RU2408728C1 - Method to produce immunogenic recombinant extracellular fragment connexin-43 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention may find application in methodology for creation of biologically active antibodies joined with live cell, address delivery of medicinal agents to target cells. Proposed method includes cloning of the second extracellular loop E2 Cx43, representing a fragment of connexin-43 Q173 -1208 with aminoacid sequence QWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTI, into two plasmid vectors pCBDQ and pHPMLQ. Then transformation of E.coli cells is carried out with produced plasmid DNAs. Then transformed cells are cultivated up to optical density of 0.7-0.9. Then plasmid expression 0.9-1.1 mM is induced with isopropyl thiogalactoside. Afterwards recombinant fused proteins and their release in presence of 7.9-8.1 M of urea by method of chromatography at Ni-NTA agarose.

EFFECT: proposed invention makes it possible to develop method to produce immunogenic recombinant extracellular fragment of rat connexin-43 in composition of highly immunogenic chimeric protein suitable to develop monoclonal antibodies.

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Description

The invention relates to the field of molecular biotechnology, in particular to the problem of producing small recombinant extracellular domains of transmembrane proteins with immunogenicity, and can be used in the methodology of creating biologically active antibodies that bind to a living cell, developing cell sorting systems using flow fluorimetry, targeted drug delivery preparations for target cells.

Connexin-43 (Cx43, CXA1_RAT) is an integral membrane protein that forms nexus or gap junction between astrocytes in the definitive nervous tissue, as well as between cardiomyocytes and cells of the cardiac conduction system. Using these contacts, the transmission of intercellular signals regulating proliferation, differentiation, apoptosis and cell migration is carried out both during normal ontogenesis and during invasion of tumor cells of glial origin [Prochnow N., et al., 2008]. The latter causes a high interest in connexin-43 as a potential molecular target in the treatment of gliomas [Huang R., et al., 2002, Fu CT, et al., 2004 Oliveira R., et al. 2005].

Connexin-43 has four transmembrane domains and two short extracellular loops; The N- and C-terminal regions of this protein are immersed in the cytoplasm [Prochnow N., et al., 2008]. In the membrane, connexin-43 forms hexamers - connexons. In the organization of functionally active gap junctions, the extracellular domains of Cx43 play a crucial role. These structures, due to interaction with similar fragments on a neighboring cell, facilitate the assembly of a full-fledged gap junction from connexin “hemicanals”. Cells interacting in this way are able to exchange certain intracellular compounds, including ions and secondary messengers [Sin WC., Et al., 2008, Decrock E., et al. 2009]. There is evidence that blocking these fragments, in particular using polyclonal antibodies, can disrupt the assembly of gap junctions and, as a result, the exchange of intracellular compounds [Lin J.H., et al. 2002]. In this regard, it seems highly relevant to obtain immunogenic preparations of recombinant extracellular fragments of Cx43 and monoclonal antibodies to them.

Known work on the creation of recombinant extracellular fragments of Cx43 and obtain polyclonal antibodies to them [Lin J.H., et al. 2002]. The disadvantage of this approach is the extreme heterogeneity of polyclonal antibodies and the inability to standardize them. The resulting recombinant extracellular fragments, due to their small size, are weakly immunogenic and are not suitable for creating monoclonal antibodies.

Commercial preparations of monoclonal antibodies to Cx43 are also known [Zymed, USA]. These monoclonal antibodies are obtained to the recombinant C-terminal region of connexin-43. Since the C-terminal portion of Cx43 is localized in the cytoplasm, these antibodies cannot be used to visualize living cells.

The technical task of the invention is to develop a method for producing an immunogenic recombinant extracellular fragment of rat connexin-43 // Cx43 (second extracellular loop E2) as part of a highly immunogenic chimeric protein suitable for creating monoclonal antibodies.

The solution of this problem is achieved in the claimed method for producing an immunogenic recombinant extracellular fragment of connexin-43, including the cloning of a second extracellular loop E2 Cx43, which is a fragment of connexin-43 Q173-1208 with the amino acid sequence QWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTI. pI = 7.87), into two plasmid vectors pCBDQ and pHPMLQ, transformation of the obtained plasmid DNA of E. coli cells, culturing the transformed cells to an optical density of 0.7-0.9, followed by the expression of plasmids 0.9-1.1 mM isopropylthiogalactoside, generation of recombinant fusion proteins and their allocation in the presence of 7.9-8.1 M urea by chromatography on Ni-NTA agarose.

The Cx43 membrane topology is evaluated using the NMMTOR, TMNMM and TMPred programs (www.expasy.org). Analysis of nucleotide and protein sequences and design of primers is carried out using the software package "DNAStar" (Lasergene). As a result of this analysis, the amino acid sequences of the two extracellular fragments involved in the formation of gap-junction are determined. For further work, the Cx43 Q173-1208 fragment (QWYIYGFSLSAVYTCK RDPCPHQVDCFLSRPTEKTI, 36 a.o., M.w. 4.28 kDa, pI = 7.87) corresponding to the second extracellular loop (E2) Cx43 is selected. Amplification of the nucleotide sequence encoding this fragment from the cDNA of the rat brain library is performed using primers: CX43_173F: 5′-GATCAGATCTCAGTGGTACATCTATGGGT-3 ′; CX43_173B: 5'-GATCAAGCTTAGATGGTTTTCTCCGTGGGAC-3 '(SibEnzyme, Novosibirsk) containing restriction enzyme recognition sites Bg1II and HindIII, respectively.

To increase the expression level of the extracellular fragment of Cx43 and increase the immunogenicity of the recombinant polypeptide, a hybrid is created consisting of the sequence of the desired fragment and a larger “carrier”. As such “carriers”, two different Ca-ATPase domains of the hPMCA4b plasma membrane of human cells are chosen: the C-terminal calmodulin binding domain (CBD, a.o. 1057-1205, 17 kDa) and the first cytoplasmic loop (HPML, a.o. . 166-371, 22.5 kDa). In relation to these domains, immunogenicity sufficient for the production of At and a high level of expression in prokaryotic cells are shown [Ter K. K. 2003, Restov N. B., et al. 2007, Dmitriev R.I., 2007].

Cloning of the E2 Cx43 nucleotide sequence into pCBDQ and pHPMLQ vectors [Dmitriev R.I., 2007] and subsequent transformation of E. coli with the created plasmid DNAs make it possible to obtain two strains producing chimeras of the second extracellular Cx43 fragment with the N-terminal CBD and HPML polypeptides (Table 1). The protein yield during overproduction under standard conditions (+ 37 ° C, 4 hours in the presence of 1 mM IPTG in LB medium) was 10 and 15 mg / L culture for Cx43_173CBD and Cx43_173HPML, respectively. Purification of the recombinant proteins is carried out using affinity chromatography on Ni-NTA agarose in the presence of 7.9-8.1 M urea.

Using disk electrophoresis in SDS page with SDS, the apparent electrophoretic mobility and purity of the obtained recombinant proteins were determined. Electrophoretic mobility for Cx43_173CBD and Cx43_173HPML corresponded to molecular weights of 25 and 30 kDa, respectively. The purity of the obtained proteins according to disk electrophoresis was at least 90%.

The resulting preparation Cx43_173CBD was used to immunize mice in the process of obtaining monoclonal antibodies. An enzyme-linked immunosorbent assay with Cx43_173HPML as an antigen showed a high level of immune response in immunized animals and the production of antibodies that recognize the amino acid sequence of the extracellular fragment of Cx43.

EXAMPLE

Isolation of total RNA from rat brain and the first cDNA strands were obtained using the SV Total RNA Isolation System (Promega) kit, according to the manufacturer's protocol. To 2 μg of the obtained RNA, 500 ng of oligo primer (dT) 18 was added, incubated at + 70 ° C for 3 min. The mixture was placed on ice, 40 units were added. Act. human placenta ribonuclease inhibitor "iRNAsin" (Promega), reverse transcriptase buffer M-MLV (Promega), dNTP up to 0.4 mM and 200 units Act. reverse transcriptase M-MLV (Promega). After incubation for 2 h at 370 ° С, nucleic acids were sequentially purified from proteins by extraction with a mixture of phenol-chloroform (1: 1), chloroform and precipitated with ethanol. The resulting precipitate was resuspended in 50 μl of deionized water.

The cDNA fragments encoding the selected region of the Cx43 protein were amplified by PCR using Taq polymerase and primers CX43_173F: 5'-GATCAGATCTCAGTGGTACAT-CTATGGGT-3 '; CX43_73B: 5'-GATCAAGCTTAGATGGTTTTCT-CCGTGGGAC-3 '(SibEnzyme, Novosibirsk), and cloned into the plasmid vector pGEM-T (Promega). The obtained plasmid DNA was used to determine the correctness of the structure of cDNA fragments by sequencing (Genome Center, Moscow, Russia). Plasmid DNAs that did not contain errors in the coding sequence were used for cloning into HPMLQ and CBDQ expression vectors, using BglII and HindIII restriction enzyme recognition sites to embed. For expression by plasmid DNA, HPMLQ-Cx43, CBDQ-Cx43 transformed E. coli SG13009 cells (Qiagen).

Transformed cells were cultured at + 37 ° C in 100 ml of LB medium containing 50 μg / ml carbenicillin and 25 μg / ml kanamycin, until the optical density (600 nm) of 0.7-0.9; after which expression of the plasmid was induced using 0.9-1.1 mM isopropylthiogalactoside (IPTG) and incubated under the same conditions for another 4 hours. Then the cells were besieged at 3000g for 20 minutes, frozen and stored at -70 ° C.

Protein isolation was performed under denaturing conditions in the presence of 7.9-8.1 M urea on Ni-NTA agarose according to the protocol recommended by Qiagen. The resulting protein preparations were analyzed by electrophoresis in 12.5% SDS page with 0.1% SDS.

Using disk electrophoresis in SDS page with SDS, the apparent electrophoretic mobility and purity of the obtained recombinant proteins were determined. Electrophoretic mobility for Cx43_173CBD and Cx43_173HPML corresponded to molecular weights of 25 and 30 kDa, respectively. The purity of the obtained proteins according to disk electrophoresis was at least 90% (Figure 1).

Purified recombinant proteins were used to immunize mice (20 μg per animal). Conducted 4-5 cycles of subcutaneous immunization with complete Freund's adjuvant (DiaM, RF) with an interval of 10-12 days. Two weeks later, an intraperitoneal booster immunization was carried out with a purified preparation and a further seven days later blood was taken.

Mice with a confirmed immune response were used to produce monoclonal antibodies. Isolation of splenocytes and fusion with SP / 20 myeloma cells was carried out according to the appropriate method [Chekhonin V.P. et al.,

2007]. Hybridomas were screened by enzyme-linked immunosorbent assay with Cx43_173HPML immobilized on a solid phase. Goat antimouse peroxidase conjugated Ab (A 9917, Sigma) at a dilution of 1: 20,000 was used as second antibodies. For the manifestation of the immunoperoxidase reaction, the prepared TMB solution (Zymed) was used. ELISA positive clones were tested by immunoblotting with the same antigens, followed by visualization using the ECL-advance kit (GE Healthcare) according to the manufacturer's method. At the last stage, hybridoma supernatants were tested in immunocytochemical analysis on fixed preparations of glioma C6 cells and on sections of rat brain in order to select hybridomas interacting with native connexin-43.

The purification of monoclonal antibodies from ascites was carried out using affinity chromatography on agarose with immobilized protein G (Invitrogen) according to the manufacturer's protocol.

Immunohistochemical analysis of antibodies was performed on frozen sections of the rat brain and on preparations of fixed and live rat and human cell cultures. For fixation, monolayer cell cultures were washed from PBS serum medium, incubated with a 4% neutral solution of paraformaldehyde pH 7.4 (30 minutes at + 4 ° C), and then washed again with PBS. For double staining, a cocktail of monoclonal and polyclonal antibodies was prepared in the required dilution (usually 1-5 μg / ml), incubated with cell preparations overnight, washed and incubated with a cocktail of secondary antibodies (a combination of Goat antimouse Alexa Fluor 488 and Goat antirabbit was usually used Alexa Fluor 594 or 350, Invitrogen), in the dilution of each type of antibody 1: 1000. Cell staining: actin filaments and nuclei was performed using Phalloidin-TRITC (Fluka) and DAPI or TOTO 633 (Invitrogen), respectively. All dilutions and washing were performed using PBS (pH 7.4) with the addition of 0.2% Tween-20 and Triton X-100 and 1% normal goat serum.

During immunocytochemical analysis of a live culture of C6 glioma, monoclonal monoclonal antibodies to the extracellular fragment of Cx43 to a concentration of 10 μg / ml were sterilized by filtration (0.22 μm filter, Costar) in a well with live attached cells in a serum medium, incubated for one hour, after which was washed in PBS, fixed with 4% paraformaldehyde and stained as described above. In an alternative variant of fluorescence imaging, monoclonal antibodies to Cx43_173 were used, biotinylated using the ProtOn kit (Vector Lab) and streptavidin (Imtek) conjugated to Alexa 532 using the Alexa Fluor 532 Protein Labeling kit (Invitrogen). Biotinylation and conjugation with fluorophore was performed according to the recommendations of manufacturers. Biotinylated anti-Cx43 antibodies were sterilized by filtration and added to a culture of living C6 glioma cells to a concentration of 10 μg / ml. After incubation for one hour, the culture medium was changed twice and streptavidin-Alex532 (1-5 µg / ml) was added and the fluorescence of living cells was recorded using a Leica DM 6000 reversed fluorescence microscope.

As a result of cloning the insert into plasmid vectors pCBDQ and pHPML and subsequent transformation of E. coli, two strains were produced that produced chimeras of the second extracellular fragment of Cx43 (E2, a.o. 173-208) with N-terminal hexahistidine tag and CBD and HPML polypeptides ( Cx43_173CBD: 22.5 kDa, pI 6.02 and Cx43_173HPML: 27.9 kDa, pI 8.36). The following procedures for purification of recombinant proteins, immunization of mice, fusion and selection of hybrid cells allowed us to obtain two clones producing monoclonal antibodies that recognize the E2 fragment of native Cx43 both on fixed preparations and in live rat cell cultures (normal astrocytes, C6 glioma cells) and human (cells of glioblastoma U251 and embryonic renal epithelium line 293) (Figure 2).

Information sources

1. Huang R, Lin Y, Wang CC, Gano J. et al. Connexin 43 suppresses human glioblastoma cell growth by down-regulation of monocyte chemotactic protein 1, as discovered using protein array technology. // Cancer Res. 2002. - 62 (10): 2806-12.

2. Oliveira R, Christov C, Guillamo JS, DeBoüard S, Palfi S, Venance L, Tardy M and Peschanski M. Contribution of gap junctional communication between tumor cells and astroglia to the invasion of the brain parenchyma by human glioblastomas. BMC Cell Biology 2005, 6: 7.

3. Fu CT, Bechberger JF, Ozog MA, Perbal B, and CC Naus. CCN3 (NOV) interacts with connexin43 in c6 glioma cells. Possible mechanism of connexin-mediated growth suppression. J. Biol. Chem., Vol. 279. - 35. - 36943-36950, 2004

4. Sin WC, Bechberger JF, Rushlow WJ, Naus CC. Dose-dependent differential upregulation of CCN1 / Cyr61 and CCN3 / NOV by the gap junction protein Connexin43 in glioma cells. J Cell Biochem. 2008., Vol. 15; 103 (6): 1772-82.

5. Decrock E, De Vuyst E, Vinken M, Van Moorhem M, Vranckx K, Wang N, Van Laeken L, De Bock M, D'Herde K, Lai CP, Rogiers V, Evans WH, Naus CC, Leybaert L. Connexin 43 hemichannels contribute to the propagation of apoptotic cell death in a rat C6 glioma cell model. Cell Death Differ. 2009 Jan; 16 (1): 151-63.

6. Prochnow N, Dermietzel R. Connexons and cell adhesion: a romantic phase. Histochem Cell Biol. 2008 Jul; 130 (1): 71-7.

7. Lin J. H., Takano T., Cotrina M.L. et al. Connexin 43 enhances the adhesivity and mediates the invasion of malignant glioma cells. // J Neurosci. - 2002 .-- Vol.22 (11). - P. 4302-4311.

8. Terpe K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2003 Jan; 60 (5): 523-33.

9. Pestov NB, Rydstrom J. Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase. Nat Protoc. 2007; 2 (1): 198-202.

10. Dmitriev RI, Korneenko TV, Bessonov AA, Shakhparonov MI, Modyanov NN, Pestov NB. Characterization of hampin / MSL1 as a node in the nuclear interactome. Biochem Biophys Res Commun. 2007 20; 3 55 (4): 1051-7.

11. Chekhonin V.P., Gurina O.I., Dmitrieva T.B. Monoclonal antibodies to neurospecific proteins. M., "Medicine", 2007., 344 p.

Table 1 Physico-chemical properties of the obtained recombinant proteins Title Mw (kD a) Mw according to the SDS page electrophoresis, (kDa) pI Primary structure Cx43_173 CBD 22.5 25 6.02 MRGSHHHHHHLKEAGHGTTKEEITKDAEGLDEIDHAE-MELRRGQILWFRGLNRIQTQIKVVKAFHSSLHESIQK-PYNQKSIHSFMTHPE-FAIEEELPRTPLLDEEEEENPDKASKFGTRVLLLDGEVT-PYANTNNNAVDCNQVQLPQSDSSLQSLETSVRQWYIYGFSLSAVYT-CKRDPCPHQVDCFLSRPTEKTI Cx43_173 HPML 27.9 thirty 8.36 MRGSHHHHHHTAFNDWSKEKQFRGLQCRIE-QEQKFSIIRNGQLIQLPVAEIVVGDIAQVKYGDLLPADGI-LIQGNDLKI-DESSLTGESDHVKKSLDKDPMLLSGTHVMEGSGRMVVTAVGVNSQT-GIILTLLGVNEDDE-GEKKKKGKKQGVPENRNKAKTQDGVALEIQPLNSQEGID-NEEKDKKAVKVPKKEKSVLQGKLTRLAVQIG-KAGLLMSQWYIYGFSLSAVYTCKRDPCPHQVDCFLS-RPTEKTI

Claims (1)

A method for producing an immunogenic recombinant extracellular fragment of connexin-43, comprising cloning a second extracellular loop of E2 Cx43, which is a fragment of connexin-43 Q173-1208 with the amino acid sequence QWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTI, in two plasmid plasmids obtained with E. coli pCBQQD cells obtained with pCQQQDL DNA transformed cells to an optical density of 0.7-0.9 with subsequent induction of expression of the plasmid 0.9-1.1 mm isopropylthiogalactoside, the production of recombinant fusion proteins and their isolation in the presence of 7.9-8.1 M urea by Ni-NTA agarose chromatography.

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