L E Petrovskaya

Russian Academy of Sciences, Moskva, Moscow, Russia

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Publications (31)74.7 Total impact

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    ABSTRACT: The review outlines progress and problems in the design of non-natural antibodies for clinical applications over the past 10-15 years. The modular structure of natural antibodies and approaches to its targeted modifications and combination with other structural elements and effector molecules are considered. The review covers modern methods for immunoglobulin engineering and promising strategies for the creation and applications of monoclonal antibodies, their derivatives and analogues, including abzymes and scaffolds, oriented to the use in the diagnosis and targeted therapy of cancer and other socially significant diseases. The bibliography includes 225 references.
    Russian Chemical Reviews 01/2015; 84(1):1-26. DOI:10.1070/RCR4459 · 2.58 Impact Factor
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    ABSTRACT: The present study reports metagenomic shotgun sequencing of microbial communities of two ancient permafrost horizons of the Russian Arctic. Results demonstrate a significant difference in microbial community structure of the analyzed samples in general and microorganisms of the methane cycle in particular. Copyright © 2015 Krivushin et al.
    Genome Announcements 01/2015; 3(1). DOI:10.1128/genomeA.01380-14
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    ABSTRACT: We have cloned the gene coding for AT877-a new predicted member of the autotransporter protein family with an esterase passenger domain from permafrost bacterium Psychrobacter cryohalolentis K5(T). Expression of AT877 gene in Escherichia coli resulted in accumulation of the recombinant autotransporter in the outer membrane fraction and at the surface of the induced cells. AT877 displayed maximum hydrolytic activity toward medium-chain p-nitrophenyl esters (C8-C10) at 50 °C and was resistant to the presence of several metal ions, organic solvents and detergents. Previously, we have described a cold-active esterase EstPc from the same bacterium which possesses high activity at low temperatures and relatively high thermal stability. To construct a cell surface display system for EstPc, the hybrid autotransporter gene coding for EstPc with the α-helical linker and the translocator domain from AT877 was constructed and expressed in E. coli. According to the results of the cell fractionation studies and esterase activity measurements, the EstPc passenger was successfully displayed at the surface of the induced cells. It demonstrated a temperature optimum at 15-25 °C and a substrate preference toward p-nitrophenyl butyrate (C4). Obtained results provide a new example of the biotechnologically relevant enzyme from the permafrost microbial community with potential applications for the conversion of short- and medium-chain ester substrates and a basis for the construction of a new cell surface display platform.
    Extremophiles 09/2014; DOI:10.1007/s00792-014-0695-0 · 2.17 Impact Factor
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    ABSTRACT: Hybrid molecules of a new type bearing a red fluorescent protein mCherry and one of the alternative scaffold proteins, the 10th human fibronectin type III domain (10Fn3), which can be used for the construction of antibody mimics with various binding specificity, were obtained. Different variants of the gene encoding the hybrid fluorescent protein were constructed and their expression in Escherichia coli cells was studied. The mCherry N-terminal position and the modification of its N-terminal amino acid sequence proposed were shown to promote the efficient bacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construct a hybrid fluorescent protein ChIBF containing an αVβ3-integrin binding variant of 10Fn3 was obtained, and its use for the visualization of αVβ3-integrin at the surface of MDCK epithelial cells was demonstrated by confocal microscopy.
    Russian Journal of Bioorganic Chemistry 07/2014; 40(4):375-382. DOI:10.1134/S1068162014030121 · 0.62 Impact Factor
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    ABSTRACT: We describe cloning and expression of genes coding for lipase Lip2Pc and lipase-specific foldase LifPc from a psychrotrophic microorganism Psychrobacter cryohalolentis K5(T) isolated from a Siberian cryopeg (the lense of overcooled brine within permafrost). Upon expression in E. coli Lip2Pc accumulated in inclusion bodies while chaperone was synthesized in a soluble form. An efficient protocol for solubilization and subsequent refolding of the recombinant lipase in the presence of the truncated chaperone was developed. Using this procedure Lip2Pc with specific activity of 6900 U/mg was obtained. Contrary to published data on other lipase-chaperone complexes, refolded Lip2Pc was mostly recovered from the complex with chaperone by metal-affinity chromatography. Recombinant Lip2Pc displayed maximum lipolytic activity at 25°C and pH 8.0 with p-nitrophenyl palmitate (C16) as a substrate. Activity assays conducted at different temperatures revealed that the recombinant Lip2Pc is a cold-adapted lipase with ability to utilize substrates with long (C10-C16) hydrocarbon chains in the temperature range from +5 to +65°C. It demonstrated relatively high stability at temperatures above 60°C and in the presence of various metal ions or organic solvents (ethanol, methanol, etc.). Non-ionic detergents, such as Triton X-100 and Tween 20 decreased Lip2Pc activity and SDS completely inhibited it.
    Protein Expression and Purification 07/2013; DOI:10.1016/j.pep.2013.07.011 · 1.51 Impact Factor
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    ABSTRACT: Light-driven proton pumps are present in many organisms. Here, we present a high-resolution structure of a proteorhodopsin from a permafrost bacterium, Exiguobacterium sibiricum rhodopsin (ESR). Contrary to the proton pumps of known structure, ESR possesses three unique features. First, ESR's proton donor is a lysine side chain that is situated very close to the bulk solvent. Second, the α-helical structure in the middle of the helix F is replaced by 310- and π-helix-like elements that are stabilized by the Trp-154 and Asn-224 side chains. This feature is characteristic for the proteorhodopsin family of proteins. Third, the proton release region is connected to the bulk solvent by a chain of water molecules already in the ground state. Despite these peculiarities, the positions of water molecule and amino acid side chains in the immediate Schiff base vicinity are very well conserved. These features make ESR a very unusual proton pump. The presented structure sheds light on the large family of proteorhodopsins, for which structural information was not available previously.
    Proceedings of the National Academy of Sciences 07/2013; 110(36). DOI:10.1073/pnas.1221629110 · 9.81 Impact Factor
  • FEBS Journal 07/2013; 280:138. · 3.99 Impact Factor
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    ABSTRACT: The photocycle of the retinal protein from Exiguobacterium sibiricum, which differs from bacteriorhodopsin in both its primary donor and acceptor, is characterized by visible and infrared spectroscopy. At pH above pKa ~6.5 we find a bacteriorhodopsin-like photocycle, which originates from excitation of the all-trans retinal chromophore with K-, L-, M- and N-like intermediates. At pH below pKa ~6.5 the M-state, which reflects Schiff base deprotonation during proton pumping, is not accumulated. However, using the infrared band at ~1760 cm-1 as a marker for transient protonation of the primary acceptor, we find that Schiff base deprotonation must have occurred at pH not only above but also below the pKa ~6.5. Thus, the M state is formed but not accumulated for kinetic reasons. Further, chromophore reisomerization from the 13-cis to the all-trans conformation occurs very late in the photocycle. The strongly red-shifted states that dominate the second half of the cycle are produced before the reisomerization step, and, by this criterion, they are not O-like but rather N-like states. The assignment of photocycle intermediates enables reevaluation of the photocycle; its specific features are discussed in relation to the general mechanism of proton transport in retinal proteins.
    The Journal of Physical Chemistry B 05/2013; 117(24):7235-7253. DOI:10.1021/jp402430w · 3.38 Impact Factor
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    ABSTRACT: A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum, ESR. The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than two orders of magnitude. In these mutants the rate constant of the M decay linearly decreases with decrease in proton concentration, as expected if reprotonation is limited by uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5±0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with pKa of 8.1±0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ε-amino group of Lys96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.
    Journal of Biological Chemistry 05/2013; 288(29). DOI:10.1074/jbc.M113.465138 · 4.60 Impact Factor
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    ABSTRACT: A gene coding for cold-active lipase from the psychrotrophic Gram-negative bacterium Psychrobacter cryohalolentis K5T isolated from a Siberian cryopeg has been cloned and expressed in Escherichia coli. The recombinant protein Lip1Pc with a 6× histidine tag at its C-terminus was purified by nickel affinity chromatography. With p-nitrophenyl dodecanoate (C12) as a substrate, the purified recombinant protein displayed maximum lipolytic activity at 25°C and pH 8.0. Increasing the temperature above 40°C and addition of various metal ions and organic solvents inhibited the enzymatic activity of Lip1Pc. Most nonionic detergents, such as Triton X-100 and Tween 20, slightly increased the lipase activity, while SDS completely inhibited it. To investigate the functional significance of the Lip1Pc N-terminal domain, we constructed five deletion mutants of this protein. The ND1 and ND2 mutants displayed specific activity reduced by 30–35%, while other truncated proteins were completely inactive. Both mutants demonstrated increased activity towards p-nitrophenyl decanoate (C10) and impaired utilization of C16 substrate. Although optimum reaction temperature of ND2 lowered to 20°C, it displayed enhanced stability by 44% after incubation at 40°C. The results prove that the N-terminal domain of Lip1Pc has a fundamental impact on the activity and stability of the protein.
    Biochemistry (Moscow) 04/2013; 78(4). DOI:10.1134/S000629791304007X · 1.35 Impact Factor
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    ABSTRACT: Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the SDS denatured state was achieved in the nanodiscs containing anionic saturated lipid DMPG and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid POPC. The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (< 20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.
    Biochimica et Biophysica Acta 11/2012; 1828(2). DOI:10.1016/j.bbamem.2012.11.005 · 4.66 Impact Factor
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    ABSTRACT: G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts fromE. colicells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cell-free production of the human β2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin fromExiguobacterium sibiricum(ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein fromB. subtilisallows to increase the CF synthesis of the target GPCRs by 5-38 times, resulting in yields of 0.6-3.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.
    10/2012; 4(4):58-64.
  • Doklady Biological Sciences 07/2012; 445:279-82. DOI:10.1134/S0012496612040035
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    ABSTRACT: One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in Escherichia coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept a proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH-dependent properties of ESR. In the H57M mutant, a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum with an increase in the pH from 5 to 8 indicate deprotonation of the counterion with a pK(a) of 6.3, which is also the pK(a) at which the M intermediate is observed in the photocycle of the protein solubilized in detergent [dodecyl maltoside (DDM)]. This is in contrast with the case for the wild-type protein, for which the same experiments show that the major fraction of Asp85 is deprotonated at pH >3 and that it protonates only at low pH, with a pK(a) of 2.3. The M intermediate in the wild-type photocycle accumulates only at high pH, with an apparent pK(a) of 9, via deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR, the pK(a) values for M formation and spectral shifts are 2-3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild-type protein versus the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pK(a) of Asp85 by stabilizing its deprotonated state.
    Biochemistry 06/2012; 51(29):5748-62. DOI:10.1021/bi300409m · 3.19 Impact Factor
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    ABSTRACT: A psychrotrophic gram-negative bacterium Psychrobacter cryohalolentis K5(T) was previously isolated from a cryopeg within Siberian permafrost and its genome has been completely sequenced. To clone and characterize potential cold-active lipases/esterases produced by P. cryohalolentis K5(T) , we have identified their potential genes by alignment with amino acid sequences of lipases/esterases from related bacteria. One of the targets, EstPc, was cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant protein was produced with a 6x histidine tag at its C-terminus and purified by nickel affinity chromatography. Purified recombinant protein displayed maximum esterolytic activity with p-nitrophenyl butyrate (C4) as a substrate at 35 °C and pH 8.5. Activity assay conducted at different temperatures revealed that EstPc is a cold-adapted esterase which displayed more than 90% of its maximum activity at 0-5 °C. In contrast to many known cold-active enzymes, it possesses relatively high thermostability, preserving more than 60% of activity after incubation for 1 h at 80 °C. It was activated by Ca(2+) , Mn(2+) , and EDTA whereas Zn(+2) , Cu(+2) , Co(+2) , Ni(+2) , and Mg(+2) inhibited it. Various organic solvents (ethanol, methanol and others) inhibited the enzyme. Most non-ionic detergents, such as Triton X-100 and Tween 20 increased the lipase activity while SDS completely inhibited it.
    FEMS Microbiology Ecology 04/2012; 82(2):367-75. DOI:10.1111/j.1574-6941.2012.01385.x · 3.88 Impact Factor
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    ABSTRACT: Tumor necrosis factor (TNF) plays a key role in the pathogenesis of various diseases. To study the possibility of constructing TNF-binding proteins by grafting hypervariable regions of immunoglobulins (CDR), we have replaced amino acid sequences of loops from the tenth type III domain of human fibronectin ((10)Fn3) by amino acid sequences of CDR from the light and heavy chains of the anti-TNF antibody F10. The assessment of TNF-binding properties of the resulting proteins by ELISA has revealed the highest activity of Hd3 containing sequences CDR-H1 and CDR-H2 of the antibody F10 and of Hd2 containing sequences CDR-H1 and CDR-H3. The proteins constructed by us on the fibronectin domain scaffold specifically bound TNF during Western blotting and also weakened its cytotoxic effect on L929 line cells. The highest neutralizing activity was demonstrated by the proteins Hd2 and Hd3, which induced, respectively, 10- and 50-fold increase in the EC(50) of TNF.
    Biochemistry (Moscow) 01/2012; 77(1):62-70. DOI:10.1134/S0006297912010075 · 1.35 Impact Factor
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    ABSTRACT: Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.
    Biochimica et Biophysica Acta 10/2011; 1818(3):349-58. DOI:10.1016/j.bbamem.2011.10.020 · 4.66 Impact Factor
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    ABSTRACT: This review is devoted to the challenging direction of modern molecular biology and bioengineering—the properties of alternative scaffold proteins (ASP) and methods for obtaining ASP binding molecules. ASP binding molecules are a combination of conservative protein cores and hypervariable regions that provide the function of specific binding of the ligand. Structural classification of ASPs includes several types that differ in their molecular targets and potential applications. Construction of artificial binding proteins on the basis of ASPs includes a combinatorial library design with subsequent selection of high-affinity variants by phage display or the more modern cell-free systems. Binding molecules on the basis of ASPs are widely used in various fields of biotechnology and molecular medicine. Keywordsalternative scaffold proteins–combinatorial library selection–affinity–phage display
    Russian Journal of Bioorganic Chemistry 09/2011; 37(5):517-526. DOI:10.1134/S1068162011050141 · 0.62 Impact Factor
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    ABSTRACT: The predicted Exigobacterium sibiricum bacterirhodopsin gene was amplified from an ancient Siberian permafrost sample. The protein bacteriorhodopsin from Exiguobacterium sibiricum (ESR) encoded by this gene was expressed in Escherichia coli membrane. ESR bound all-trans-retinal and displayed an absorbance maximum at 534nm without dark adaptation. The ESR photocycle is characterized by fast formation of an M intermediate and the presence of a significant amount of an O intermediate. Proteoliposomes with ESR incorporated transport protons in an outward direction leading to medium acidification. Proton uptake at the cytoplasmic surface of these organelles precedes proton release and coincides with M decay/O rise of the ESR.
    FEBS letters 10/2010; 584(19):4193-6. DOI:10.1016/j.febslet.2010.09.005 · 3.34 Impact Factor
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    ABSTRACT: To elaborate a high-performance system for expression of genes of G-protein coupled receptors (GPCR), methods of direct and hybrid expression of 17 GPCR genes in Escherichia coli and selection of strains and bacteria cultivation conditions were investigated. It was established that expression of most of the target GPCR fused with the N-terminal fragment of OmpF or Mistic using media for autoinduction provides high output (up to 50 mg/liter).
    Biochemistry (Moscow) 07/2010; 75(7):881-91. DOI:10.1134/S0006297910070102 · 1.35 Impact Factor

Publication Stats

270 Citations
74.70 Total Impact Points

Institutions

  • 2001–2015
    • Russian Academy of Sciences
      • Institute of Physicochemical and Biological Problems of Soil Science
      Moskva, Moscow, Russia
    • University of Reading
      • Department of Animal Science
      Reading, England, United Kingdom
  • 2013
    • Moscow State Forest University
      Mytishi, Moskovskaya, Russia
  • 2005–2013
    • Pacific Institute of Bioorganic Chemistry
      Wladiwostok, Primorskiy, Russia