RU2683221C1 - Recombinant el1 peptide having cytotoxic activity relative to human cancer cells - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, specifically to the production of recombinant lactaptine analogues, and can be used in medicine. Plasmid DNA pEL1 is engineered to provide synthesis of a recombinant EL1 peptide in mammalian cells, and the recombinant EL1 peptide having a molecular weight of 14.1 kDa is obtained.EFFECT: invention provides preparing a recombinant anti-tumor EL1 peptide, having high cytotoxic activity relative to human cancer cells.1 cl, 4 dwg, 4 ex

Description

The group of inventions relates to biotechnology, genetic and protein engineering, specifically to obtaining a highly active recombinant peptide EL1.

Today, oncological diseases are one of the main causes of death both in Russia and abroad [1].

It is known that one of the reasons for the development of malignant tumors in the body is a violation of the regulation of apoptosis (programmed cell death). The development of new inducers and modulators of cancer cell apoptosis is one of the most productive approaches for creating modern oncotherapeutic agents [2]. It is also known that, in comparison with prokaryotic expression systems, the production of genetically engineered recombinant human proteins and peptides in a mammalian cell culture allows their correct folding and the native spectrum of translational modifications, which in turn favorably affects the level of functional activity of such recombinant proteins, and thereby is a promising direction for improving the therapeutic agents for the treatment of malignant neoplasms.

Earlier, a proteolytic fragment of kappa-casein, lactaptin protein (~ 8.6 kDa), capable of inducing apoptotic death of cancer cells in culture, was isolated and characterized from human milk [3, 4]. A number of recombinant analogues of the natural peptide were obtained [5, 6] and it was shown that the lactaptine analog, the recombinant peptide RL2, induces apoptosis of human cancer cells in culture and inhibits the growth and metastasis of animal and human tumors in vivo. Mouse hepatoadenocarcinoma-1 was one of the most sensitive tumors to the action of RL2 in vitro and in vivo [7, 8]. Currently, preclinical trials of the drug "Lactaptin", created on the basis of the recombinant peptide RL2, have been performed, the safety and antitumor efficacy of the drug have been shown.

However, Lactaptin, like other protein therapeutic drugs, has a number of significant drawbacks. It is evenly distributed over the organs and tissues of the body, which reduces its concentration in the tumor and, accordingly, reduces the effectiveness of the antitumor effect [9]. In addition, the existing process for the production and purification of the lactaptine analog, the recombinant peptide RL2, in the E. coli system for the preparation of the dosage form is based on its isolation in the predominantly denatured form from inclusion bodies, followed by dialysis and partial refolding, as well as chromatographic purification, due to which the specific activity of this drug is significantly reduced compared to the original lactaptin from human milk.

Closest to the claimed group of inventions, the prototype is recombinant plasmid DNA pFK2, which provides the synthesis of a recombinant peptide RL2, which is an analogue of a human kappa-casein fragment and a recombinant peptide RL2, an analogue of a human kappa-casein fragment, having a molecular weight of 14.1 kDa, consisting of the methionine residue, a fragment of human kappa-casein from 24 to 134 amino acid residues and the C-terminal histidine tract having cytotoxic activity against cancer cells, including aminokis otnuyu sequence encoded by the nucleotide sequence SEQ ID NO: 1 [5].

The known plasmid pFK2 has a molecular weight of 2.1 MDa, size 3164 bp, contains the following elements: plasmid vector pGSDI, hydrolyzed at SalI and BglII sites and containing a replication initiation site for plasmid pBR322, T7 phage promoter and unique EcoRI restriction sites (1595 ), BglII (1613), SalI (1985), PstI (1995), HindIII (2000), ClaI (2053); BglII-SalI, a 372 bp fragment comprising a start codon, a segment encoding a fragment of human 24 kappa casein amino acid residue, an oligopeptide with the sequence GGSHHHHHH and a stop codon; genetic markers: AMPr is a beta-lactamase gene that determines ampicillin resistance during Escherichia coli transformation [5].

A disadvantage of the known plasmid pFK2 is that it does not ensure the production of the recombinant peptide RL2 in mammalian cells.

A disadvantage of the known recombinant peptide RL2 produced in the Escherichia coli system is that the effectiveness of its antitumor effect is many times reduced compared to the native fragment of human kappa-casein.

The objective of the group of inventions is the creation of a recombinant plasmid that provides the production of the EL1 peptide, and the preparation of a recombinant EL1 peptide having increased cytotoxic activity against human cancer cells.

The technical result of the group of inventions is the creation of a recombinant plasmid pEL1, which provides the synthesis of a recombinant EL1 peptide, and the preparation of a recombinant EL1 peptide having increased cytotoxic activity against human cancer cells.

This technical result is achieved by constructing the plasmid pEL1 by inserting into the plasmid vector pCDH-EF1a-GlucSP-MCS-IRES-copGFP a DNA fragment encoding the recombinant EL1 peptide in a single translation frame with the GlucSP signal peptide, transfecting the cultured human cell culture plasmid pEL1 obtained biomass of transfected cells, followed by the use of conditioned supernatants of such cells.

The essence of the claimed group of inventions is as follows.

Genetically engineered to obtain the plasmid pEL1 carrying the gene encoding the recombinant analog of lactaptine (EL1 peptide) fused to the GlucSP signal sequence. The pEL1 construct is designed such that the cloned GlucSP-EL1 gene is controlled by the constitutive promoter of the human EF1a gene.

The starting genetic material for the construction of the recombinant plasmid pEL1 is:

a) a third-generation lentiviral vector pCDH-EF1a-GlucSP-MCS-IRES-copGFP, which is a derivative of the pCDH-EF1a-MCS-IRES-copGFP vector (SystemBio, USA) and allows the insertion of a DNA fragment encoding the recombinant EL1 peptide, its expression under control the constitutive promoter of the human EF1a gene and secretion into the extracellular space by using the GlucSP signal sequence;

b) a DNA fragment flanked by the AgeI and EcoRI restriction sites encoding the recombinant EL1 peptide, which is obtained in the polymerase chain reaction using oligonucleotide primers: lactap_AgeF 5'-gcaccggtatgaaccagaaacaaccagca-3gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt -PE / L-Pat [10]. The lactap_AgeF primer ensures the presence of an AgeI restriction site in the amplification fragment. The resulting plasmid pEL1 (Fig. 1) is characterized by the following features:

- has a size of 8248 bp .;

- encodes a recombinant GlucSP-EL1 peptide containing a methionine residue, a GlucSP signal peptide (GVKVLFALICIAVAEAKPT), a glycine spacer (G), and a recombinant EL1 peptide that, when exported to the extracellular space, loses the signal and spacer sequences and converts to the recombinant peptide EL1;

- consists of the following elements:

a) a 426 bp DNA fragment encoding the recombinant peptide GlucSP-EL1;

b) a pUC replication initiation site providing high copy maintenance of the construct in Escherichia coli cells;

C) the site of initiation of replication of the SV40 virus, providing stable maintenance of the construct in an episomal state in mammalian cells expressing the large antigen T of the SV40 virus;

d) a promoter of the human EF1a gene, which provides constitutive and strong expression of recombinant fusion proteins of interest in mammalian cells;

e) genetic markers:

AMPr - ampicillin resistance gene (bla), which determines ampicillin resistance during Escherichia coli transformation;

and

copGFP is the reporter gene of the green fluorescent protein of copepod Pontellina plumata, which determines the luminescence of transformed or transduced mammalian cells when they are irradiated in the blue-green UV range;

f) a hybrid RSV-5'LTR promoter, consisting of sequences of Rauss sarcoma virus (RSV) and HIV-1, providing TAT-independent synthesis of viral transcripts in cells-packers of lentiviral particles;

g) modified genetic elements of HIV-1 (cpp, gag, env, RRE) necessary for the correct processing of viral RNA and assembly of lentiviral particles;

h) 5'LTR and 3'dLTR sequences of HIV-1, ensuring stable integration of the sequences enclosed between them into the genome of mammalian cells without the possibility of their further re-mobilization;

i) the sequence of WPRE, a regulatory element of the marmot hepatitis virus, which provides increased stability and transcription of transcripts;

j) an IRES element, an internal site for the insertion of the cardiovirus A ribosome, providing cap-independent translation initiation of the copGFP linked reporter;

k) SV40 polyadenylation site, which provides efficient termination of transcription and processing of recombinant transcripts.

Thus, pEL1 plasmid DNA was obtained for the first time, which ensures the synthesis of recombinant peptide GlucSP-EL1 in human cells, consisting of the signal peptide GlucSP cleaved from export from the cell and the antitumor recombinant peptide EL1, which has cytotoxic activity against human cancer cells.

Mammalian cells, for example, HEK293 human embryonic renal epithelium cells expressing the temperature-sensitive allele of the SV40 virus large T antigen, are transformed with the constructed plasmid pEL1 and grown for 6 h in IMDM medium with the addition of fetal bovine serum up to 10%, 100 μg / ml 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. After that, the supernatants of the conditioned medium are filtered through 0.45 μm PES filters and used to quantify the level of secreted recombinant peptide and its cytotoxic activity against cancerous and conditionally normal human cell lines.

The recombinant EL1 peptide present in the conditioned transfected cell supernatant has cytotoxic activity against human breast cancer cells MDA-MB-231, human glioblastoma cells T98G, and human prostate cancer cells PC3. The recombinant EL1 peptide has the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 (Fig. 2).

The invention is illustrated by the following drawings:

FIG. 1. Map of the genetic design of plasmid pEL1, where: Fragment is a DNA sequence encoding the recombinant peptide GlucSP-EL1; EF1a is the promoter of the human EF1a gene, GaussiaSP is the signal peptide of the lussiferase Gaussia princeps, IRES is the internal landing site of the cardiovirus A ribosome, copGFP is the green fluorescent copepod protein Pontellina plumata, RSV is the hybrid promoter, HIV LTR is the long terminal repeat of HIV-1, gag shortened gag gene sequence HIV-1, RRE - REV-binding element of HIV-1, WPRE regulatory element of marmot hepatitis virus, 3'dLTR - truncated long terminal repeat of HIV-1, AmpR - ampicillin resistance gene, pUCori - pUC replication origin, SV40ori - SV40 virus replication initiation site, SV40polyA - polyadeni site ation of the SV40 virus.

FIG. 2. The nucleotide sequence of a fragment of plasmid pEL1 and its encoded amino acid sequence of the recombinant peptide EL1.

FIG. 3. Western blot analysis of the content of recombinant EL1 peptide in conditioned medium of HEK293T cells transfected with plasmid pEL1 and pCDH-EF1-MCS-IRES-copGFP, where lanes: M - a set of marker proteins of known molecular weight, 1 - recombinant RL2 purified from Escherichia coli (RL2 _coli ), 2 - medium conditioned with HEK293T cells transfected with the empty vector pCDH-EF1-MCS-IRES-copGFP (negative control); 3 - medium conditioned by HEK293T cells transfected with plasmid pEL1.

FIG. 4. Changes in cell viability of human breast adenocarcinoma MDA-MB-231, human glioblastoma T98G, and PC3 prostate cancer when incubated with the recombinant peptide RL2 obtained in the Escherichia coli system (RL2 _coli ), and with the recombinant peptide EL1 during its production in cell culture Human HEK293T. Cell viability was determined relative to the viability of the control cells (incubation without proteins) ± standard deviation (M ± s) according to the results of three independent experiments.

For a better understanding of the essence of the proposed group of inventions, the following are specific examples of their implementation.

Example 1. Construction of a recombinant plasmid pEL1.

The nucleotide sequence encoding the recombinant lactaptin analog (EL1 recombinant peptide) is amplified from the plasmid pVGF-FR2-PE / L-Pat using the primers lactap_AgeF 5′-gcaccggtatgaaccagaaacaaccagca-3 ′ and U30R 5′-gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtt. The reaction is carried out in an Eppendorf Mastercycler thermocycler. The composition of the reaction mixture with a volume of 50 μl includes: 1xSEBuffer Pfu DNA polymerase (SibEnzyme, Russia), a mixture of deoxynucleoside triphosphates (dATP, dGTP, dCTP, dGTP) (dNTP mixture) (2.5 μM), plasmid pVGF-FR2-PE / L-Pat (10 ng), lactap_AgeF primer (0.3 nM), U30R primer (0.3 nM), Pfu polymerase (2.5 act units) (SibEnzyme, Russia). Conditions for PCR: preliminary denaturation - 2 minutes at 96 ° C; 35 cycles - 15 seconds at 94 ° C, 15 seconds at 54 ° C, 20 seconds at 72 ° C; final stage - 2 minutes at 72 ° C.

At the end of the PCR, 5 μl of 3M NaAc pH 5.0 and 125 μl of 96% ethanol are added to the reaction and DNA is precipitated for several hours at a temperature of minus 20 ° С. The precipitate obtained after centrifugation (13000 g, 10 min) is washed twice with 500 μl of 70% ethanol (centrifugation 13000 g, 5 min), dried in a vacuum evaporator and dissolved in 40 μl of autoclaved bidistilled water.

The vector DNA pCDH-EF1a-GlucSP-MCS-IRES-copGFP (2 μg) and the reprecipitated PCR product (1 μg) encoding the recombinant EL1 peptide are treated with AgeI and EcoRI restriction endonucleases (10 units of each enzyme) (Thermo Scientific, Lithuania) in a reaction volume of 50 μl. The reaction is carried out under the conditions recommended by the manufacturer.

After treatment with AgeI and EcoRI restriction enzymes, the vector DNA pCDH-EF1a-GlucSP-MCS-IRES-copGFP and the cloned PCR fragment were purified by electrophoresis on 1.0% TAE agarose gel (40 mM Tris-HCl, 20 mM acetic acid, 1 mM EDTA (pH = 8.4)) and extracted from the gel with a QIAquick Gel Extraction Kit (Qiagen, USA) according to the manufacturer's protocol.

The T4 ligation reaction with DNA ligase (SibEnzyme, Russia) of the linearized vector pCDH-EF1a-GlucSP-MCS-IRES-copGFP and the cloned fragment previously treated with restriction endonucleases AgeI and EcoRI, is carried out in a reaction with a volume of 10 μl and a composition of 50 mM Tris-HCl ( pH = 7.8); 10 mM MgCl ₂ ; 10 mM DTT; 1 mM rATP, at + 4 ° C overnight. The reaction uses 50 ng of the linear shape of the vector and the cloned fragment in a three-fold molar excess with respect to the vector.

Example 2. Obtaining transfected human HEK293T cells secreting the recombinant EL1 peptide.

HEK293T cells were cultured in IMDM medium (Gibco) with 10% fetal bovine serum (HyClone, USA), 100 μg / ml streptomycin and 100 units / ml penicillin in an atmosphere of 5% CO2 at 37 ° C. Upon reaching confluence, the cells are subcultured onto a new medium by diluting the cell suspension 1: 5 (or once every two days) or 16 hours before transfection so that at the time of transfection the confluency level is about 50-60%.

The plasmid preparation pEL1 and pCDH-EF1a-MCS-IRES-copGFP were prepared using the QIAGEN Plasmid Maxi plasmid DNA purification kit according to the manufacturer's recommendations and the DNA was dissolved in autoclaved deionized water. HEK293T cells are then transfected with plasmid preparations using calcium phosphate transfection. For transfection of 5 million cells in a 10 cm diameter dish, 10 μg of DNA is used. Plasmid DNA pEL1 or pCDH-EF1a-MCS-IRES-copGFP (negative control) was mixed with 31.2 μl of CaCl2 and water up to 250 μl was added. After this, the mixture is gradually added to 250 μl of sterile HBS (150 mm NaCl, 20 mm HEPES, pH 7.0;), vigorously mixing it. The thus obtained suspension of calcium phosphate complexes and plasmid DNA is carefully digested on the surface of a plate with transfected HEK293T cells. 6 hours after transfection, the medium in the plates is replaced with a new one and the cells are incubated for 48 hours. After that, supernatants of conditioned media obtained by centrifugation for 5 min at 800 g are filtered through 0.45 μm PES filters and used for further analysis.

Example 3. Detection and evaluation of the concentration of recombinant EL1 peptide in a conditioned medium.

To confirm the production of the full-length recombinant EL1 peptide, Western blot analysis of the conditioned medium is carried out. Aliquots of the conditioned medium are added 1/4 part (by volume) of a buffer composition of 100 mm Tris-HCl pH = 6.8, 400 mm 2-mercaptoethanol, 4% SDS, 40% glycerol, bromphenol blue (300 ng / ml), xylene cyanolic (300 ng / ml), heated in a boiling water bath for 1 minute, centrifuged for 1 minute at 13000 g and applied to a polyacrylamide gel (5% concentrating and 15% separating) [11]. As a molecular weight marker, a ready-made set of pre-stained proteins with a molecular weight of 10-260 kDa is used (Thermo Scientific Spectra Multicolor Broad range Protein Ladder, Thermo Scientific, Lithuania). At the end of electrophoresis, proteins and peptides from the gel are transferred onto a nitrocellulose membrane (Bio-Rad, USA) by wet transfer at 150 V for 1 h in NuPAGE buffer (Novex Life Technologies, USA). The nitrocellulose membrane was incubated in the iBind Solution buffer (Novex Life Technologies, USA) on an iBind Western device (Life Technologies) for 18 h with the addition of antibodies to RL2 conjugated with horseradish peroxidase (clone F14, Biosan, Russia), a 1: 1000 dilution. After that, the membrane was incubated with a chemiluminescent substrate (Novex® ECL, Invitrogen, USA) for 1 min and the data was visualized on a C-Digit fluorescent scanner (LI-COR, USA). Data is processed using the Image Studio Ver 4.0 digits software package (LI-COR, USA). The experimental results are shown in FIG. 3 and indicate that the HEK293T cells were transfected with plasmid DNA pEL1, actually produce the recombinant peptide EL1, secreted into medium: electrophoretic mobility of the detectable anti-RL2 antibodies recombinant peptide EL1 corresponds mobility recombinant RL2, obtained in Escherichia coli system (RL2 _coli).

Determination of the concentration of recombinant EL1 peptide in conditioned media is carried out according to the following protocol. Specific monoclonal antibodies to RL2 (100 μl) (clone F21, ZAO Biosan, RF) in carbonate-bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH = 9.6.), Immobilized in a 96-well ELISA sorption plate ( Costar, USA) and incubated successively for 60 min at 37 ° C and then 18 h at 4 ° C. The contents of the wells are removed, the plate is washed twice with 1 x washing buffer (150 mm NaCl, 25 mm Tris-HCl pH = 7.5, 0.05% Tween-20) and 200 μl of blocking buffer (150 mm NaCl, 25 mm Tris are added to the wells -HCl pH = 7.5, 0.05% Tween-20, 0.9% BSA), incubated for 1 h at 37 ° C. The wells are washed twice with wash buffer and samples of conditioned medium diluted 2, 5, and 10 times are applied in triplicate. To build a calibration curve, samples of the recombinant peptide RL2 (prototype) prepared in the Escherichia coli (RL2 _coli ) system (30 ng / ml, 60 ng / ml, 90 ng / ml, 120 ng / ml, 300 ng / ml, 600 ng / ml) and place 100 μl in triplicate in a prepared ELISA plate. The plate is incubated for 1 h at 37 ° C, after which the contents of the wells are removed and 100 μl of monoclonal antibodies to RL2 (clone F15, ZAO Biosan, RF) conjugated with horseradish peroxidase are added to each well and incubated for 1 h at 37 ° C. The wells are washed with wash buffer, 100 μl of substrate (0.05 M citrate-phosphate buffer, 0.1 mg / ml TMB, 0.006% H2O2 pH = 5.0.) Is added and incubated for 20 min in the dark at 24 ° C. The reaction was stopped by the addition of 100 μl of 2 M H2SO4. The optical density of the solution in the wells was measured on an Apollo LB 912 plate spectrophotometer (Berthold Technologies, Germany) at λ = 450 nm, reference wavelength λ = 620 nm. Based on the results obtained, a calibration curve is constructed and the content of the recombinant EL1 peptide in the conditioned medium is determined by its linear section.

Example 4. Evaluation of the cytotoxic activity of the recombinant peptide EL1 in relation to human cancer cells.

For a comparative analysis of the cytotoxic effect of RL2 (prototype), purified from Escherichia coli cells of strain BL21 (DE3) / pFK2, deposited in the Collective Use Center “Collection of Extremophilic Microorganisms and Typical Cultures” of the Institute of Chemical Biology and Fundamental Medicine of the SB RAS under number 1020 (RL2 _coli ), and EL1 secreted by HEK293T human cells transfected with pEL1 plasmid DNA use MDA-MB-231 human breast adenocarcinoma cells and T98G human glioma cells obtained from the cell culture collection of Research Institute of Chemical Diversity "(Khimki, Moscow region), and human prostate cancer PC3 cell line (ATCC, USA).

MDA-MB-231 cells were 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 were cultured at 37 ° C in an atmosphere of 5% CO ₂ .

As a method for quickly and accurately assessing the cytotoxic activity of the inventive recombinant EL1 peptide obtained in the eukaryotic expression system and RL2 _coli (prototype), an MTT test is used. Cells are plated on 96-well cell culture plates at a concentration of 4-5 * 10 ³ cells / well in 100 μl RPMI-1640 culture medium (Gibco, Life Technologies, UK) containing 10% FBS by addition of an antibiotic-antimycotic solution composition: 100 units / ml penicillin G, 100 μg / ml streptomycin sulfate, 0.25 μg / ml amphotericin, and incubated for 24 hours at a temperature of 37 ° C in an atmosphere of 5% CO ₂ .

A series of successive dilutions of the RL2 _coli reference recombinant peptide and conditioned media containing EL1 are prepared in RPMI-1640 culture medium containing no FBS. Pipette 100 μl of the resulting dilutions into the wells of a plastic plate with cells. After 48 hours of incubation at 37 ° C in an atmosphere of 5% CO2, the medium is removed from the wells and 200 μl of RPMI-1640 medium and 10 μl of MTT solution (Sigma, USA) with a concentration of 5 mg / ml are added to each well, after which continue incubation under the same conditions for 4 hours. Then, the MTT medium is removed from the wells and the resulting MTT-formazan crystals are dissolved by adding 150 μl of DMSO. The optical density of MTT-formazan dissolved in DMSO was measured on a multi-channel flatbed scanner at λ = 570 nm.

The experimental results are shown in FIG. 4 and indicate that incubation of cancer cell lines in the presence of a recombinant peptide RL2 (prototype) obtained in the Escherichia coli system (RL2 _coli ) and in the presence of a recombinant peptide EL1 in conditioned media from HEK293T cells transfected with pEL1 leads to a dose-dependent decreased cancer cell viability. The concentration of the recombinant peptide RL2 _coli (prototype), leading to a twofold decrease in the viability of the tested cancer cell lines (IC50) corresponds to ~ 200 μg / ml, while the IC50 for recombinant EL1 peptide produced by human HEK293T cells transfected with pEL1, not higher 400 ng / ml, i.e. at least 500-fold differences in IC50 are observed.

Thus, the constructed plasmid DNA pEL1 provides the production of the antitumor recombinant EL1 peptide in the human HEK293T cell line, and the recombinant EL1 peptide thus obtained has at least 500 times more pronounced cytotoxic activity against human cancer cells of the MDA-MB-231 lines, T98G and PC3.

Information sources

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Claims (1)

Recombinant EL1 peptide having cytotoxic activity against human cancer cells, having a molecular weight of 14.1 kDa, having the amino acid sequence of SEQ ID NO: 2 shown in FIG. 2.

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