Roman S. Esipov

Selected publications

  1. Timofeev V.I., Sinitsyna E.V., Kostromina M.A., Muravieva T.I., Makarov D.A., Mikheeva O.O., Kuranova I.P., Esipov R.S. (2017). Crystal structure of recombinant phosphoribosylpyrophosphate synthetase 2 from Thermus thermophilus HB27 complexed with ADP and sulfate ions. Acta Crystallogr F Struct Biol Commun 73 (Pt 6), 369–375 [+]

    Phosphoribosylpyrophosphate synthetase (PRPPS) from the thermophilic bacterial strain Thermus thermophilus HB27 catalyzes the synthesis of phosphoribosylpyrophosphate from ribose 5-phosphate and ATP, and belongs to the class I PRPPSs. The three-dimensional structure of the recombinant enzyme was solved at 2.2 Å resolution using crystals grown in microgravity from protein solution containing ATP, magnesium and sulfate ions. An ADP molecule was located in the active site of each subunit of the hexameric enzyme molecule and sulfate ions were located in both the active and allosteric sites. It was found that the catalytic loop that restricts the active-site area and is usually missing from the electron-density map of class I PRPPSs adopts different conformations in three independent subunits in T. thermophilus PRPPS. A closed conformation of the active site was found in one of subunits where the highly ordered catalytic β-hairpin delivers the Lys and Arg residues that are essential for activity directly to the ADP molecule, which occupies the ATP-binding site. A comparison of the conformations of the catalytic loop in the three independent subunits reveals a possible mode of transition from the open to the closed state of the active site during the course of the catalyzed reaction.

    ID:1806
  2. Esipov R.S., Makarov D.A., Stepanenko V.N., Miroshnikov A.I. (2016). Development of the intein-mediated method for production of recombinant thymosin β4 from the acetylated in vivo fusion protein. J. Biotechnol. 228, 73–81 [+]

    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.

    ID:1808
  3. Макаров Д.А., Есипов Р.С. (2016). Разработка способов получения аналогов тимозин-бета 4 в виде коньюгатов, устойчивых к деградации в токе крови. Биотехнология 32 (2), 57–71 [+]

    В работе представлен способ получения моноконъюгатов рекомбинантного тимозин-бета 4 (Т|34) человека с капроновой кислотой, полисиаловой кислотой и полиэтиленгликолем (ПЭГ). Реакцию ацилирования проводили с использованием ангидрида гексановой кислоты, сиалирования - бутиральдегидного производного полисиаловой кислоты (14 кДа) и ПЭГилирования - пропиональдегидного производного ПЭГ (10 кДа). В результате многофакторных экспериментов были определены компоненты реакционной смеси и оптимизированы условия проведения данных реакций. Для каждого из аналогов разработана схема одностадийной очистки с помощью ОФ-ВЭЖХ; степень чистоты при этом составила не менее 98%. Результаты пептидного картирования в совокупности с хромато-масс-спектрометрическим и электрофоретическим методами анализа показали, что полученные аналоги тимозин-бета 4 являются моноконъюгатами, в которых исходный пептид модифицирован по N-концевому серину. Полученные аналоги обладают повышенной устойчивостью к деградации в плазме крови по сравнению с немодифицированным тимозином-бета 4 и рассматриваются как перспективные кандидаты для дальнейших биологических испытаний.

    ID:1980
  4. Fateev I.V., Kharitonova M.I., Antonov K.V., Konstantinova I.D., Stepanenko V.N., Esipov R.S., Seela F., Temburnikar K.W., SeleyRadtke K.L., Stepchenko V.A., Sokolov Y.A., Miroshnikov A.I., Mikhailopulo I.A. (2015). Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base-Modified Nucleosides. Chemistry 21 (38), 13401–19 [+]

    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.

    ID:1809
  5. Moiseeva E.V., Beirakhova K.A., Semushina S.G., Aronov D.A., Makarov D.A., Esipov R.S. (2015). Efficiency of recombinant thymosin β4 in spontaneous mouse model of chronic dermatitis. Bull. Exp. Biol. Med. 158 (5), 670–2 [+]

    The therapeutic efficiency of recombinant thymosin β4 (rTβ4) synthesized by us was studied in vivo on spontaneous CBRB mouse model that is adequate to human chronic dermatitis. Three applications of the drug during a week significantly alleviated symptoms of the disease in female mice, and in complex with subsequent antibacterial and antifungal therapy led to a pronounced and lasting (2 months) therapeutic effect. The results attest to a possibility of using rTβ4 in combination with the known treatment protocols for chronic inflammatory diseases of the skin.

    ID:1281
  6. Esipov R.S., Kostromina M.A. (2015). Comparative Analysis of the Effectiveness of C-terminal Cleavage Intein-Based Constructs in Producing a Recombinant Analog of Anophelin, an Anticoagulant from Anopheles albimanus. Appl. Biochem. Biotechnol. 175 (5), 2468–88 [+]

    Production of small recombinant peptides by expressing them as fusion proteins, with subsequent proteolytic or chemical cleavage of the latter, is a widespread approach in modern biotechnology. An alternative method is to produce such peptides as self-cleaving fusion proteins with inteins. To date, only a small proportion of known inteins have been used for this purpose, and analysis of other inteins for the ability to cleave off the target polypeptide can significantly expand the range of intein-based transgenic constructs available to researchers. Most interesting in practical terms are С-terminal cleavage constructs for producing target polypeptides without an N-terminal methionine residue. We prepared two new such constructs with mini-inteins GyrA from Mycobacterium xenopi and RIR1 from Methanobacterium thermoautotrophicum. Together with the previous construct based on the artificial mini-intein derived from Synechocystis sp. DnaB intein, they were used to produce a recombinant analog of anophelin, the naturally occurring thrombin inhibitor from the mosquito Anopheles albimanus. The effectiveness of the constructs with Ssp DnaB and Mth RIR1 proved to be relatively low because of spontaneous fusion protein cleavage during the producer strain culturing in the former case and a low degree of its cleavage upon purification in the latter case. The most effective Mxe GyrA construct was used to develop a semipreparative procedure for producing recombinant anophelin, with its yield reaching 91 ± 2 mg protein per liter of culture medium. As determined by an amidolytic assay, the antithrombin activity and K i of recombinant anophelin were 3362.8 ATU/mg and 87 ± 3 рМ, respectively.

    ID:1249
  7. Poltavtseva R.A., Nikonova Y.A., Selezneva I.I., Yaroslavtseva A.K., Stepanenko V.N., Esipov R.S., Pavlovich S.V., Klimantsev I.V., Tyutyunnik N.V., Grebennik T.K., Nikolaeva A.V., Sukhikh G.T. (2014). Mesenchymal stem cells from human dental pulp: isolation, characteristics, and potencies of targeted differentiation. Bull. Exp. Biol. Med. 158 (1), 164–9 [+]

    We studied cell cultures isolated from the pulp of third molar germ of an adult human and from the skin of a human fetus on gestation day 10. Both cultures expressed similar repertoire of surface markers typical of multipotent mesenchymal cells (CD44, CD90, and CD105). Under in vitro conditions, dental pulp cells were more susceptible to factors inducing their differentiation into adipogenic, chondrogenic, and osteogenic lineage cells.

    ID:1895
  8. Timofeev V., Abramchik Y., Zhukhlistova N., Muravieva T., Fateev I., Esipov R., Kuranova I. (2014). 3'-Azidothymidine in the active site of Escherichia coli thymidine phosphorylase: the peculiarity of the binding on the basis of X-ray study. Acta Crystallogr. D Biol. Crystallogr. 70 (Pt 4), 1155–65 [+]

    The structural study of complexes of thymidine phosphorylase (TP) with nucleoside analogues which inhibit its activity is of special interest because many of these compounds are used as chemotherapeutic agents. Determination of kinetic parameters showed that 3'-azido-3'-deoxythymidine (3'-azidothymidine; AZT), which is widely used for the treatment of human immunodeficiency virus, is a reversible noncompetitive inhibitor of Escherichia coli thymidine phosphorylase (TP). The three-dimensional structure of E. coli TP complexed with AZT was solved by the molecular-replacement method and was refined at 1.52 Å resolution. Crystals for X-ray study were grown in microgravity by the counter-diffusion technique from a solution of the protein in phosphate buffer with ammonium sulfate as a precipitant. The AZT molecule was located with full occupancy in the electron-density maps in the nucleoside-binding pocket of TP, whereas the phosphate-binding pocket of the enzyme was occupied by phosphate (or sulfate) ion. The structure of the active-site cavity and conformational changes of the enzyme upon AZT binding are described in detail. It is found that the position of AZT differs remarkably from the positions of the pyrimidine bases and nucleoside analogues in other known complexes of pyrimidine phosphorylases, but coincides well with the position of 2'-fluoro-3'-azido-2',3'-dideoxyuridine (N3FddU) in the recently investigated complex of E. coli TP with this ligand (Timofeev et al., 2013). The peculiarities of the arrangement of N3FddU and 3'-azidothymidine in the nucleoside binding pocket of TP and correlations between the arrangement and inhibitory properties of these compounds are discussed.

    ID:1250
  9. Fateev I.V., Antonov K.V., Konstantinova I.D., Muravyova T.I., Seela F., Esipov R.S., Miroshnikov A.I., Mikhailopulo I.A. (2014). The chemoenzymatic synthesis of clofarabine and related 2'-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases. Beilstein J Org Chem 10, 1657–69 [+]

    Two approaches to the synthesis of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (1, clofarabine) were studied. The first approach consists in the chemical synthesis of 2-deoxy-2-fluoro-α-D-arabinofuranose-1-phosphate (12a, (2F)Ara-1P) via three step conversion of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranose (9) into the phosphate 12a without isolation of intermediary products. Condensation of 12a with 2-chloroadenine catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of clofarabine in 67% yield. The reaction was also studied with a number of purine bases (2-aminoadenine and hypoxanthine), their analogues (5-aza-7-deazaguanine and 8-aza-7-deazahypoxanthine) and thymine. The results were compared with those of a similar reaction with α-D-arabinofuranose-1-phosphate (13a, Ara-1P). Differences of the reactivity of various substrates were analyzed by ab initio calculations in terms of the electronic structure (natural purines vs analogues) and stereochemical features ((2F)Ara-1P vs Ara-1P) of the studied compounds to determine the substrate recognition by E. coli nucleoside phosphorylases. The second approach starts with the cascade one-pot enzymatic transformation of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a, followed by its condensation with 2-chloroadenine thereby affording clofarabine in ca. 48% yield in 24 h. The following recombinant E. coli enzymes catalyze the sequential conversion of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a: ribokinase (2-deoxy-2-fluoro-D-arabinofuranose-5-phosphate), phosphopentomutase (PPN; no 1,6-diphosphates of D-hexoses as co-factors required) (12a), and finally PNP. The substrate activities of D-arabinose, D-ribose and D-xylose in the similar cascade syntheses of the relevant 2-chloroadenine nucleosides were studied and compared with the activities of 2-deoxy-2-fluoro-D-arabinose. As expected, D-ribose exhibited the best substrate activity [90% yield of 2-chloroadenosine (8) in 30 min], D-arabinose reached an equilibrium at a concentration of ca. 1:1 of a starting base and the formed 2-chloro-9-(β-D-arabinofuranosyl)adenine (6) in 45 min, the formation of 2-chloro-9-(β-D-xylofuranosyl)adenine (7) proceeded very slowly attaining ca. 8% yield in 48 h.

    ID:1894
  10. Timofeev V., Smirnova E., Chupova L., Esipov R., Kuranova I. (2012). X-ray study of the conformational changes in the molecule of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis during the catalyzed reaction. Acta Crystallogr. D Biol. Crystallogr. 68 (Pt 12), 1660–70 [+]

    Structures of recombinant phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium tuberculosis (PPATMt) in the apo form and in complex with the substrate ATP were determined at 1.62 and 1.70 Å resolution, respectively, using crystals grown in microgravity by the counter-diffusion method. The ATP molecule of the PPATMt-ATP complex was located with full occupancy in the active-site cavity. Comparison of the solved structures with previously determined structures of PPATMt complexed with the reaction product dephosphocoenzyme A (dPCoA) and the feedback inhibitor coenzyme A (CoA) was performed using superposition on C(α) atoms. The peculiarities of the arrangement of the ligands in the active-site cavity of PPATMt are described. The conformational states of the PPAT molecule in the consequent steps of the catalyzed reaction in the apo enzyme and the enzyme-substrate and enzyme-product complexes are characterized. It is shown that the binding of ATP and dPCoA induces the rearrangement of a short part of the polypeptide chain restricting the active-site cavity in the subunits of the hexameric enzyme molecule. The changes in the quaternary structure caused by this rearrangement are accompanied by a variation of the size of the inner water-filled channel which crosses the PPAT molecule along the threefold axis of the hexamer. The molecular mechanism of the observed changes is described.

    ID:1810
  11. Esipov R., Beyrakhova K., Likhvantseva V., Stepanova E., Stepanenko V., Kostromina M., Abramchik Y., Miroshnikov A. (2012). Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model. Biochimie 94 (6), 1368–75 [+]

    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.

    ID:1251
  12. Esipov R.S., Abramchik Y.A., Fateev I.V., Konstantinova I.D., Kostromina M.A., Muravyova T.I., Artemova K.G., Miroshnikov A.I. (2009). A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides. Acta Naturae 8 (4), 82–90 [+]

    We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.

    ID:1807
  13. Kostromina M.A., Esipov R.S., Miroshnikov A.I. (2009). [Biotechnological production of recombinant analogs of hirudin-1 from Hirudo medicinalis]. Bioorg. Khim. 38 (2), 166–76 [+]

    Hirudin-1 is a highly selective inhibitor of thrombin secreted by salivary glands of the medicinal leech Hirudo medicinalis. This direct anticoagulant is used for the treatment and prevention of disorders in blood coagulation system. Apart from the existing recombinant analog of hirudin-1 (63-desulfatohirudin-1, desirudin) its modified analogs possessing higher activity and stability are of medical value. In this study artificial genes of hirudin and two its analogs (hirudin-1, [Leu1, Thr2]-hirudin-1 and [Leu1, Thr2]-hirudin-1/3) were synthesized and cloned in an expression vector pTWIN1 in frame with the gene of mini-intein SspDnaB from Synechocystis sp. Producing strains of the corresponding fusion proteins were constructed using E. coli strain ER2566. Biotechnological schemes for the production of 63-desulfatohirudin-1 and its analogs were developed. The scheme includes the following stages: isolation of the fusion protein after the desintegration of the cell biomass, refolding of the target peptide within the fusion protein, pH-inducible cleavage of the fusion protein, and chromatographic purification of the target product. Antithrombotic activity of the obtained peptides was determined by a standard amidolytic assay. The developed methods for the production of 63-desulfatohirudin-1, [Leu1, Thr2]-desulfatohirudin-1 [Leu1, Thr2]-desulfatohirudin-1/3 allowed to obtain these peptides with high yields (14, 25 and 24 mg per liter of cell culture respectively) and high activity (13423, 33333 and 19802 ATU/mg respectively).

    ID:1811
  14. Esipov R.S., Gurevich A.I., Stepanenko V.N., Chupova L.A., Chuvikovskiĭ D.V., Miroshnikov A.I. (2009). [Recombinant thymosin alpha1]. Bioorg. Khim. 30 (5), 481–6 [+]

    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.

    ID:1815
  15. Kommer A.A., Dashkova I.G., Esipov R.S., Miroshnikov A.I., Spirin A.S. (2009). Synthesis of functionally active human proinsulin in a cell-free translation system. Dokl. Biochem. Biophys. 401, 154–8 ID:1816
  16. Konstantinova I.D., Leonteva N.A., Galegov G.A., Ryzhova O.I., Chuvikovskiĭ D.V., Antonov K.V., Esipov R.S., Taran S.A., Verevkina K.N., Feofanov S.A., Miroshnikov A.I. (2009). [Biotechnological synthesis of ribavirin. Effect of ribavirin and its various combinations on the reproduction of Vaccinia virus]. Bioorg. Khim. 30 (6), 613–20 [+]

    The biotechnological method of synthesis of ribavirin, vidarabin, and 6-azauridine by the use of immobilized recombinant enzymatic preparations of nucleoside phosphorylase was improved. The effect of ribavirin and its combinations with the other synthesized nucleosides on the reproduction of Vaccinia virus was studied using cultures of Vero cells. The combination of ribavirin and vidarabin was shown to provide an antiviral effect at lesser concentrations than when these compounds were taken separately. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

    ID:1817
  17. Panova N.G., Shcheveleva E.V., Alekseev K.S., Mukhortov V.G., Zuev A.N., Mikhailov S.N., Esipov R.S., Chuvikovskiĭ D.V., Miroshnikov A.I. (2009). [Using of 4-thiouridine and 4-thiothymidine for pyrimidine nucleoside phosphorylase studing]. Mol. Biol. (Mosk.) 38 (5), 907–13 [+]

    4-Thiouridine and 4-thiothymidine were developed as efficient substrates for spectrophotometric determination of uridine phosphorylase and thymidine phosphorylase activity. 4-Thiouridine has maximum absorbance at 330 nm (pH 7.5). The change in extinction coefficient for 4-thiouridine/4-thiouracil, deltaepsilon is 3000 M(-1) x cm(-1). It appeared that 4-thiouridine is a good substrate for uridine phosphorylase with Michaelis-Menten constant 130 microM and kcat 49 s(-1). In the case of 4-thiothymidine/4-thiothymine deltaepsilon is even larger: 5000 M(-1) x cm(-1) at 336 nm.

    ID:1818
  18. Stepannenko V.N., Esipov R.S., Gurevich A.I., Chupova L.A., Miroshnikov A.I. (2009). [Recombinant oxyntomodulin]. Bioorg. Khim. 33 (2), 245–50 [+]

    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.

    ID:1893
  19. Esipov R.S., Stepanenko V.N., Chupova L.A., Boyarskikh U.A., Filipenko M.L., Miroshnikov A.I. (2008). Production of recombinant human epidermal growth factor using Ssp dnaB mini-intein system. Protein Expr. Purif. 61 (1), 1–6 [+]

    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.

    ID:1812
  20. Roivainen J., Elizarova T., Lapinjoki S., Mikhailopulo I.A., Esipov R.S., Miroshnikov A.I. (2007). An enzymatic transglycosylation of purine bases. Nucleosides Nucleotides Nucleic Acids 26 (8-9), 905–9 [+]

    An enzymatic transglycosylation of purine heterocyclic bases employing readily available natural nucleosides or sugar-modified nucleosides as donors of the pentofuranose fragment and recombinant nucleoside phosphorylases as biocatalysts has been investigated. An efficient enzymatic method is suggested for the synthesis of purine nucleosides containing diverse substituents at the C6 and C2 carbon atoms. The glycosylation of N(6)-benzoyladenine and N(2)-acetylguanine and its O(6)-derivatives is not accompanied by deacylation of bases.

    ID:200
  21. Panova N.G., Alexeev C.S., Kuzmichov A.S., Shcheveleva E.V., Gavryushov S.A., Polyakov K.M., Kritzyn A.M., Mikhailov S.N., Esipov R.S., Miroshnikov A.I. (2007). Substrate specificity of Escherichia coli thymidine phosphorylase. Biochemistry Mosc. 72 (1), 21–8 [+]

    Substrate specificity of Escherichia coli thymidine phosphorylase to thymidine derivatives modified at 5' -, 3' -, and 2' ,3' - positions of the sugar moiety was studied. Equilibrium and kinetic constants (K(m), K(I), k(cat)) of the phosphorolysis reaction have been determined for 20 thymidine analogs. The results are compared with X-ray and molecular dynamics data. The most important hydrogen bonds in the enzyme-substrate complex are revealed.

    ID:1813
  22. Chuvikovsky D.V., Esipov R.S., Skoblov Y.S., Chupova L.A., Muravyova T.I., Miroshnikov A.I., Lapinjoki S., Mikhailopulo I.A. (2006). Ribokinase from E. coli: expression, purification, and substrate specificity. Bioorg. Med. Chem. 14 (18), 6327–32 [+]

    Ribokinase (RK) was expressed in the Escherichia coli ER2566 cells harboring the constructed expression plasmid encompassing the rbsK gene, encoding ribokinase. The recombinant enzyme was purified from sonicated cells by double chromatography to afford a preparation that was ca. 90% pure and had specific activity of 75 micromol/min mg protein. Catalytic activity of RK: (i) is strongly dependent on the presence of monovalent cations (potassium>>>ammonium>cesium), and (ii) is cooperatively enhanced by divalent magnesium and manganese ions. Besides D-ribose and 2-deoxy-D-ribose, RK was found to catalyze the 5-O-phosphorylation of D-arabinose, D-xylose, and D-fructose in the presence of ATP, and potassium and magnesium ions; L-ribose and L-arabinose are not substrates for the recombinant enzyme. A new radiochemical method for monitoring the formation of D-pentofuranose-5-[32P]phosphates in the presence of [gamma-32P]ATP and RK is reported.

    ID:201
  23. Esipov R.S., Stepanenko V.N., Gurevich A.I., Chupova L.A., Miroshnikov A.I. (2006). Production and purification of recombinant human glucagon overexpressed as intein fusion protein in Escherichia coli. Protein Pept. Lett. 13 (4), 343–7 [+]

    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.

    ID:1897
  24. Esipov R.S., Chupova L.A., Shvets S.V., Chuvikovsky D.V., Gurevich A.I., Muravyova T.I., Miroshnikov A.I. (2003). Production and purification of recombinant human oxytocin overexpressed as a hybrid protein in Escherichia coli. Protein Pept. Lett. 10 (4), 404–11 [+]

    The plasmid DNA pERilox4 containing the gene of the recombinant protein, which included the leader sequence and the oxytocinoyl lysine tetramer, was constructed. The high level of gene expression in E. coli was achieved. The method for purification of the recombinant protein and its isolation in the soluble form was developed. The conditions for digestion of the hybrid protein by trypsin and carboxypeptidase B were matched. The effective method for transformation of oxytocinic acid to oxytocin was worked out. The scheme suggested allowed obtaining oxytocin in high yield.

    ID:1814
  25. Esipov R.S., Gurevich A.I., Chuvikovsky D.V., Chupova L.A., Muravyova T.I., Miroshnikov A.I. (2002). Overexpression of Escherichia coli genes encoding nucleoside phosphorylases in the pET/Bl21(DE3) system yields active recombinant enzymes. Protein Expr. Purif. 24 (1), 56–60 [+]

    The Escherichia coli genes encoding purine nucleoside phosphorylase, uridine phosphorylase, and thymidine phosphorylase were cloned into pET plasmids to generate highly effective E. coli BL21(DE3) strains producing each of these enzymes. Optimum conditions for biosynthesis of each enzyme as a soluble protein with intact biological activity were found. The crude preparations are approximately 80% pure and can be used immediately for enzymatic transglycosylation. The enzyme preparations were purified to homogeneity by two steps including fractional precipitation with ammonium sulfate and subsequent chromatography on Sephadex G-100 and DEAE-Sephacel.

    ID:203
  26. Esipov R.S., Gurevich A.I., Kaiushin A.L., Korosteleva M.D., Miroshnikov A.I., Shevchenko L.V., Pluzhnikov K.A., Grishin E.V. (1997). [Recombinant proteins containing amino acid sequences of two ectatomin chains]. Bioorg. Khim. 23 (12), 949–52 [+]

    Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors. Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB). Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained.

    ID:1896
  27. Gurevich A.I., Esipov R.S., Kaiushin A.L., Korosteleva M.D. (1997). [Construction of artificial genes by PCR methods using the synthetic template]. Bioorg. Khim. 23 (6), 492–6 [+]

    Artificial genes were synthesized by the PCR method. Single-stranded DNA contained in an unpurified mixture of oligodeoxynucleotides after automated synthesis was used as a template. The features of this approach were studied.

    ID:1892
  28. Gurevich A.I., Tuzova T.P., Shpak E.D., Starkova N.N., Esipov R.S., Miroshnikov A.I. (1996). [Mechanism of action of the plant hormone jasmonate. 1. Jasomonate-interacting proteins that regulate transcription of the p. pinII potato gene]. Bioorg. Khim. 22 (2), 101–7 [+]

    A fragment containing the regulatory region of the p. pinII gene was isolated from potato DNA by polymerase chain reaction. Interactions of this DNA region with jasmonate determines the transcriptional activation. The isolated DNA fragment was cloned into the pTE2pb plasmid, which was used for preparing an affinity sorbent. Using this sorbent, four proteins were isolated from the total protein capable of desorption at physiological concentration of jasmonate. These proteins are likely to be subunits of two transcription repressors, whereas jasmonate serves as an inducer. Three sequences of the regulatory regions (boxes G, I, and III) are binding sites for repressors; similar sequences were found in various plant genes activated by jasmonate.

    ID:1891
  29. Gurevich A.I., Esipov R.S., Kachalina T.A., Kaiushin A.L. (1995). [Dependence of the level of gene expression in E. coli on the structure of the translation initiation segment (TIR)]. Bioorg. Khim. 21 (4), 282–8 [+]

    The expression levels of genes that are transcribed to give mRNAs with identical leader sequences and even with identical extended coding regions may differ considerably. In order to determine the mechanism of this phenomenon, secondary structures of some mRNAs synthesized from a series of expression plasmids were studied. It was shown that the effect of the mRNA secondary structure in the translation initiation region on the initiation efficiency is due not only to the hairpin formation in this region but also to long-range interactions. When complementary structures tighter than those resulted from the interaction of regions SD, UB1, UB2, and DB with 16S rRNA are formed, the efficiency of the translation initiation and, consequently, the expression level decrease.

    ID:1890
  30. Gurevich A.I., Esipov R.S., Kachalina T.A., Kaliushin A.L., Korosteleva M.D. (1995). [Dependence of the level of gene expression in E. coli on the structure of the translation initiation segment (TIR). I. Primary structure of TIR]. Bioorg. Khim. 21 (2), 117–23 [+]

    The comparison of expression levels of two genes-interleukin-3 (il3) and epidermal growth factor connected to leader peptide of OmpF (lompegf)-was carried out using specially constructed plasmids contained various structures of translational enhancers. It was shown that besides already known binding sites from mRNA translation initiation region (TIR) to 16S rRNA (SD, UB1 and DB), there is additional binding site of TIR disposed from -30 to -60 nt upstream of start codon AUG (UB2) having significant increasing effect on translation initiation and correspondingly on expression level.

    ID:1889