RU2693251C1 - Method for extracting and purifying recombinant protein, an analogue of a human kappa-casein fragment having cytotoxic activity towards human cancer cells - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, genetic and protein engineering. Method for extraction and purification of recombinant protein, analogue of human kappa-casein fragment, involves cultivation of HEK293T-producing cells expressing recombinant EL1 protein, concentration and purification of the culture medium first using cation-exchange chromatography on SP-sepharose, and then using nickel-affinity chromatography on IMAC-sepharose, successive elution from impurity proteins and target product sorbents using a system of eight buffer solutions and subsequent purification of the end product by dialysis against 150 mM NaCl.EFFECT: invention increases specific activity and purity of the end product.1 cl, 7 dwg, 4 ex

Description

The invention relates to biotechnology, genetic and protein engineering, specifically, to a method for isolating and purifying a recombinant protein, an analogue of a human kappa-casein fragment (recombinant EL1 protein), which has cytotoxic activity against human cancer cells.

Reducing mortality from malignant tumors is an important task of modern medicine. One of the directions in solving this problem is the development and implementation of modern anticancer drugs. The development of drugs that induce apoptosis of cancer cells deserves special attention. Currently, several such protein drugs are already used in clinics for the treatment of certain oncological diseases. These are mainly drugs based on the tumor necrosis factor (TNF) and other proteins of the TNF family (TRIAL, FasL).

There is a method for isolation and purification of recombinant TNF-beta protein, including cultivation of E.coli producer strain SG20050 / pLT 21, sonication of cells, removal of cell debris, chromatographic purification on columns with diethylaminoethylcellulose (DEAE-cellulose) and hydroxylapate and an additional stage of the stage. on hydroxyapatite in the concentration gradient of potassium phosphate monosubstituted at pH values of 7.0-7.2 (Patent RU 2132385 C1, op. 06.26.1999).

A known method for the isolation and purification of recombinant antitumor proteins TRAIL person, consisting in the following. The destroyed cell mass is precipitated by centrifugation at 28000 rpm for 40 minutes. The soluble fraction is applied to a chromatographic column with nickel agarose (Ni-NTA High Performance). Trx-DR5-A or Trx-DR5-B fusion proteins are eluted in a buffer containing 25 mM NaH ₂ PO ₄ , 500 mM NaCl, 500 mM imidazole (pH 7.4). Purified preparations were dialyzed against a buffer containing 50 mM Tris / HCl (pH 8.0), 80 mM NaCl and 1 mM DTT at 4 ° C for 18 hours. After dialysis, the Trx-DR5-A or Trx-DR5-B proteins are cleaved with the recombinant light chain of human enteropeptidase for 18 hours at room temperature. After cleavage, the residual activity of enteropeptidase is removed on a column with STI (soybean trypsin inhibitor, soybean trypsin inhibitor) agarose. Mutant proteins DR5-A or DR5-B are separated from thioredoxin on a nickel agarose column. At the last stage, the preparations are dialyzed against a buffer containing 50 mM sodium phosphate (pH 7.5) and 150 mM NaCl, then sterilized through a filter and stored at 4 ° C for further use (patent RU 2405038 C1, op. 27.11.2010).

A method of obtaining a hybrid protein TNFRl-Fc, including the cultivation of HEK293 cells sequentially transformed by the introduction of the pFIG-hTNFr vector and the psiRNA-h7SK-miR7 vector in a liquid nutrient medium, followed by isolation and purification of the TNFRl-Fc protein using affinity chromatography on a GH-ROS (using a GH-Fc protein) (by purification using TNFRl-Fc using GHG 293) ). For this, 5 ml columns are equilibrated with working buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, the resulting supernatants are passed through the column at a rate of 1 ml / min, then the columns are washed with working buffer (10 V columns) and protein 0 is eluted , 1M solution of glycine pH 2.5 in tubes containing 100 mm solution of Tris-HCl, pH 9.0 (patent RU 2625010 C1, op. 11.07.2017).

However, drugs based on anti-inflammatory cytokines cause serious side effects, which limits their use, therefore, the search for new proteins that can inhibit the growth and cause apoptotic death of cancer cells is an important task.

A natural peptide called lactaptin, which has a cytotoxic effect on cancer cells, was previously discovered and studied (patent RU 2317304 C1, op. 02/20/2008). This peptide, which is a fragment of kappa-casein from human milk, has a molecular weight of about 8.6 kDa and contains an established sequence of 74 amino acid residues, which includes a proteolytic fragment of kappa-casein from 63 to 123 amino acid residue. The known protein possesses apoptotic activity against the cultures of MCF-7 breast adenocarcinoma cells (Necipelae VV and others. Lactaptin is a human milk peptide inducing apoptosis of MCF-7 adenocarcinoma cells (Dokl. AN. - 2008, - T. 419. - p. 268-2712)).

The main disadvantage of the drug based on natural human kappa-casein is the high cost and limited source of raw materials for its production.

Later, a series of recombinant analogues of the natural protein was obtained (Semenov DV et al., The Protein Journal. - 2010. - Vol. 29. - N. 3. - P. 174-180; patent EAPO 023475, op. 06/30/2016; patent EAPO 023387, op. 31.05.2016) and it has been shown that recombinant analogs of lactaptin induce apoptosis of human cancer cells in culture and inhibit the growth and metastasis of animal and human tumors in the in vivo system.

The closest to the claimed method is a prototype, is a method of isolation and purification of the analogue of lactaptin, recombinant protein RL2, in the system of E. coli, which consists in the following.

The culture medium obtained from the stage of culturing E. coli XL-blu / pFK2 producing cells is purified using Ni-NTA (Nickel Nitrilotriacetic Acid) nickel agarose affinity chromatography. To this end, a chromatographic column with Ni-NTA agarose, equilibrated with buffer A, containing 50 mM Na-phosphate buffer pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, 10 mM 2-mercaptoethanol, is applied to the lysate of induced cells with flow rate 1 ml / min. To remove non-specifically sorbed E.coli proteins, 20 ml of buffer A containing 50 mM imidazole are eluted. The recombinant peptide RL2 was eluted with 10 ml of buffer B containing 250 mM imidazole, and then additional elution under denaturing conditions was carried out with 10 ml of buffer B containing 50 mM Tris-HCl pH 8.0, 6 M guanidine-HCl. The resulting protein fractions were dialyzed twice for 18 hours at 5 ° C against a solution containing 150 mM NaCl and Tris-HCl pH 7.5. The fractions containing the recombinant RL2 protein are concentrated by ion-exchange chromatography on diethylaminoethylcellulose (DEAE-cellulose). The total yield of RL2 is 150 µg from 500 ml of a culture of Escherichia coli cells (patent RU 2401307 C1, op. 10.10.2010)

The disadvantages of the prototype are low specific activity and insufficient purity of the target product due to contamination with co-secreted lipopolysaccharides (LPS) of producing bacteria, which can cause nonspecific immune responses when administered to mammals.

The objective of the invention is to develop a method for the isolation and purification of recombinant EL1 protein, an analogue of the human kappa-casein fragment produced by eukaryotic HEK293T cells, which has a high cytotoxic activity against human cancer cells.

Technical result: increased specific activity and purity of the target product.

The task is achieved by the proposed method, which consists in the following.

The culture medium obtained from human embryonic renal epithelium (HEK293T) cells is concentrated and purified using sulfopropyl sepharose ion exchange chromatography (SP-Sepharose), for which two volumes of 50-100 mM acetate buffer pH 5.5 are added to the culture medium sample and applied to the chromatographic column equilibrated with the first buffer solution containing 50-75 mM NaCl, 50 mM Na-acetate buffer pH 6.0. After applying the sample, the sorbent is washed with the first buffer solution. To remove non-specifically sorbed proteins of the culture medium, they are sequentially eluted first with a second buffer solution containing 150-175 mM NaCl, 50 mM Na-acetate buffer pH 6.0, then a third buffer solution containing 50-75 mM NaCl, 50 mM Na-acetate buffer pH 5.5 and then the fourth buffer solution containing 50-75 mm NaCl, 50 mm Tris-HCl pH 7.0. The fraction of positively charged proteins, presumably containing recombinant EL1 protein, is eluted with a fifth buffer solution containing 300-400 mM NaCl, 50 mM Tris-HCl pH 7.0.

The fractions containing the recombinant EL1 protein are further purified on a chromatographic column with an IMAC sorbent Sepharose (Immobilized metal ion affinity chromatography), with nickel ions, and equilibrated with a fifth buffer solution. The proteins not bound to the sorbent are eluted with a fifth buffer solution.

To remove proteins weakly bound to the sorbent, the elution is carried out first with a sixth buffer solution containing 20-25 mM imidazole, 50 mM Tris-HCl pH 7.0, and then a seventh buffer solution containing 500-600 mM NaCl, 50 mM Na-acetate buffer pH 6.0 . The elution of proteins strongly bound to the sorbent is carried out with an eighth buffer solution containing 300-400 mM imidazole. The fraction containing the protein with an electrophoretic mobility corresponding to 16 kDa (the final purification step) was then dialysed twice against a solution of 150 mM NaCl for 18 hours at 5 ° C. At all stages of purification, all the fractions obtained are analyzed by electrophoresis on a 12% polyacrylamide gel (PAG) according to Laemmli (Nature. - 1970. - V.227. -. P. 680-685). The yield of the target product is 17 μg from 100 ml of culture fluid from the cell-producers HEK293T.

The defining differences of the proposed method from the prototype are:

one). HEK293T cells expressing the recombinant EL1 protein are used as producer cells, which makes it possible to improve the quality of the target product by increasing its cytotoxic activity.

2) The first stage of chromatographic purification of recombinant EL1 protein from components of the culture medium is carried out using cation-exchange chromatography on SP-Sepharose, and the second stage of purification is carried out using Nickel-affinity chromatography on IMAC-Sepharose, which improves the quality of the target product.

3) For the elution of poorly and non-specifically associated with the sorbent proteins and the target product using experimentally selected optimal system of eight buffer solutions, which allows to increase the purity and homogeneity of the target product.

The invention is illustrated by the following graphic materials:

FIG. 1 shows the chromatogram (peaks 1-5) of the culture medium conditioned by HEK293T cells expressing the recombinant EL1 protein on ion-exchange SP-sepharose.

FIG. 2 shows the electrophoregram in 12% SDS-PAGE of the fractions of the proteins obtained by isolating the recombinant EL1 protein by SP-Sepharose ion exchange chromatography (peaks 1-5), where the lanes: 1 protein markers, molecular weight in kDa on the left; 2 - Proteins of the culture medium from HEK293T producing cells expressing EL1 protein; 3 - peak 1 - proteins not sorbed on SP-Sepharose; 4 - peak 2 non-specific sorbed proteins, eluted with a solution of 150 mM NaCl, at pH 6.0; 5 - peak 3 - proteins eluted with 50 mM NaCl at pH 5.5; 6-peak 4-proteins eluted with 50 mM NaCl at pH 7.0; 7 - 5-protein peak, eluted with 300 mM NaCl at pH 7.0.

FIG. 3 shows the chromatogram (peaks 6-9) on the IMAC-sepharose protein fraction, eluted previously with a fifth buffer solution from SP-sepharose and containing recombinant EL1 protein.

FIG. 4 shows the electrophoregram separation in 12% SDS-PAG of protein fractions obtained by affinity chromatography on IMAC-sepharose (samples of peaks 5-9), where the tracks: 1 - protein markers (molecular weight in kDa is shown on the left); 2 — peak 5 — fraction eluted from SP-Sepharose with 300 mM NaCl; 3 - peak 6 - proteins not sorbed on IMAC-Sepharose; 4 — peak 7 — proteins eluted by 20 mM imidazole at pH 7.0; 5 — peak 8 — proteins eluted with 600 mM NaCl at pH 6.0: 6 — peak 9 — proteins eluted with 250 mM imidazole.

FIG. 5 shows the analysis of purified EL1 protein after the dialysis stage versus 150 mM NaCl (final purification stage) for purity by the RP-HPLC method in a gradient from 0 to 100% acetonitrile in 0.1% H ₃ PO ₄ , peak 3 - EL1 protein, peak 1 - NaCl, 2.4 peaks are system peaks.

FIG. 6 shows the analysis of the purified EL1 protein after the dialysis stage against 150 mM NaCl (final purification stage) for authenticity by the Western blot method.

FIG. 7 shows the analysis of the cytotoxic activity of the EL1 protein after the final purification stage for human breast adenocarcinoma cells MDA-MB-231.

For a better understanding of the essence of the invention, the following are examples of its implementation.

Example 1. Obtaining transfected human HEK293T cells secreting recombinant EL1 protein.

HEK293 cells are transformed with the constructed plasmid pEL1 (Vavilovsky Journal of Genetics and Selection. - 2017. - 21 (7): 764-769) using calcium phosphate transfection and grown for 6 hours in IMDM (Iscove's Modified Dulbecco's Medium) supplemented with fetal bovine serum up to 10%, 100 µg / ml of streptomycin and 100 units / ml of penicillin in an atmosphere of 5% CO2 at 37 ° C. 6 hours after transfection, the medium in the plates is replaced with a new one and the cells are incubated for 48 hours.

Example 2. Isolation and purification of recombinant EL1 protein.

The culture medium obtained from HEK293T-producing cells was concentrated and purified by ion-exchange chromatography on SP-Sepharose, according to the manufacturer's instructions. To this end, two volumes of 100 mM acetate buffer pH 5.5 were added to the culture medium sample and applied at a rate of 1 ml / min to a chromatographic column packed with 5 ml of SP-Sepharose (SP Sepharose Fast Flow 6) and equilibrated with buffer solution 1 containing 50 mM NaCl, 50 mM Na-acetate buffer pH 6.0. After applying the sample, the sorbent was washed with buffer solution 1. To remove non-specifically sorbed proteins of the culture medium, sequential elution was performed with buffer solutions 2, 3 and 4, containing respectively 150 mM NaCl, 50 mM Na-acetate buffer pH 6.0; 50 mM NaCl, 50 mM Na-acetate buffer pH 5.5 and 50 mM NaCl, 50 mM Tris-HCl pH 7.0. The fraction of positively charged proteins, presumably containing the recombinant EL1 protein, was eluted with buffer solution 5 containing 300 mM NaCl, 50 mM Tris-HCl pH 7.0.

All the chromatographic fractions obtained were analyzed by electrophoresis in 12% PAAG with SDS according to Laemmli under reducing conditions to identify the fraction containing the recombinant EL1 protein.

The fractions containing the recombinant EL1 protein were further purified by affinity chromatography at a rate of 1 ml / min. on a chromatographic column packed with 5 ml of IMAC-Sepharose sorbent (IMAC Sepharose Fast Flow 6), with nickel ions, and equilibrated with buffer solution 5.

The proteins not bound to the sorbent were eluted with buffer solution 5. To remove proteins that were weakly bound to the sorbent, elution was performed with buffer solutions 6 and 7 containing 20 mM imidazole, 50 mM Tris-HC1 pH 7.0 and 600 mM NaCl, 50 mM Na-acetate buffer pH 6.0, respectively. The elution of proteins strongly bound to the sorbent was performed with buffer solution 8 containing 250 mM imidazole. All fractions were analyzed by electrophoresis in 12% PAG with SDS according to Laemmli under reducing conditions. The fraction containing protein with electrophoretic mobility corresponding to 16 kDa was further dialyzed against a solution of 150 mM NaCl (two changes for 18 h each at 5 ° C). The target product (ndialized sample) was analyzed by chromatography by HPLC.

FIG. 5 that in comparison with the gradient without a sample, only one peak is distinguished in chromatography. Thus, the purified sample contains only one protein product. The correspondence of a protein with a molecular mass of about 16 kDa to the target protein — the recombinant protein EL1 — was analyzed by the method of western blot using monoclonal antibodies to the recombinant protein RL2. FIG. 6 that a single band is visualized on a Western blot. This band corresponds to a protein - a fragment of human kappa-casein with a mobility corresponding to a protein with a molecular mass of about 16 kDa. As a control sample, RL2 protein was applied, two bands of which correspond to the monomeric and dimeric forms of the kappa-casein fragment. Thus, the protein obtained after chromatographic purification corresponds to the target recombinant EL1 protein.

The concentration of the target protein in the sample was determined by the method of Bradford (Anal. Biochem. - 1976. - V. 72. - P. 248-254). Bovine serum albumin (Serva, USA) was used to build a calibration curve. The yield of the target product was 17 μg of 100 ml of culture fluid from the cells producing HEK293T.

Example 3. Isolation and purification of recombinant EL1 protein.

The culture medium obtained from the HEK293T producer cells was concentrated and purified by ion exchange chromatography on a SP Sepharose column (GE HealthCare, Sweden). For this, two volumes of 50 mM acetate buffer pH 5.5 were added to the sample of culture medium and applied at a rate of 1 ml / min to a chromatographic column packed with 5 ml of SP-Sepharose and equilibrated with buffer solution 1 containing 75 mM NaCl, 50 mM Na -acetate buffer pH 6.0. After applying the sample, the sorbent was washed with buffer solution 1. To remove the nonspecifically sorbed proteins of the culture medium, sequential elution was performed with buffer solutions 2, 3 and 4, containing respectively 175 mM NaCl, 50 mM Na-acetate buffer pH 6.0; 75 mM NaCl, 50 mM Na-acetate buffer pH 5.5 and 75 mM NaCl, 50 mM Tris-HCl pH 7.0. The fraction of positively charged proteins, presumably containing the recombinant EL1 protein, was eluted with solution 5 containing 400 mM NaCl, 50 mM Tris-HCl pH 7.0.

All the chromatographic fractions obtained were analyzed by electrophoresis in 12% PAAG with SDS according to Laemmli under reducing conditions to identify the fraction containing the recombinant EL1 protein. The fractions containing the recombinant protein EL1 were further purified by affinity chromatography at a rate of 1 ml / min. on a chromatographic column packed with 5 ml of IMAC Sepharose sorbent (IMAC Sepharose Fast Flow 6), with nickel ions, and balanced with solution 5.

The proteins not bound to the sorbent were eluted with solution 5. To remove proteins weakly bound to the sorbent, elution was performed with buffer solutions 6 and 7 containing 30 mM imidazole, 50 mM Tris-HCl pH 7.0 and 500 mM NaCl, 50 mM Na-acetate buffer pH 6.0 , respectively. The elution of proteins strongly bound to the sorbent was performed with buffer solution 8 containing 400 mM imidazole. All fractions were analyzed by electrophoresis in 12% PAG with SDS according to Laemmli under reducing conditions. The fraction containing protein with electrophoretic mobility corresponding to 16 kDa was further dialyzed against a solution of 150 mM NaCl (two changes for 18 h each at 5 ° C). The removed sample was analyzed by chromatography by HPLC. The correspondence of the protein with a molecular mass of about 16 kDa to the target protein — the recombinant protein EL1 was analyzed by the method of a western blot using monoclonal antibodies to the recombinant protein RL2. The concentration of the target protein in the sample was determined by the Bradford method. Bovine serum albumin (Serva, USA) was used to build a calibration curve. The yield of the target product was 17 μg of 100 ml of culture fluid from the cells producing HEK293T.

Example 4. Evaluation of the cytotoxic activity of the recombinant EL1 protein in relation to human cancer cells MDA-MB-231 using the MTT-test.

To assess the cytotoxic activity of the recombinant EL1 protein purified by the proposed method, human MDA-MB-231 human breast adenocarcinoma cells obtained from the Cell Culture Collection of the Research Institute of Chemical Diversity (Khimki, Moscow Region) are used.

MDA-MB-231 cells are cultured in L15 medium (Gibco, Life Technologies, UK) in the presence of 10% FBS, 2 mM L-glutamine, 250 mg / ml amphotericin B and 100 units / ml penicillin / streptomycin. T98G and PC3 cells were cultured in IMDM medium (Gibco, Life Technologies, UK) in the presence of 10% FBS, 2 mM L-glutamine and 100 units / ml penicillin / streptomycin. Cells are cultured at 37 ° C in an atmosphere of 5% CO ₂ .

As a way to quickly and accurately assess the cytotoxic effect of the recombinant EL1 protein obtained in a pro- or eukaryotic expression system, the MTT test is used. Cells are planted on 96-well cell culture plates at a concentration of 4-5 * 10 ³ cells / well in 100 μl of RPMI-1640 culture medium (Gibco, Life Technologies, UK) containing 10% by volume of FBS with the addition of antimycotic antibiotics composition: 100 units / ml penicillin G, 100 μg / ml of streptomycin sulfate, 0.25 μg / ml of amphotericin B, and incubated for 24 hours at 37 ° C in an atmosphere of 5% CO ₂ .

A recombinant protein RL2, an analogue of the human kappa-casein fragment obtained in the prokaryotic system (E.coli), is used as a reference drug. From the comparator drug RL2 and EL1, a series of consecutive dilutions are prepared in RPMI-1640 nutrient medium containing no FBS. 100 μl of the obtained protein dilutions are introduced into the wells of a plastic plate with cells. After 48 hours of incubation at 37 ° C in an atmosphere of 5% CO _2, the medium is removed from the wells and 200 μl of RPMI-1640 nutrient medium and 10 μl of MTT solution (Sigma, USA) with a concentration of 5 mg / ml are added to each well, after then continue incubation under the same conditions for 4 hours. Then remove the MTT medium from the wells and dissolve the formed MTT-formazan crystals by adding 150 μl of DMSO. The optical density of MTT formazan dissolved in DMSO is measured on a multichannel flatbed scanner at λ = 570 nm.

The results of the experiment are presented in FIG. 7 and indicate that incubation of cells in the presence of a comparator drug - recombinant RL2 protein, obtained in the Escherichia coli system, and in the presence of recombinant EL1 protein, leads to a dose-dependent decrease in the viability of cancer cells. The concentration of recombinant purified RL2 protein, resulting in a twofold decrease in the viability of the tested cancer cell lines (IC50), corresponds to ~ 200 μg / ml, while the IC50 for EL1 secreted by human cells of the HEK293T line transfected with pEL1 does not exceed 400 ng / ml, those. there are at least 500-fold differences in the IC50 for these forms of lactaptin.

The proposed method allows to obtain a recombinant protein with high specific activity against cancer cells, which, in turn, reduces the protein load when administered as a drug to mammals.

Claims (1)

The method of isolation and purification of recombinant protein, an analogue of human kappa-casein fragment, which has cytotoxic activity against human cancer cells, including the cultivation of producer cells, concentration and purification of the culture medium using ion-exchange chromatography, sequential elution from the sorbent of impurity proteins and the target product with using buffer solutions, purification of the obtained protein fractions by twofold dialysis for 18 h at 5 ° С against a solution containing 150 mM NaCl, I differ Because HEK293T cells expressing recombinant EL1 protein are used as producer cells, the culture medium is purified using cation-exchange chromatography on SP-Sepharose, and fractions containing recombinant EL1 protein are purified using nickel-affinity chromatography on IMAC-Sepharose , sequential elution from the sorbent of impurity proteins and the target product is carried out using a system of eight buffer solutions, while two volumes of 50-100 mM acetate buffer pH 5 are added to the sample of culture medium .5 and applied to a chromatographic column with SP-Sepharose, equilibrated with the first buffer solution containing 50-75 mM NaCl, 50 mM Na-acetate buffer pH 6.0, elution from SP-Sepharose sorbent of nonspecifically sorbed proteins of the culture medium is first carried out with a second buffer solution containing 150-175 mM NaCl, 50 mM Na-acetate buffer pH 6.0, then a third buffer solution containing 50-75 mM NaCl, 50 mM Na-acetate buffer pH 5.5 and then a fourth buffer solution containing 50-75 mM NaCl, 50 mM Tris-HCl pH 7.0, elution from the SP-Sepharose sorbent protein fraction containing rivers the om1 protein EL1 is carried out with a fifth buffer solution containing 300–400 mM NaCl, 50 mM Tris-HCl pH 7.0, and elution of weakly bound proteins with an IMAC Sepharose sorbent is first carried out with a sixth buffer solution containing 20–25 mM imidazole, 50 mM Tris– HCl pH 7.0, and then the seventh buffer solution containing 500-600 mm NaCl, 50 mm Na-acetate buffer pH 6.0, and the elution from the sorbent IMAC-sepharose firmly bound proteins hold the eighth buffer solution containing 300-400 mm imidazole.

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