using p-Nitroanisole (a compound similar to PMSF) to induce cell cycle arrest, activate the ATM/CHK2 pathway and induce autophagy of colon cancer cells. Furthermore, studies indicate that PMSF may lead to structural disruption of cell membranes, as Xu et al (2015) studies found that pH-responsive nanocomposites can cause mitochondrial-mediated apoptosis by generating ROS (reactive oxygen species), which is also similar to the mechanism of action of PMSF, and as such PMSF may trigger autophagic death (Zhou et al, 2019), a process by which cells themselves eliminate, if uncontrolled, may promote cell death. PMSF may inactivate membrane proteins and eventually lead to cell death by altering the acid-base balance (pH) of the cell's internal environment. Therefore, the improvement of the cell lysate is also an important link for the extraction and purification of Gp 96.

Disclosure of Invention

The invention provides an extraction and purification lysate and an extraction and purification method of Gp96, which can efficiently obtain natural Gp 96-containing extract, and the protein extraction amount and the purity are obviously improved.

The invention provides a cell lysate, which is a mixed solution containing NaHCO ₃ and EDTA.

Preferably, the final concentration of NaHCO ₃ in the lysate is 30mM, and the mass percentage of EDTA is 1%.

The invention also provides a Gp96 extraction and purification method, which comprises the following steps of mixing human placenta tissue or tumor tissue with the cell lysate to prepare homogenate, repeatedly freezing and thawing, centrifuging to extract supernatant, and sequentially extracting and purifying the supernatant through Gp96 monoclonal antibody-Sepharose 4B affinity chromatography and HiTrap-Q Sepharose ion exchange chromatography to obtain an extract containing Gp 96.

Preferably, the repeated freezing and thawing times are 3-5 times.

Preferably, centrifugation is performed after repeated freeze thawing, the centrifugation is performed for 1-2 times, the rotation speed of each centrifugation is 10000rpm, the time of the first centrifugation is 4 hours, and the time of the second centrifugation is 0.5 hours.

Preferably, extracting supernatant after the centrifugation, further comprising salting out the supernatant with ammonium sulfate salt, and redissolving the salting-out precipitate and then dialyzing;

The solvent for re-dissolution and the dialysate for dialysis both comprise PBS solution containing NaCl, and the pH value is 7.2.

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

Preferably, the eluent used for the Gp96 monoclonal antibody-Sepharose 4B affinity chromatography is 0.01M glycine-hydrochloric acid buffer solution, and the pH value is 2.8.

Preferably, the method comprises adjusting the pH value of an elution solution of Gp96 monoclonal antibody-Sepharose 4B affinity chromatography to neutral.

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

Preferably, after the extraction and purification, ultrafiltration concentration is further carried out on the collected eluent by adopting a membrane package of 30 KD.

The invention has the beneficial effects that the cell lysate is the mixed solution containing NaHCO ₃ and EDTA, and the conventionally used toxic PMSF is replaced by the nontoxic EDTA, so that the product safety can be ensured. The invention also provides an extraction and purification method of Gp96, which uses improved cell lysate and changes harmful substances Tritonx-100 with protein modification into repeated freezing and thawing operations to destroy cell membranes and increase yield. By using the extraction and purification method disclosed by the invention, the Gp96 protein can reach 68 mug/g tissue, the purity of protein delivery detection can reach 88%, the method is almost nontoxic to 3T3 cell proliferation, and the clinical application range of Gp96 can be obviously improved.

Drawings

FIG. 1 is a graph showing the results of comparison of the protein yields of example 1 (after improvement) and comparative example 1 (before improvement) according to the present invention;

FIG. 2 is a total ion flow chromatogram of the protein of comparative example 1;

FIG. 3 is a total ion flow chromatogram of the protein of example 1;

FIG. 4 shows the proliferation curves of HaCat cells at different Tritonx-100 concentrations;

FIG. 5 is a graph showing the effect of Gp96 on 3T3 cell proliferation obtained by various methods, wherein comparative example 1 is shown in general and example 1 is shown in modified form;

FIG. 6 is a verification graph of Gp96 obtained by extraction and purification in example 1 of the present invention.

Detailed Description

The final concentration of NaHCO ₃ in the lysate is 30mM, and the mass percentage of EDTA is 1%. In the invention, EDTA (pH 8.0) with corresponding concentration and NaHCO ₃ (pH 7.2) are mixed before the lysate is used.

The invention takes human isolated placenta tissue or tumor tissue as raw material, when taking isolated placenta tissue as raw material, it is preferable to remove envelope material and connective tissue, when taking frozen isolated placenta as raw material, it is preferable to defrost first, then remove envelope material and connective tissue, and wash the tissue repeatedly to near white, when taking tumor tissue as raw material, it is preferable that the tumor tissue includes liver cancer or kidney cancer.

In the invention, the raw materials are preferably sheared and then mixed with the pyrolysis liquid to prepare homogenate. The invention mixes the cracking liquid with the sheared raw materials and then directly grinds the mixture. The invention is preferably prepared by grinding into a homogenate until no distinct bulk particles are present. The invention can destroy cell membranes and increase yield by repeatedly freezing and thawing the homogenate, wherein the times of repeated freezing and thawing are preferably 3-5 times, the homogenate is directly placed in an environment of-20 ℃ when being frozen, and the homogenate is placed in an environment of 4 ℃ when being thawed.

The invention preferably carries out centrifugation after repeated freeze thawing, the centrifugation is preferably carried out for 1-2 times, the rotating speed of each centrifugation is preferably 10000rpm, the time of the first centrifugation is preferably 4h, and the time of the second centrifugation is preferably 0.5h. The centrifugation according to the invention is preferably carried out at 4℃and the supernatant is collected after the centrifugation and subjected to the next operation.

The invention extracts supernatant after the centrifugation, preferably further comprises salting out by ammonium sulfate salt, redissolving the salting-out precipitate and dialyzing. In the present invention, the salting-out of the ammonium sulfate salt is preferably performed twice, and the operation of salting-out of the ammonium sulfate salt is not particularly limited, and the salting-out may be performed by a conventional method in the art. The salting-out precipitate is preferably redissolved according to the invention, the redissolved solvent preferably comprises 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 invention preferably further comprises performing dialysis after said reconstitution, said dialysis fluid preferably comprises a PBS solution containing NaCl, having a pH of 7.2, and said PBS buffer preferably has a concentration of 0.01M, wherein the final concentration of NaCl is preferably 150mM. The composition ratio of the redissolved solvent and the dialysate can ensure that the Gp96 protein is in a physiological environment close to nature and the integrity of the protein, and the added EDTA can be dialyzed by adopting a dialysis bag with 30KD during dialysis, and the safety of a molecular weight product with about 90KD is improved.

The invention loads the dialyzed clear liquid into a Gp96 monoclonal antibody-Sepharose 4B affinity chromatography column, and the loading flow rate of the clear liquid is preferably 0.5mL/min. When the Gp96 monoclonal antibody-Sepharose 4B affinity chromatographic column is used for elution, the chromatographic column is preferably eluted by adopting a Tris-HCl buffer solution containing NaCl, and then the chromatographic column is eluted by adopting a glycine-hydrochloric acid buffer solution as an eluent, wherein the concentration of the glycine-hydrochloric acid buffer solution is preferably 0.01M, and the pH value is 2.8. The final collected eluate is preferably adjusted to neutral pH, and in the example, 0.5mol/L TrisHCl buffer containing NaCl is preferably used to adjust pH to 7.0.

The invention applies the eluent which is regulated to be neutral to a HiTrap-Q Sepharose ion exchange chromatographic column, the loading speed is preferably 1.5mL/min, and then the chromatographic column is eluted by adopting 400mM Tris-HCl buffer solution containing NaCl.

The invention preferably further comprises subjecting the collected eluate to ultrafiltration concentration to obtain an extract containing Gp96, preferably using a 30KD membrane pack.

In order to further illustrate the present invention, the following examples are provided to illustrate the extraction and purification method of Gp96 of the present invention, but they should not be construed as limiting the scope of the present invention.

All buffer solutions used in the examples of the present invention were formulated as follows:

30mMNaHCO ₃ pH7.2,1% EDTA (pH 8.0);

100mM PBS pH7.2,150mM NaCl of dialysate;

affinity chromatography eluent of 100mM glutamic acid-hydrochloric acid pH7.2;

Ion exchange chromatography eluate 150mM Tris-HCl pH7.2,600mM NaCl.

Example 1

(1) Pretreatment of human placenta tissue comprises removing coating substance and connective tissue or taking tumor tissue as raw material, cutting placenta tissue or tumor tissue, adding lysate (30 mMNaHCO ₃, 1% EDTA), grinding to obtain homogenate, repeatedly freezing and thawing for 3-5 times, centrifuging the homogenate twice (the first centrifugation speed is 10000rpm for 1 hr at 4deg.C, the second centrifugation speed is 10000rpm for 0.5 hr at 4deg.C), collecting supernatant

Salting out the precipitate twice with ammonium sulfate salt, re-dissolving in 150mM NaCl 0.01M, pH 7.2PBS, and dialyzing with 150mM NaCl 0.01M, pH 7.2.2 PBS for use;

(2) Gp96 monoclonal antibody-Sepharose 4B affinity chromatography, wherein the supernatant obtained in the step (1) is dialyzed by 0.01M, pH 7.2.2 PBS containing 150mM NaCl, and then is loaded on a Gp96 monoclonal antibody-Sepharose 4B affinity chromatography column at a speed of 0.5mL/min, repeatedly washed by 0.01M, pH 7.2.2 PBS containing 150mM NaCl with about 5 column volumes until the running-through liquid does not contain protein, and then the chromatography column is eluted by using pH 2.8, 0.01M glycine-hydrochloric acid buffer solution, and the collected eluent is immediately adjusted to pH=7.0 by using 0.5M TrisHCl containing NaCl;

(3) HiTrap-Q Sepharose ion exchange chromatography, wherein the eluate obtained in step (2) was applied to HiTrap-Q Sepharose ion exchange chromatography column at a rate of 1.5mL/min, and then eluted with Tris-HCl buffer solution containing NaCl, and the collected eluate was subjected to ultrafiltration concentration using a 30KD membrane pack to obtain an extract containing Gp96 (FIG. 6).

Comparative example 1

The extraction and purification of Gp96 were carried out by referring to Chinese patent CN112111016A (a method for extracting and purifying Gp 96-polypeptide complex (CTL-OT), and Hongshan medical (Chengdu) Biotechnology Co., ltd., 2020, 24 th month).

Protein yield relative to tissue was determined using BCA protein concentration assay and protein purity by total ion chromatography, as shown in FIGS. 1,2 and 3, with comparative example 1 having a protein yield of 9. Mu.g/g tissue and a purity of 48% and example 1 having a protein yield of 67. Mu.g/g tissue and a purity of 88%.

Experimental example 1

Determination of Gp96 cytotoxicity Using CCK-8 method

1. Tritonx-100 effects on HaCat cell proliferation:

1) Preparation was prepared by resuscitating HaCat (human immortalized keratinocytes, purchased from Changsha Bai memory Biotech Co., ltd.) and performing expansion culture using DMEM medium containing 10% FBS. Cells in the logarithmic growth phase were collected, digested with 0.25% trypsin containing 1mg/ml EDTA, collected cell suspension was blown with DMEM medium containing 10% FBS, washed by centrifugation at 1000g, the supernatant was discarded, cell pellet was suspended with DMEM medium containing 10% FBS, and cell concentration was adjusted to 5X 10 ⁴ cells/ml.

2) Inoculated into 96-well cell culture plates, 100. Mu.L per well, 37℃in a 5% CO ₂, saturated humidity carbon dioxide incubator, and cultured for 24 hours.

3) The method comprises the following steps:

(1) Under aseptic conditions Tritonx-100 (purchased from chinese drug groups) were diluted ：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) in different gradients using DMEM medium containing 10% fbs;

(2) Transferring Tritonx-100 after gradient dilution into 96-well cell culture plate in the process of 'preparation', mixing 100 μl of each well with 3 multiple wells of each concentration by gentle shaking;

(3) Placing the mixture in a incubator with the temperature of 37 ℃ and the CO ₂ percent and the saturated humidity, and culturing the mixture for 20 hours;

(4) 4 hours before the end of the culture, 10 mu L of CCK8 detection reagent is added into each hole;

(5) After the completion of the culture, OD ₄₅₀ was measured by an ELISA reader.

4) As a result, proliferation curves are produced by taking the OD ₄₅₀ mean value of three compound holes corresponding to HaCat cells at the concentrations of Tritonx-100 as the abscissa and taking the OD ₄₅₀ mean value of three compound holes corresponding to HaCat cells at the concentrations of different Tritonx-100 as the ordinate, and the results show that the components Tritonx-100 in the lysate have obvious toxicity to the proliferation of the HaCat cells in the results shown in FIG. 4.

2. Effect of Gp96 obtained in example 1 and Gp96 obtained in comparative example on proliferation of 3T3 cells (mouse fibrosarcoma cells, purchased from longsand hundreds of memristive ltd):

1) Preparation was performed by resuscitating 3T3 (mouse fibrosarcoma cell line) and performing expansion culture in DMEM medium containing 10% FBS. Cells in the logarithmic growth phase were collected, digested with 0.25% trypsin containing 1mg/ml EDTA, collected cell suspension was blown with DMEM medium containing 10% FBS, washed by centrifugation at 1000g, the supernatant was discarded, cell pellet was suspended with DMEM medium containing 10% FBS, and cell concentration was adjusted to 5X 10 ⁴ cells/ml.

Inoculated into 96-well cell culture plates, 100. Mu.L per well, 37℃in a 5% CO ₂, saturated humidity carbon dioxide incubator, and cultured for 24 hours.

2) The method comprises the following steps:

(1) GP96 prepared conventionally and in the modification method was diluted in gradient of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0 (cell control) μg/ml under aseptic conditions;

(2) Transferring the GP96 sample prepared by the conventional and improved method after gradient dilution into a 96-well cell culture plate in the process of preparation, and lightly shaking and uniformly mixing 100 mu L of each well and 3 compound wells of each concentration;

3) As a result, the proliferation curve was prepared with the protein concentration on the abscissa and the OD ₄₅₀ mean value of the triplicate wells corresponding to the GP96 samples prepared in example 1 and comparative example 1 on the ordinate, as shown in FIG. 5, and Gp96 prepared in example 1 was almost non-toxic to proliferation of 3T3 cells, whereas GP96 prepared in comparative example 1 had significant toxicity to 3T3 cells at a concentration of 2.5. Mu.g/ml and above.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. A cell lysate, which is characterized in that the lysate is a mixed solution containing NaHCO ₃ and EDTA.

2. The cell lysate of claim 1, wherein the final concentration of NaHCO ₃ in the lysate is 30mM and the mass percentage of EDTA is 1%.

3. A Gp96 extraction and purification method is characterized by comprising the steps of mixing isolated placenta tissues or tumor tissues of human sources with the cell lysate of claim 1 or 2 to prepare homogenate, repeatedly freezing and thawing, centrifuging to extract supernatant, and sequentially extracting and purifying the supernatant through Gp96 monoclonal antibody-Sepharose 4B affinity chromatography and HiTrap-Q Sepharose ion exchange chromatography to obtain an extract containing Gp 96.

4. The extraction and purification method according to claim 3, wherein the number of repeated freeze thawing is 3 to 5.

5. The method according to claim 3 or 4, wherein the centrifugation is performed 1 to 2 times, each time the rotational speed of the centrifugation is 10000rpm, the time of the first centrifugation is 4 hours, and the time of the second centrifugation is 0.5 hours.

6. The method according to claim 3, further comprising salting out the supernatant with ammonium sulfate salt, redissolving the salting-out precipitate, and dialyzing;

7. The method according to claim 3, wherein the Gp96 monoclonal antibody-Sepharose 4B affinity chromatography is performed using 0.01M glycine-hydrochloric acid buffer at a pH of 2.8.

8. The method according to claim 3 or 7, comprising adjusting the pH of the elution solution of Gp96 monoclonal antibody-Sepharose 4B affinity chromatography to neutral.

9. The method according to claim 3, wherein the eluent of HiTrap-Q Sepharose ion exchange chromatography comprises Tris-HCl buffer solution containing NaCl.

10. The method according to claim 3, further comprising ultrafiltration concentration of the collected eluate using a 30KD membrane pack after the extraction and purification.