RU2527171C1 - RECOMBINANT PLASMID DNA ENCODING HYBRID POLYPEPTIDES WITH PROPERTIES OF RED FLUORESCENT PROTEIN mCherry TO PRODUCE HYBRID FLUORESCENT PROTEINS IN Escherichia coli - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is recombinant plasmid DNA encoding hybrid polypeptides with the properties of red fluorescent protein mCherry. The recombinant plasmid DNA pChFN3 encodes the hybrid protein ChFN3 which has the properties of red fluorescent protein mCherry and 10 domain of human fibronectin of type III, and the recombinant plasmid DNA pChTNF encodes the hybrid protein ChTNF which has the properties of red fluorescent protein mCherry and TNF. The present invention provides efficient production of hybrid proteins pChFN3 and ChTNF with the properties of red fluorescent protein mCherry in the strain E.coli BL21 (DE3).

EFFECT: invention enables to obtain the target protein in high yield by adding a smaller amount of inducer.

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Description

The invention relates to biotechnology, in particular to genetic engineering. It can be used to produce hybrid fluorescent proteins in Escherichia coli cells.

Fluorescent proteins are an effective tool for studying the localization of proteins and the dynamics of intracellular processes [Chudakov D., Lukyanov S., Lukyanov K. Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 2005. V.23. P.605-613; Giepmans B., Adams S., Ellisman M., Tsien R. The fluorescent toolbox for assessing protein location and function. Science. 2006. V.312. P.217-224]. Hybrid fluorescent proteins can be used for molecular visualization of target proteins for in vivo diagnostics [Desplancq D., Rinaldi AS, Stoessel A., Sibler AP, Busso D., Oulad-Abdelghani M., Van Regenmortel MH, Weiss E. Single- chain Fv fragment antibodies selected from an intrabody library as effective mono- or bivalent reagents for in vitro protein detection. J. Immunol. Methods 2011. V.369. P.42-50], to study factors that influence protein interactions, using the FRET method [Huang J., Koide S. Rational conversion of affinity reagents into label-free sensors for peptide motifs by designed allostery. ACS Chem. Biol. 2010. V.5. P.273-277], for immunoassay with a single-component detection system [Sakamoto S., Taura F., Pongkitwitoon B., Putalun W., Tsuchihashi R., Kinjo J., Tanaka H., Morimoto S. Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin. Anal, and Bioanal. Chemistry. 2010. V.356. P.2955-2963].

Recombinant plasmid DNA is known for producing EGFP-scFv fusion protein in E. coli cells [Casey J.L., Coley A.M., Tilley L.M., Foley M. Green fluorescent antibodies: novel in vitro tools. Protein Eng. 2000. V.13. P.445-452]. The EGFP green fluorescent protein gene is cloned in front of the single chain antibody gene of anti-HepBsAg in the pGC vector. Induction is carried out in Sure strain cells by adding 1 mM IPTG, then cultivation is continued for 4 hours at 25 ° C. The output of the recombinant protein, provided through the use of this design, is 0.2 mg / l of culture. The disadvantage of this design is the large amount of inducer required for expression, and the low yield of the hybrid fluorescent protein.

Known recombinant plasmid DNA that determines the production of a hybrid fluorescent protein REDantibody in E. coli cells [Markiv A., Anani B., Durvasula R., Kang A. Module based antibody engineering: A novel synthetic REDantibody. J. of Immunol. Meth. 2011. V.364. P.40-49]. The genes of VH and VL fragments of antibodies are combined in a single reading frame with the gene of the red fluorescent protein mRFP using cloning under the control of the T71ac promoter in the vector plasmid pET26b. The presence of the coding sequence of the pelB signal peptide ensures the secretion of expression products into the periplasmic space of bacteria. Induction is carried out in the cells of strain BL21 (DE3) by adding 0.3 mm IPTG, then cultivation is continued for 20 hours at 20 ° C. The yield of recombinant proteins provided through the use of this design is 1-9 mg / l of culture. The disadvantage of this design is the low yield of hybrid fluorescent proteins.

The closest to the claimed recombinant plasmid DNA is known, which ensures the production of the zif268-mCherry hybrid fluorescent protein in E. coli cells [Ree J., Kim S. Kit including target sequence-binding protein and method of detecting target nucleic acid by using the kit. US Patent 2012/0064510 A1]. The zinc finger domain (zif) DNA binding protein gene is cloned in front of the mCherry red fluorescent protein gene, separated by a glycine-serine linker coding sequence. The expression of the hybrid gene is regulated by the T71ac promoter in the vector plasmid pET21b. Induction is carried out in the cells of strain BL21 (DE3) by adding 0.5 mM IPTG, after which cultivation is continued at 25 ° C for 16 hours. The disadvantage of the described construction is the location of the mCherry coding sequence at the 3'-end of the hybrid protein gene, which puts the efficiency of hybrid expression protein depending on the nucleotide sequence of the 5'-terminal partner.

An object of the invention is the production of recombinant plasmid DNA, which ensures the efficient production of hybrid fluorescent proteins in E. coli cells. The problem is solved by constructing a recombinant plasmid DNA pChFN3 encoding a hybrid ChFN3 polypeptide with the properties of the red fluorescent protein mCherry and 10 domain of type III human fibronectin ( ¹⁰ FN3), mol. weighing 4.2 Md (6.373 kbp), consisting of NdeI / XhoI - a DNA fragment of plasmid pET32a (+) with a length of 5.366 kbp, including the T71ac promoter, T7 bacteriophage transcription terminator, bla β- gene lactamase, which determines the resistance of ampicillin transformed by the pChFN3 plasmid to plasmid, replication initiation site ori; and NdeI / XhoI, a 1.007 kbp DNA fragment containing the chfn3 gene encoding the amino acid sequence of the ChFN3 fusion polypeptide with the properties of the red fluorescent protein mCherry and ¹⁰ FN3; containing unique recognition sites by restriction endonucleases having the following coordinates: NdeI - 367, BamHI - 1068, XhoI - 1374, as well as recombinant plasmid DNA pChTNF encoding the hybrid ChTNF polypeptide with the properties of red fluorescent protein mCherry and human tumor necrosis factor (TNF) pier mass of 4.24 Md (6.539 kbp), consisting of NdeI / XhoI - a 5.289 kbp DNA fragment of plasmid pET28a, including the T7 bacteriophage promoter, T71ac bacteriophage transcription terminator, KanR gene that determines the stability of transformed plasmid pChTNF cells to kanamycin, site of replication initiation ori; and NdeI / HinDIII - a 1,250 kbp DNA fragment containing the chtnf gene encoding the amino acid sequence of the ChTNF fusion polypeptide with the properties of the red fluorescent protein mCherry and TNF containing unique recognition sites by restriction endonucleases having the following coordinates: NdeI - 238, BamHI - 939, HinDIII - 1442.

Recombinant plasmid DNAs pChFN3 and pChTNF encode the inducible synthesis of hybrid ChFN3 / ChTNF polypeptides with the properties of the red fluorescent protein mCherry and ¹⁰ FN3 / TNF, respectively. The red fluorescent protein mCherry, obtained on the basis of Discosoma DsRed protein, is characterized by high maturation rate and photostability, retains its properties in the N- and C-terminal hybrids [ShanerN.C, Campbell RE, Steinbach P.A., Giepmans BN, Palmer AE, Tsien RY Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp.red fluorescent protein. Nat. Biotechnol. 2004. V.22. P.1567-72]. In contrast to the green fluorescent protein GFP, prone to aggregation during bacterial expression [Chang N., Kaiser C., Hartl F., Barral J. De novo folding of GFP fusion proteins: high efficiency in eukaryotes but not in bacteria. J. Mol. Biol. 2005. V.353. P.397-409], mCherry is a monomeric protein that does not form aggregates.

It is known that the efficiency of gene expression in bacterial cells is largely determined by the efficiency of translation initiation, which in turn depends on the primary and corresponding secondary structure of the 5'-terminal region of the mRNA containing the Shine-Dalgarno sequence and the initiating codon [de Smiy M., van Duin J. Control of prokaryotic translational initiation by mRNA secondary structure. Prog. Nucl. Acid Res. Mol. Biol. 1990. V.38. P.1-35]. A feature of the proposed plasmid constructs is the location of the mCherry red fluorescent protein gene at the 5'-end of the hybrid gene, as well as the modification of its 5'-terminal sequence, which contributes to a more efficient initiation of mRNA translation. The proposed modification does not affect the fluorescent properties of the protein, while contributing to a high level of expression of the mCherry gene and the hybrid protein gene as a whole, regardless of which sequence is cloned at the 3'-terminal position.

To obtain producer strains of the hybrid polypeptides ChFN3 and ChTNF, competent E. coli cells of strain BL21 (DE3) are transformed with the recombinant plasmid pChFN3 and pChTNF, respectively. As a result of the transformation of E. coli strain BL21 (DE3) by the obtained plasmid DNA and the induction of ITPG, the expression level of these polypeptides is not less than 15% of the total cellular protein. A high inducible level of synthesis is ensured by the fact that the plasmids pChFN3 and pChTNF contain the T71ac promoter and the translation amplification gene 10 of the bacteriophage T7, as well as a modification of the 5'-terminal sequence of the gene for the red fluorescent protein mCherry.

The use of the modified coding sequence of the hybrid polypeptides ChFN3 and ChTNF (SEQ ID NO 1, SEQ ID NO 7), under the control of a strong regulated T71ac promoter, in combination with the presence of the LacI repressor gene and the bacteriophage T7 RNA polymerase gene in the chromosome of the host strain, provides an inducible synthesis of target proteins (SEQ ID NO 2, SEQ ID NO 8) with reliable regulation and high yield achieved at low concentrations of inducer. At the 3'-end of the chfn3 gene and at the 5'-end of the chtnf gene, a sequence encoding six histidine residues is located to facilitate purification of the fusion proteins by metal affinity chromatography.

The proposed design can be used to obtain recombinant plasmid DNA containing the coding sequence of other hybrid proteins. By treating plasmid DNAs with pChFN3 or pChTNF with restriction enzymes BamHI and XhoI or BamHI and HinDIII, respectively, cloning vectors are obtained that are combined in a ligation reaction with target coding sequences resulting from PCR and / or treatment with the same restriction enzymes of the plasmid or genomic DNAs containing them. The choice of one of the proposed vectors is determined by the preferred arrangement of the hexahistidine sequence at the N- or C-terminus of the fusion protein (in the case of plasmid DNAs pChTNF and pChFN3, respectively).

The technical result of the claimed invention lies in the fact that the obtained plasmid constructs pChTNF and pChFN3 provide efficient production of hybrid ChFN3 and ChTNF polypeptides with the properties of the red fluorescent protein mCherry in strain E. coli BL21 (DE3) in an amount of at least 15% of the total cellular protein at a concentration inductor 0.2 mm. In contrast to the prototype, the use of these structures provides a high yield of the target protein (at least 100 mg / l) when 2.5 times less amount of inducer (IPTG) is added. The combination of these properties determines the high adaptability of the process of obtaining hybrid fluorescent proteins.

The invention is illustrated by graphic materials.

Figure 1. Physical map of the recombinant plasmid pChFN3. Unique restriction endonuclease sites are indicated. T7 promotor is the T71ac promoter, T7 terminator is the T7 bacteriophage transcription terminator, lad is the lac repressor gene, Ap is the ampicillin resistance gene, ori is the plasmid replication initiation site, chfn3 is the recombinant gene encoding the amino acid sequence of the ChFN3 hybrid polypeptide.

Figure 2. Electrophoregram of cell lysates of E. coli producing strains BL21 (DE3) / pChFN3 (lane 1 - before induction, lane 2 - after induction) and BL21 (DE3) / pChTNF (lane 3) after induction in 13% polyacrylamide gel (M - protein markers of molecular weight); arrows indicate hybrid ChFN3 and ChTNF polypeptides.

Figure 3. Physical map of the recombinant plasmid pChTNF. Unique restriction endonuclease sites are indicated. T7 promotor - T71ac promoter, T7 terminator - T7 bacteriophage transcription terminator, lad - lac repressor gene, KanR - kanamycin resistance gene, ori - plasmid replication initiation site, chtnf recombinant gene encoding the amino acid sequence of the ChTNF hybrid polypeptide.

The invention is illustrated by the following examples.

Example 1

Construction of recombinant plasmid DNA pChFN3.

To amplify the mCherry gene, a polymerase chain reaction (PCR) was performed using pmCherry-Cl plasmid DNA (Clontech, USA) as a template. A 50 μl PCR reaction mixture contains 50 pmol of NdeCh primer (SEQ ID NO 3), 50 pmol of CheBam primer (SEQ ID NO 4), 2 mM of each of the four dNTPs, 2 ng of the DNA template, 5 μl of 10x Pfu DNA buffer polymerases (Fermentas, Lithuania) and 2.5 ea Pfu DNA polymerase (Fermentas). PCR is carried out in the following mode: denaturation - 1 min, 94 ° C; annealing - 40 s, 52 ° C; completion - 40 s, 72 ° C; the number of cycles is 25. 2 μg of the PCR product is treated with restriction enzymes NdeI and BamHI (Fermentas) under the conditions recommended by the manufacturer of the enzymes, a fragment of 0.701 kb in length. isolated from 1% agarose gel using the MinElute Gel Extraction Kit (Qiagen, Germany).

For amplification of the ¹⁰ FN3 gene, PCR is performed under similar conditions using BamFN primers (SEQ ID NO 5) and FN_Xho (SEQ ID NO 6) and plasmid DNA pETFN3 [L.E. Petrovskaya, L.N. Shingarova, E.A. Kryukova , E.F. Boldyreva, S.A. Yakimov, S.V. Guryanova, V.N. Novoseletsky, D.A. Dolgikh, M.P. Kirpichnikov. Construction of TNF-binding proteins based on the fibronectin domain by transplantation of hypervariable regions of the F10 antibody. Biochemistry. 2012.V.77. S.79-89] as a matrix. 2 μg of the PCR product is treated with restriction enzymes BamHI and XhoI (Fermentas) under the conditions recommended by the manufacturer of the enzymes, a fragment of 0.306 kbp. isolated from 1.5% agarose gel using the MinElute Gel Extraction Kit (Qiagen).

1 μg of the obtained fragments and 0.2 μg of the plasmid vector pET32a (+) linearized by treatment with restriction enzymes NdeI and XhoI, 5366 bp in length ligated for 3 hours at 13 ° C in 10 μl of a solution containing 40 mM Tris-HCl (pH 7.8), 10 mM MgCl ₂ , 10 mM dithiothreitol, 0.5 mM adenosine triphosphate and 5 ea. T4 DNA ligases (Fermentas). 5 μl of the ligase mixture is used to transform competent E. coli XL-1 Blue cells (Stratagene, USA). Transformants are plated on LB-arap containing 100 μg / ml ampicillin.

Plasmid DNA was isolated from the grown clones and treated with restriction enzymes NdeI and XhoI followed by electrophoretic analysis of the lengths of restriction fragments on a 1% agarose gel. The structure of the cloned gene in the selected clones is confirmed by nucleotide sequence determination. The result is the target plasmid DNA pChFN3 (Figure 1).

Example 2

Obtaining a producer strain of the hybrid ChFN3 polypeptide.

Recombinant plasmid DNA pChFN3 was transformed with competent E. coli BL21 cells (DE3) (Novagen) and, after growing recombinant clones on LB-arape with ampicillin (100 μg / ml) at 37 ° C, a producer strain of the hybrid ChFN3 polypeptide was obtained.

Example 3

Determination of the productivity of the producer strain of the hybrid ChFN3 polypeptide.

To determine productivity, E. coli BL21 (DE3) / pChFN3 cells were grown at 37 ° C in 5 ml of LB liquid medium containing 100 μg / ml ampicillin for 16 hours on a shaker at 250 rpm. The resulting night culture in a volume of 100 μl was transferred to 5 ml of LB liquid medium (dilution to an optical density of 0.15-0.20 at a wavelength of 560 nm) containing 100 μg / ml ampicillin and grown to an optical density of 0.8 (wavelength 560 nm) at 37 ° C and stirring 250 rpm IPTG was added to a final concentration of 0.2 mM, after which the cells were grown for 4 hours at 27 ° C with stirring at 250 rpm. A culture sample was taken in an amount of 1 optical unit (wavelength 560 nm) and centrifuged for 5 minutes at a speed of 7000 rpm. The precipitated cells are suspended in 100 μl of a buffer containing 125 mM Tris-HCl, pH 6.8, 20% glycerol, 3% sodium dodecyl sulfate, 3% mercaptoethanol, 0.005% bromophenol blue, incubated for 10 min in a boiling water bath, samples with a volume of 10 μl analyzed by electrophoresis in 13% polyacrylamide gel with sodium dodecyl sulfate. At the end of electrophoresis, the gel is stained with Coomassie R-250. After washing the dye, the gel is scanned and the results are mathematically processed using the Scion Image program (Scion Corp., USA). According to the scan, the content of the hybrid ChFN3 polypeptide is 15% of the total cellular protein. Figure 2 presents the electrophoregram of cell lysates of the producer strain E. coli BL21 (DE3) / pChFN3 in a 13% polyacrylamide gel before (lane 1) and after induction of IPTG (lane 2) (M - protein molecular weight markers; arrow indicated ChFN3 polypeptide).

Example 4

Isolation and purification of the hybrid ChFN3 polypeptide.

The biosynthesis of the hybrid ChFN3 polypeptide is induced in the cells of the E. coli producer strain BL21 (DE3) / pChFN3 IPTG (final concentration 0.2 mM) for 4 hours. Cells are centrifuged for 15 minutes at 7000 rpm.

1 g of wet biomass-induced cells are resuspended in 10 ml of buffer (50 mM Tris-HCl pH 8.0; 200 mM NaCl; 1 mM PMSF) and then the cells are destroyed by treatment in a Branson Sonifier 450 ultrasonic disintegrator, preventing heating above 10 ° C. The resulting suspension is centrifuged for 15 minutes at 17,000 rpm. Isolation and purification of the desired polypeptide from the supernatant is carried out using Ni-affinity chromatography on a Ni-Sepharose FastFlow column (GE Healthcare) with a volume of 2 ml in a concentration gradient of imidazole (0.1-0.5 M) in a buffer (20 mM Tris-HCl pH 8.0; 200 mM NaCl). The combined fractions containing purified protein and having a purity greater than 90% according to protein electrophoresis are dialyzed against a buffer (20 mM Tris-HCl pH 8.0; 50 mM NaCl) and sterilized by filtration. The average yield of the thus purified ChFN3 hybrid polypeptide is 120 mg per 1 liter of culture.

Example 5

Construction of recombinant plasmid DNA pChTNF.

To amplify the TNF gene, a polymerase chain reaction (PCR) is performed using plasmid DNA pTNF331 [L.N. Shingarova, L.N. Sagaidak, R.L. Turetskaya, S.A. Nedospasov, D.S. Esipov, V.G. .Korobko. Mutants of tumor necrosis factor alpha human: obtaining and some properties. Bioorgan, chemistry. 1996.V.22. S.243-251] as a matrix. A 50 μl PCR reaction mixture contains 50 pmol of Bam_TNF primer (SEQ ID NO 9), 50 pmol of TNFHIND primer (SEQ ID NO 10), 2 mM of each of the four dNTPs, 2 ng of the DNA template, 5 μl of 10x Pfu DNA buffer polymerases (Fermentas, Lithuania) and 2.5 e.a. Pfu DNA polymerase (Fermentas). PCR is carried out in the following mode: denaturation - 1 min, 94C; annealing - 40 s, 54 ° C; completion - 45 s, 72 ° C; the number of cycles is 25. 2 μg of the PCR product is treated with restriction enzymes BamHI and HindIII (Fermentas) under the conditions recommended by the manufacturer of the enzymes, a fragment of 0.503 kb in length. isolated from 1% agarose gel using the MinElute Gel Extraction Kit (Qiagen, Germany).

A DNA fragment containing the mCherry gene is obtained by treating plasmid DNA with pChFN3 restriction enzymes NdeI and BamHI (Fermentas), a fragment of 0.701 kb in length. isolated from 1% agarose gel using the MinElute Gel Extraction Kit (Qiagen, Germany).

1 μg of the obtained fragments and 0.2 μg of the plasmid vector pET28a (+) linearized by treatment with restriction enzymes NdeI and HinDIII, 5289 bp in length. ligated for 3 hours at 13 ° C in 10 μl of a solution containing 40 mM Tris-HCl (pH 7.8), 10 mM MgCl ₂ , 10 mM dithiothreitol, 0.5 mM adenosine triphosphate and 5 ea. T4 DNA ligases (Fermentas). 5 μl of the ligase mixture is used to transform competent E. coli XL-1 Blue cells (Stratagene, USA). Transformants are plated on LB-arap containing 25 μg / ml kanamycin.

Plasmid DNA was isolated from the grown clones and treated with restriction enzymes NdeI and HinDIII followed by electrophoretic analysis of the lengths of restriction fragments on a 1% agarose gel. The structure of the cloned gene in the selected clones is confirmed by nucleotide sequence determination. The result is the target plasmid DNA pChTNF (Figure 3).

Example 6

Obtaining a producer strain of the hybrid ChTNF polypeptide.

The recombinant plasmid DNA pChTNF was transformed with competent E. coli BL21 (DE3) cells (Novagen) and, after growing the recombinant clones on an LB-arape with kanamycin (25 μg / ml) at 37 ° C, a producer strain of the ChTNF hybrid polypeptide was obtained.

Example 7

Determination of the productivity of the producer strain of the hybrid ChTNF polypeptide.

To determine productivity, E. coli BL21 (DE3) / pChTNF cells were grown at 37 ° C in 5 ml of LB liquid medium containing 25 μg / ml kanamycin for 16 hours on a shaker at 250 rpm. The resulting night culture in a volume of 100 μl was transferred to 5 ml of LB liquid medium (dilution to an optical density of 0.15-0.20 at a wavelength of 560 nm) containing 25 μg / ml kanamycin, and grown to an optical density of 0.8 (wavelength 560 nm) at 37 ° C and stirring 250 rpm IPTG was added to a final concentration of 0.2 mM, after which the cells were grown for 4 hours at 27 ° C with stirring at 250 rpm. A culture sample was taken in an amount of 1 optical unit (wavelength 560 nm) and centrifuged for 5 minutes at a speed of 7000 rpm. The precipitated cells are suspended in 100 μl of a buffer containing 125 mM Tris-HCl, pH 6.8, 20% glycerol, 3% sodium dodecyl sulfate, 3% mercaptoethanol, 0.005% bromophenol blue, incubated for 10 min in a boiling water bath, samples with a volume of 10 μl analyzed by electrophoresis in 13% polyacrylamide gel with sodium dodecyl sulfate. At the end of electrophoresis, the gel is stained with Coomassie R-250. After washing the dye, the gel is scanned and the results are mathematically processed using the Scion Image program (Scion Corp., USA). According to the scan? the content of the hybrid ChTNF polypeptide is 20% of the total cellular protein. Figure 2 presents the electrophoregram of cell lysates of the producer strain E. coli BL21 (DE3) / pChTNF in 13% polyacrylamide gel after induction of IPTG (lane 3) (M - protein molecular weight markers; arrow indicates the ChTNF polypeptide).

Example 8

Isolation and purification of the hybrid ChTNF polypeptide.

The biosynthesis of the hybrid ChTNF polypeptide is induced in the cells of the E. coli producer strain BL21 (DE3) / pChTNF IPTG (final concentration 0.2 mM) for 4 hours. The cells are centrifuged for 15 minutes at 7000 rpm.

1 g of wet biomass-induced cells are resuspended in 10 ml of buffer (50 mM Tris-HCl pH 8.0; 200 mM NaCl; 1 mM PMSF) and then the cells are destroyed by treatment in a Branson Sonifier 450 ultrasonic disintegrator, preventing heating above 10 ° C. The resulting suspension is centrifuged for 15 minutes at 17,000 rpm. Isolation and purification of the recombinant polypeptide from the supernatant is carried out using Ni-affinity chromatography on a Ni-Sepharose FastFlow column (GE Healthcare) with a volume of 2 ml in an imidazole concentration gradient (0.1-0.5 M) in a buffer (20 mM Tris-Hcl pH 8.0; 200 mM NaCl). The combined fractions containing purified protein and having a purity greater than 90% according to protein electrophoresis are dialyzed against a buffer (20 mM Tris-HCl pH 8.0; 50 mM NaCl) and sterilized by filtration. The average yield of the thus purified ChTNF hybrid polypeptide is 150 mg per 1 liter of culture.

The list of sequences presented in the description of the invention

SEQ ID NO 1.

The nucleotide sequence of the gene of the hybrid ChFN3 polypeptide in plasmid pChFN3. The initiating codon is in bold.

SEQ ID NO 2.

Amino acid sequence of the hybrid ChFN3 polypeptide encoded by the recombinant plasmid pChFN3.

The nucleotide sequence of the primer used to amplify the mCherry gene.

The nucleotide sequence of the primer used to amplify the mCherry gene.

The nucleotide sequence of the primer used to amplify the ¹⁰ FN3 gene.

The nucleotide sequence of the primer used to amplify the ^I0 FN3 gene.

The nucleotide sequence of the gene of the hybrid ChTNF polypeptide in the plasmid pChTNF. The initiating codon is in bold.

Amino acid sequence of the hybrid ChTNF polypeptide encoded by the recombinant plasmid pChTNF.

The nucleotide sequence of the primer used to amplify the TNF gene.

The nucleotide sequence of the primer used to amplify the TNF gene.

Claims (2)

1. Recombinant plasmid DNA pChFN3 encoding a hybrid ChFN3 polypeptide having the amino acid sequence of SEQ ID NO: 2, with the properties of the red fluorescent protein mCherry and 10 domain of type III human fibronectin, mol. mass of 4.2 Md (6.373 kbp), consisting of NdeI / XhoI - a DNA fragment of plasmid pET32a with a length of 5.366 kbp, including the T7 bacteriophage promoter, T71ac bacteriophage transcription terminator, β-lactamase bla gene, determining the resistance of ampicillin transformed by pChFN3 plasmid cells, replication initiation site ori; and NdeI / XhoI, a 1.007 kbp DNA fragment containing the chfn3 gene encoding the amino acid sequence of the ChFN3 fusion polypeptide (SEQ ID NO: 2) with the properties of the red fluorescent protein mCherry and type 10 human fibronectin domain III. 2. Recombinant plasmid DNA pChTNF encoding a hybrid ChTNF polypeptide having the amino acid sequence of SEQ ID NO: 8, with the properties of the red fluorescent protein mCherry and TNF, mol. mass of 4.24 Md (6.539 kbp), consisting of NdeI / HinDIII - a DNA fragment of plasmid pET28a with a length of 5.289 kbp, including the T7 bacteriophage promoter, the T7lac bacteriophage transcription terminator, the KanR gene that determines the stability of transformed plasmid pChTNF cells to kanamycin, site of replication initiation ori; and NdeI / HinDIII, a 1,250 kbp DNA fragment containing the chtnf gene encoding the amino acid sequence of the ChTNF fusion polypeptide (SEQ ID NO: 8) with the properties of the red fluorescent protein mCherry and TNF.

Images