11 results on '"Vasily N. Stepanenko"'
Search Results
2. Biotechnological Method for Production of Recombinant Peptide Analgesic (Purotoxin-1) from Geolycosa sp. Spider Poison
- Author
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Vasily N. Stepanenko, Roman S. Esipov, Tatyana I. Muravyova, T. I. Kostromina, Anatoly I. Miroshnikov, I. O. Zvereva, M. A. Kostromina, Dmitry Makarov, and E. V. Grishin
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0301 basic medicine ,Drug ,Toxin ,media_common.quotation_subject ,Organic Chemistry ,Analgesic ,Purinergic receptor ,Pharmacology ,Biology ,urologic and male genital diseases ,medicine.disease_cause ,Biochemistry ,female genital diseases and pregnancy complications ,law.invention ,body regions ,03 medical and health sciences ,030104 developmental biology ,Spider poison ,law ,Recombinant peptide ,medicine ,Recombinant DNA ,Intein ,media_common - Abstract
Severe chronic and sometimes incurable diseases are frequently accompanied by a pain syndrome. Purotoxin-1, isolated from the poison of the Central Asian Geolycosa sp. spider and selectively inhibiting the purinergic P2X3 receptor (which is considered as a target for the control of the pain states), is one potentially highly effective drug with an analgesic effect. To produce the recombinant purotoxin-1, we created four genetically engineered constructions with different carrier proteins for the expression in E. coli: pTRX-PT1, pCBD-PT1, pGyrA-PT1, pDnaB-PT1. The construction with mini-intein DnaB from the Synechocystis sp. was the most efficient. Using the E. coli C3030/pDnaB-PT1 producer strain, the laboratory method, based on which a pilot technology of recombinant purotoxin-1 production was developed as a result of optimization and scaling, was created. Six grams of recombinant PT1 preparation with the confirmed pharmacological purity was developed for preclinical trials.
- Published
- 2018
3. Development of the intein-mediated method for production of recombinant thymosin β4 from the acetylated in vivo fusion protein
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Dmitry Makarov, Roman S. Esipov, Anatoly I. Miroshnikov, and Vasily N. Stepanenko
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0301 basic medicine ,Recombinant Fusion Proteins ,Bioengineering ,Peptide ,Target peptide ,Biology ,Applied Microbiology and Biotechnology ,Inteins ,03 medical and health sciences ,Escherichia coli ,Protein biosynthesis ,Humans ,Cloning, Molecular ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Thymosin ,Acetylation ,General Medicine ,Fusion protein ,Thymosin beta-4 ,030104 developmental biology ,Biochemistry ,chemistry ,Intein ,Protein Processing, Post-Translational ,Plasmids ,Biotechnology - Abstract
Thymosin β4 is a 43 amino acid long peptide with an acetylated N-terminal serin that has a high potential as a remedy for healing ulcers, wounds and burns. Although protein biosynthesis offers attractive opportunities in terms of a large-scale production, currently thymosin β4 is mainly produced by chemical synthesis. The problems that hinder the successful commercialization of the biotechnological approach are associated with the small peptides expression and N-terminal acetylation. This work presents an innovative biotechnological method for thymosin β4 production that employs the peptide acetylation in vivo. A genetically engineered construct was created, where the Tβ4 coding sequence fused with the intein Mxe GyrA sequence and chitin-binding domain was combined with the acetyltransferase coding sequence to form a polycistronic construct under a stringent control of T7 promoter. This plasmid construct provided for the expression of the Tβ4-intein fusion protein. In the process of the post-translational modification in vivo formyl methionine was completely removed from the target peptide N-terminus and followed by the Tβ4 precursor N-terminal acetylation. The use of the intein-mediated expression system made it possible to extract thymosin β4 in only 2 chromatographic runs. The method is straightforward to implement and scale up.
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- 2016
4. Recognition of Artificial Nucleobases byE. coliPurine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base-Modified Nucleosides
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Maria I. Kharitonova, Vasily N. Stepanenko, Anatoly I. Miroshnikov, Ilja V. Fateev, Konstantin V. Antonov, Igor A. Mikhailopulo, Roman S. Esipov, Irina D. Konstantinova, Yuri A. Sokolov, Frank Seela, Vladimir A. Stepchenko, Kartik Temburnikar, and Katherine L. Seley-Radtke
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Benzimidazole ,Stereochemistry ,Purine analogue ,Purine nucleoside phosphorylase ,Crystallography, X-Ray ,medicine.disease_cause ,Catalysis ,Nucleobase ,Dephosphorylation ,Structure-Activity Relationship ,chemistry.chemical_compound ,Escherichia coli ,medicine ,chemistry.chemical_classification ,Alanine ,Binding Sites ,Base Sequence ,Organic Chemistry ,Nucleosides ,General Chemistry ,Benzoxazole ,Kinetics ,Enzyme ,Purine-Nucleoside Phosphorylase ,chemistry ,Biochemistry - Abstract
A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild-type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8-aza-7-deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α-D-pentofuranose-1-phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2-deoxy-α-D-ribofuranose-1-phosphate in the trans-2-deoxyribosylation reaction. 5-Aza-7-deazaguanine manifested excellent substrate activity for both enzymes, 8-amino-7-thiaguanine and 2-aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2-amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1- and unusual N2-glycosides, respectively. 9-Deaza-5-iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9-deazaxanthine and its 2'-deoxyriboside are weak inhibitors.
- Published
- 2015
5. Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model
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Roman S. Esipov, Ksenia A. Beyrakhova, Vasily N. Stepanenko, Anatoly I. Miroshnikov, Yulia Abramchik, Evgenia V. Stepanova, Vera Likhvantseva, and M. A. Kostromina
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Collagen Type IV ,Tumstatin ,Angiogenesis ,Recombinant Fusion Proteins ,Target peptide ,Peptide ,Autoantigens ,Biochemistry ,Neovascularization ,Mice ,medicine ,Animals ,Corneal Neovascularization ,Tube formation ,chemistry.chemical_classification ,Neovascularization, Pathologic ,Chemistry ,Endothelial Cells ,General Medicine ,medicine.disease ,Fusion protein ,Peptide Fragments ,eye diseases ,Cell biology ,Corneal neovascularization ,Immunology ,Rabbits ,medicine.symptom - Abstract
Tumstatin, a cleavage fragment of collagen IV, is a potent endogenous inhibitor of angiogenesis. Tumstatin-derived peptide T8 possesses all angiostatic properties of full-length tumstatin and indirectly suppresses tumor growth. The potential of T8 to block pathological angiogenesis in the eye has not been explored yet. Here we assess antiangiogenic effects of a recombinant T8 peptide in rabbit corneal neovascularization models. The fusion protein consisting of T8 and thioredoxin was synthesized in a highly efficient Escherichia coli expression system, isolated using ion-exchange chromatography and cleaved with TEV (tobacco etch virus) protease. The target peptide was purified on an anion-exchange resin and by reversed phase high-performance liquid chromatography. The recombinant peptide suppressed the proliferation of basic fibroblast growth factor-induced SVEC-4-10 endothelial cells (simian virus 40-immortalized murine endothelial cells) and inhibited tube formation in these cells in a dose-dependent manner. In rabbit corneal neovascularization models T8 demonstrated the ability to prevent pathological angiogenesis (when injected simultaneously with the induction of neovascularization) and, moreover, to promote the regression of newly-formed blood vessels (when injected on day 8 after angiogenesis stimulation). Our results suggest that T8 may have a therapeutic potential in the treatment of ocular neovascular diseases.
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- 2012
6. Production of Recombinant Oxytocin Through Sulfitolysis of Inteincontaining Fusion Protein
- Author
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L. A. Chupova, Roman S. Esipov, Anatoly I. Miroshnikov, and Vasily N. Stepanenko
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Recombinant Fusion Proteins ,Molecular Sequence Data ,Protein aggregation ,Oxytocin ,Recombinant Oxytocin ,Biochemistry ,Protein Refolding ,Inclusion bodies ,Inteins ,chemistry.chemical_compound ,Structural Biology ,Humans ,Urea ,Trypsin ,Amino Acid Sequence ,Cysteine ,Cloning, Molecular ,Peptide sequence ,Sodium tetrathionate ,Inclusion Bodies ,Base Sequence ,Chemistry ,Lysine ,General Medicine ,Hydrogen-Ion Concentration ,Fusion protein ,Solubility ,Intein - Abstract
An artificial gene consisting of seven copies of an oxytocinoyl-lysine encoding sequence arranged in a tandem was synthesized and inserted downstream of the SspDnaB intein gene in a pTWIN1 plasmid. The corresponding fusion protein Dnab-7oxy contained 16 cysteine residues and formed inclusion bodies when expressed in E. coli. The standard protocol involving solubilization of the fusion protein and its autocatalytic cleavage on a chitin resin was not effective because of a very low yield of the cleavage reaction. Attempts to perform a refolding of the intein part of the fusion protein in solution were also unsuccessful because of a high level of protein aggregation. Sulfitolysis of cysteine residues is known to increase a solubility of proteins and peptides. Therefore we suggested a one-step approach that combines solubilization of inclusion bodies and sulfitolysis of a hybrid protein. The fusion protein was completely reduced and solubilized in 8M urea at pH 9.0 in the presence of sodium sulfite and sodium tetrathionate. The sulfitized protein was loaded onto a chitin column, an efficient cleavage was induced by a pH shift from 9.0 to 6.5, and seven successively connected oxytocinoyl- lysine units were released. The heptamer was subjected to trypsinolysis yielding sulfitized monomers of oxytocinoyllysine. Oxytocinoyl-lysine was refolded as described previously and treated by carboxypeptidase B to form the oxytocinic acid. The target oxytocin amide was then synthesized via methyl ester intermediate. Using this approach 6 mg of recombinant oxytocin can be obtained from 1 g of biomass.
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- 2012
7. Cloning, expression, isolation, and properties of thymidine kinase from herpes simplex virus type 1, strain L2
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A. Yu. Skoblov, A. A. Gus’kova, A. I. Miroshnikov, Maxim V. Yasko, V. L. Andronova, Vasily N. Stepanenko, G. A. Galegov, Roman S. Esipov, and Yu. S. Skoblov
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chemistry.chemical_classification ,Cloning ,Guanine ,Kinase ,Organic Chemistry ,medicine.disease_cause ,Biochemistry ,Molecular biology ,chemistry.chemical_compound ,Herpes simplex virus ,Enzyme ,chemistry ,Thymidine kinase ,medicine ,Thymidine ,Gene - Abstract
Thymidine kinase UL23 gene (EC 2.7.1.145) from the L2 acyclovir-sensitive strain of herpes simplex virus type 1 was cloned and expressed in E. coli. The enzyme was purified by chromatography to the purity of 90% according to PAG electrophoresis data. The Michaelis constants for the reactions with thymidine and acyclovir were determined. The enzyme was found to phosphorylate modified nucleosides, particularly 3′-deoxythymidine, 3′-deoxy-2′,3′-didehydrothymidine, 2′,3′-dideoxycytidine, 9-[(hydroxyethyl)methyl]guanine, E-5-(2-bromovinyl-2′-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, 2′,3′-dideoxydehydrothymidine, β-L-2′,3′-dideoxy-3′-thiacytidine, and 3′-fluoro-3′-deoxythymidine. Some properties of the purified enzyme were compared with those of thymidine kinases of other herpes simplex virus strains. It was shown that acyclovir H-phosphonate inhibited the enzyme.
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- 2011
8. Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system
- Author
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Roman S. Esipov, Vasily N. Stepanenko, Maxim L. Filipenko, Uljana A. Boyarskikh, L. A. Chupova, and Anatoly I. Miroshnikov
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DNA, Complementary ,Expression vector ,Base Sequence ,Epidermal Growth Factor ,Recombinant Fusion Proteins ,Protein subunit ,Molecular Sequence Data ,Biology ,medicine.disease_cause ,Fusion protein ,Inteins ,law.invention ,Biochemistry ,law ,Complementary DNA ,Recombinant DNA ,medicine ,Humans ,Electrophoresis, Polyacrylamide Gel ,Amino Acid Sequence ,Target protein ,Intein ,DnaB Helicases ,Escherichia coli ,Biotechnology - Abstract
Chemical-enzymatic synthesis of human Epidermal Growth Factor (hEGF) cDNA has been performed, following by cloning into expression vector pTWIN1 (New England Biolabs). The resulting recombinant fusion protein expressed in Escherichia coli consisted of the N-terminal chitin-binding domain, mini-intein Ssp dnaB domain and hEGF polypeptide at the C-terminus. In this construct, mini-intein Ssp dnaB played a role of catalytically active subunit capable under certain conditions of autocatalytic cleavage resulting in separation of the target protein. As the hybrid protein had several cysteins in its sequence-one in chitin-binding domain, one in mini-intein and six in hEGF, it was necessary to work out optimal scheme for refolding and purification of the recombinant hEGF. As a result of this work, two schemes of the recombinant hEGF purification have been developed: according to the first scheme, the recombinant protein with reduced cysteins is bound to the chitin column, the hEGF is cleaved off and eluted, and then refolded to form appropriate cystein bridges. In the second scheme, the entire hybrid protein is first refolded to form disulfide bonds and then loaded to affinity resin; the recombinant hEGF is cleaved off and eluted in its native state. In spite of the fact that the first scheme is more common and suitable for a variety of recombinant proteins, in case of recombinant hEGF, the second scheme proved to be more productive and cost-effective.
- Published
- 2008
9. Recombinant oxyntomodulin
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L. A. Chupova, Vasily N. Stepanenko, A. I. Miroshnikov, Gurevich Ai, and Roman S. Esipov
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Recombinant Fusion Proteins ,Molecular Sequence Data ,medicine.disease_cause ,Cleavage (embryo) ,Biochemistry ,Catalysis ,Inteins ,law.invention ,chemistry.chemical_compound ,law ,Escherichia coli ,medicine ,Amino Acid Sequence ,Gene ,dnaB helicase ,chemistry.chemical_classification ,Organic Chemistry ,Synechocystis ,Recombinant Proteins ,Oxyntomodulin ,Enzyme ,chemistry ,Mutation ,Recombinant DNA ,bacteria ,Intein ,DnaB Helicases ,Plasmids - Abstract
An artificial gene encoding oxyntomodulin was obtained using chemical and enzymatic methods and cloned into Escherichia coli. A recombinant plasmid was constructed containing a hybrid oxyntomodulin gene and Ssp dnaB intein from Synechocystis sp. The expression of the resulting hybrid gene in E. coli, its properties, and the conditions of its autocatalytic cleavage to oxyntomodulin were studied.
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- 2007
10. Production and Purification of Recombinant Human Glucagon Overexpressed as Intein Fusion Protein in Escherichia coli
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Anatoly I. Miroshnikov, Vasily N. Stepanenko, Roman S. Esipov, Gurevich Ai, and L. A. Chupova
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Electrophoresis ,endocrine system ,Recombinant Fusion Proteins ,Two-hybrid screening ,Molecular Sequence Data ,Biology ,medicine.disease_cause ,Biochemistry ,Glucagon ,Inteins ,law.invention ,Structural Biology ,law ,Escherichia coli ,medicine ,Humans ,Amino Acid Sequence ,Gene ,dnaB helicase ,General Medicine ,Fusion protein ,Molecular biology ,Recombinant DNA ,Intein ,hormones, hormone substitutes, and hormone antagonists - Abstract
Chemico-enzymatic synthesis and cloning in Esherichia coli of an artificial gene coding human glucagon was performed. Recombinant plasmid containing hybrid glucagons gene and intein Ssp dnaB from Synechocestis sp. was designed. Expression of the obtained hybrid gene in E. coli, properties of the formed hybrid protein, and conditions of its autocatalytic cleavage leading to glucagon formation were studied.
- Published
- 2006
11. Recombinant Thymosin 1
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D V Chuvikovskiĭ, Gurevich Ai, Anatoly I. Miroshnikov, L A Chupova, Roman S. Esipov, and Vasily N. Stepanenko
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Chemistry ,Organic Chemistry ,Thymosin ,medicine.disease_cause ,Biochemistry ,Molecular biology ,law.invention ,Protein splicing ,law ,Recombinant DNA ,medicine ,Bioorganic chemistry ,Thymalfasin ,Intein ,Escherichia coli ,Peptide sequence ,hormones, hormone substitutes, and hormone antagonists - Abstract
An artificial gene encoding thymosin alpha1 was obtained by the chemoenzymatic synthesis and cloned into Escherichia coli. An expressing recombinant plasmid containing the hybrid protein gene, which encodes amino acid sequences of thymosin alpha1 and the Saccharomyces cerevisiae intein Sce VMA, was constructed. The expression of the hybrid protein from the resulting hybrid gene in E. coli, the properties of the resulting hybrid protein, and the conditions for its nonenzymatic cleavage to thymosin alpha1 were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.
- Published
- 2004
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