Group of nanobioengineering

Bioengineering department

Head: Oksana Nekrasova, Ph.D.
okatja@yandex.ru+7(495)3306638

bioengineering; Membrane proteins; Ligands; Ligand-receptor interactions

The group is aimed at the bioengineering of recombinant proteins and polypeptides, developing the procedures for their purification and renaturation, studies on the properties of recombinant molecules using physico-chemical methods, as well as the approaches and methods of nanotechnology.

The group collaborates with the departments of the IBCh RAS (Laboratory of optical microscopy and spectroscopy of biomolecules, the Molecular Instruments for Neurobiology Group and the Laboratory of molecular toxinology) and with Department of Bioengineering of the Biological Faculty of the Lomonosov Moscow State University and the Laboratory of Physicochemical Foundations of Reception of the Emanuel IBHF RAS.

Bioengineering group was formed in 2010 as a part of Bioengineering department of IBCh RAS.

  • Membrane engineering in living cells – bioengineering of recombinant receptor molecules that are targeted to and inserted into bacterial membrane, study of their ligand-binding activity at the surface of the cell membrane using fluorescent methods of detection. 
  • Development of bioengineering approaches to the production of recombinant membrane photosensitive proteins in order to study their structural organization and photochemical properties.
  • Development of bioengineering methods for obtaining soluble protein and peptide ligands in the functionally active form and studying their interaction with receptor proteins.

1.     A bioengineering test system was developed to study interactions of potassium channels with ligands. The test system is based on the use of fluorescently-labeled peptide probe and hybrid potassium channels that are embedded in the membrane of whole bacterial cells. The detection of ligand-receptor interactions is carried out by the method of laser scanning confocal microscopy (LSCM). Using the test system new blockers originating from animal venoms were identified that target Kv1.1 and Kv1.3 – potassium channels of important biomedical significance. With the help of molecular modeling methods, molecular determinants of interaction of peptide toxins with potassium channels were studied, and the mutant forms of peptide toxins were constructed with increased selectivity for the target channel. The principle of engineering of genetically encoded fluorescent ligands of potassium channels was developed with the aim to use them as fluorescent probes in binding studies and also for visualization of potassium channels in cells and tissues. The work is carried out in collaboration with departments of  IBCh - Laboratory of Optical Microscopy and Spectroscopy of Biomolecules, Laboratory of Molecular Instruments for Neurobiology, Laboratory of Molecular Toxinology, as well as Department of Bioengineering of the Biological Faculty of the Lomonosov Moscow State University.

2.     Bioengineering method was worked out for over-production of recombinant bacteriorhodopsin (Halobacterium salinarum) in E.coli expression system. Photochemical properties of recombinant bacteriorhodopsin were found to be similar to those of the monomeric form of the native protein. Recombinant bacteriorhodopsin and its mutants are used to study primary events of photocycle and mechanisms of energy transfer using the method of Femtosecond Transient Absorption Spectroscopy (in collaboration with the Laboratory of Physico-Chemical Basis of Reception, N.M. Emanuel Institute of Biochemical Physics RAS).

3.     New effective bioengineering methods have been developed for producing functionally active recombinant ligands, such as: disulfide-rich peptide toxins from scorpion venoms; efrin A1 - the ligand of  Ephrine receptors. The obtained peptides and proteins are used in various studies on ligand-receptor interactions. 

NamePositionContacts
Oksana Nekrasova, Ph.D.depart. dir.okatja@yandex.ru+7(495)3306638
Kseniya Kudryashova, Ph.D.r. f.rekamoskva@mail.ru
Elena Krjukovar. f.kelen.kryukova@yandex.ru+7(495)330-69-83
Sergej Yakimovres. eng.+7(495)330-72-74
Klara Birikh, Ph.D.sen. eng.+7(495)336-42-00

Former members:

Roman Tikhonov, Ph.D.s. r. f.

Selected publications

  1. Nekrasova O., Yakimov S., Kirpichnikov M., Feofanov A. (2017). Recombinant scorpion toxins: Focus on four-disulfide peptide blockers of Kv1-channels. Bioengineered , 1–5 [+]

    We have recently developed a simple and effective bioengineering approach to large-scale production of alpha-KTx, peptide toxins from scorpion venoms, that block voltage-gated potassium channels with high affinity and specificity. This approach was successfully approved for different peptides containing three disulfide bonds. To extend this method to production of peptide toxins with four disulfide bridges, in particular, maurotoxin and hetlaxin, appropriate conditions of a cleavage reaction with tobacco etch virus (TEV) protease need to be found. For this, the interplay between efficiency of TEV hydrolysis and sensitivity of the target peptides to disulfide reducing agents was studied, and optimized protocols of TEV cleavage reaction were worked out. Maurotoxin and hetlaxin were produced in a folded form avoiding in vitro renaturation step with yields of 14 and 12 mg/liter of culture, respectively.

    ID:1978
  2. Smitienko O.A., Nekrasova O.V., Kudriavtsev A.V., Yakovleva M.A., Shelaev I.V., Gostev F.E., Dolgikh D.A., Kolchugina I.B., Nadtochenko V.A., Kirpichnikov M.P., Feldman T.B., Ostrovsky M.A. (2017). Femtosecond and Picosecond Dynamics of Recombinant Bacteriorhodopsin Primary Reactions Compared to the Native Protein in Trimeric and Monomeric Forms. Biochemistry Mosc. 82 (4), 490–500 [+]

    Photochemical reaction dynamics of the primary events in recombinant bacteriorhodopsin (light-sensitive protein from Halobacterium salinarum) was studied by femtosecond laser absorption spectroscopy. It was shown that photochemical properties of recombinant bacteriorhodopsin are similar to those of the monomeric form of the native protein. Thus, recombinant protein can be used as a convenient model for further studies of photochemical properties of bacteriorhodopsin. 

    ID:1790
  3. Nekrasova O., Kudryashova K., Fradkov A., Yakimov S., Savelieva M., Kirpichnikov M., Feofanov A. (2016). Straightforward approach to produce recombinant scorpion toxins-Pore blockers of potassium channels. J. Biotechnol. 241, 127–135 [+]

    Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEV can be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.

    ID:1690
  4. Feldman T.B., Smitienko O.A., Shelaev I.V., Gostev F.E., Nekrasova O.V., Dolgikh D.A., Nadtochenko V.A., Kirpichnikov M.P., Ostrovsky M.A. (2016). Femtosecond spectroscopic study of photochromic reactions of bacteriorhodopsin and visual rhodopsin. J. Photochem. Photobiol. B, Biol. 164, 296–305 [+]

    Photochromic ultrafast reactions of bacteriorhodopsin (H. salinarum) and bovine rhodopsin were conducted with a femtosecond two-pump probe pulse setup with the time resolution of 20-25fs. The dynamics of the forward and reverse photochemical reactions for both retinal-containing proteins was compared. It is demonstrated that when retinal-containing proteins are excited by femtosecond pulses, dynamics pattern of the vibrational coherent wave packets in the course of the reaction is different for bacteriorhodopsin and visual rhodopsin. As shown in these studies, the low-frequencies that form a wave packets experimentally observed in the dynamics of primary products formation as a result of retinal photoisomerization have different intensities and are clearer for bovine rhodopsin. Photo-reversible reactions for both retinal proteins were performed from the stage of the relatively stable photointermediates that appear within 3-5ps after the light pulse impact. It is demonstrated that the efficiency of the reverse phototransition K-form→bacteriorhodopsin is almost five-fold higher than that of the Batho-intermediate→visual rhodopsin phototransition. The results obtained indicate that in the course of evolution the intramolecular mechanism of the chromophore-protein interaction in visual rhodopsin becomes more perfect and specific. The decrease in the probability of the reverse chromophore photoisomerization (all-trans→11-cis retinal) in primary photo-induced rhodopsin products causes an increase in the efficiency of the photoreception process.

    ID:1612
  5. Nekrasova O.V., Volyntseva A.D., Kudryashova K.S., Novoseletsky V.N., Lyapina E.A., Illarionova A.V., Yakimov S.A., Korolkova Y.V., Shaitan K.V., Kirpichnikov M.P., Feofanov A.V. (2016). Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel. J Neuroimmune Pharmacol , [+]

    Potassium voltage-gated Kv1.6 channel, which is distributed primarily in neurons of central and peripheral nervous systems, is of significant physiological importance. To date, several high-affinity Kv1.6-channel blockers are known, but the lack of selective ones among them hampers the studies of tissue localization and functioning of Kv1.6 channels. Here we present an approach to advanced understanding of interactions of peptide toxin blockers with a Kv1.6 pore. It combines molecular modeling studies and an application of a new bioengineering system based on a KcsA-Kv1.6 hybrid channel for the quantitative fluorescent analysis of blocker-channel interactions. Using this system we demonstrate that peptide toxins agitoxin 2, kaliotoxin1 and OSK1 have similar high affinity to the extracellular vestibule of the K(+)-conducting pore of Kv1.6, hetlaxin is a low-affinity ligand, whereas margatoxin and scyllatoxin do not bind to Kv1.6 pore. Binding of toxins to Kv1.6 pore has considerable inverse dependence on the ionic strength. Model structures of KcsA-Kv1.6 and Kv1.6 complexes with agitoxin 2, kaliotoxin 1 and OSK1 were obtained using homology modeling and molecular dynamics simulation. Interaction interfaces, which are formed by 15-19 toxin residues and 10 channel residues, are described and compared. Specific sites of Kv1.6 pore recognition are identified for targeting of peptide blockers. Analysis of interactions between agitoxin 2 derivatives with point mutations (S7K, S11G, L19S, R31G) and KcsA-Kv1.6 confirms reliability of the calculated complex structure.

    ID:1611
  6. Kuzmenkov A.I., Nekrasova O.V., Kudryashova K.S., Peigneur S., Tytgat J., Stepanov A.V., Kirpichnikov M.P., Grishin E.V., Feofanov A.V., Vassilevski A.A. (2016). Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels. Sci Rep 6, 33314 [+]

    Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

    ID:1561
  7. Dolgikh D.A., Malyshev A.Y., Salozhin S.V., Nekrasova O.V., Petrovskaya L.E., Roshchin M.V., Borodinova A.A., Feldman T.B., Balaban P.M., Kirpichnikov M.P., Ostrovsky M.A. (2015). Anion-selective channelrhodopsin expressed in neuronal cell culture and in vivo in murine brain: Light-induced inhibition of generation of action potentials. Dokl. Biochem. Biophys. 465 (1), 424–7 [+]

    Anionic channelrhodopsin slow ChloC was expressed in the culture of nerve cells and in vivo in mouse brain. We demonstrated ability of slow ChloC to suppress effectively the activity of the neuron in response to the illumination with the visible light. It has been shown for a first time that slow ChloC works equally efficiently in both neuronal culture and in the whole brain being expressed in vivo. Thus, slow ChloC could be considered as an effective optogenetic tool capable in response to light stimulation to inhibit the generation of action potentials in the neuron.

    ID:1478
  8. Kuzmenkov A.I., Vassilevski A.A., Kudryashova K.S., Nekrasova O.V., Peigneur S., Tytgat J., Feofanov A.V., Kirpichnikov M.P., Grishin E.V. (2015). Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1: AN INTEGRATED TRANSCRIPTOMIC AND PROTEOMIC STUDY. J. Biol. Chem. 290 (19), 12195–209 [+]

    The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs.

    ID:1310
  9. Kudryashova K.S., Chertkov O.V., Nikitin D.V., Pestov N.A., Kulaeva O.I., Efremenko A.V., Solonin A.S., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. (2015). Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET. Methods Mol. Biol. 1288, 395–412 [+]

    Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.

    ID:1471
  10. Hoang A.N., Vo H.D., Vo N.P., Kudryashova K.S., Nekrasova O.V., Feofanov A.V., Kirpichnikov M.P., Andreeva T.V., Serebryakova M.V., Tsetlin V.I., Utkin Y.N. (2014). Vietnamese Heterometrus laoticus scorpion venom: evidence for analgesic and anti-inflammatory activity and isolation of new polypeptide toxin acting on Kv1.3 potassium channel. Toxicon 77, 40–8 [+]

    The scorpion Heterometrus laoticus (Scorpionidae) inhabits Indochinese peninsula and is widely distributed in South-West Vietnam. Since no human fatalities caused by H. laoticus stings were reported, no systematic characterization of the venom was earlier done. In this study we report on biological activity of the venom from H. laoticus caught in Vietnamese province An Giang. The venom manifested a very low acute toxicity with LD50 of about 190 mg/kg body weight in mice at subcutaneous (s.c.) injection and 12 mg/kg at intravenous injection. The venom analgesic effects using tail immersion and writhing tests as well as anti-inflammatory effect using carrageenan test were analyzed at doses of 9.5 and 19 mg/kg at s.c. injections. It was found that at two doses tested H. laoticus venom showed both anti-nociceptive and anti-inflammatory activity. The venom was fractionated by means of gel-filtration and reversed-phase HPLC. As a result several polypeptide toxins were isolated and new toxin hetlaxin was identified. Its amino acid sequence was determined and binding to the extracellular vestibule of the K⁺-conducting pore of Kv1.1 and Kv1.3 potassium channels was studied. Hetlaxin belongs to the scorpion alpha-toxin family and is the first toxin isolated from H. laoticus venom which possesses high affinity (K(i) 59 nM) to Kv1.3 potassium channel.

    ID:1082
  11. Shenkarev Z.O., Lyukmanova E.N., Butenko I.O., Petrovskaya L.E., Paramonov A.S., Shulepko M.A., Nekrasova O.V., Kirpichnikov M.P., Arseniev A.S. (2013). Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins. Biochim. Biophys. Acta 1828 (2), 776–84 [+]

    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 sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (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 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (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.

    ID:802
  12. Kudryashova K.S., Nekrasova O.V., Kuzmenkov A.I., Vassilevski A.A., Ignatova A.A., Korolkova Y.V., Grishin E.V., Kirpichnikov M.P., Feofanov A.V. (2013). Fluorescent system based on bacterial expression of hybrid KcsA channels designed for Kv1.3 ligand screening and study. Analytical and bioanalytical chemistry , [+]

    Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.

    ID:782
  13. Nekrasova O.V., Sharonov G.V., Tikhonov R.V., Kolosov P.M., Astapova M.V., Yakimov S.A., Tagvey A.I., Korchagina A.A., Bocharova O.V., Wulfson A.N., Feofanov A.V., Kirpichnikov M.P. (2012). Receptor-binding domain of ephrin-A1: production in bacterial expression system and activity. Biochemistry Mosc. 77 (12), 1387–94 [+]

    Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.

    ID:1479
  14. Nekrasova O.V., Wulfson A.N., Tikhonov R.V., Yakimov S.A., Simonova T.N., Tagvey A.I., Dolgikh D.A., Ostrovsky M.A., Kirpichnikov M.P. (2010). A new hybrid protein for production of recombinant bacteriorhodopsin in Escherichia coli. J. Biotechnol. 147 (3-4), 145–50 [+]
    ID:933
  15. Shenkarev Z.O., Lyukmanova E.N., Solozhenkin O.I., Gagnidze I.E., Nekrasova O.V., Chupin V.V., Tagaev A.A., Yakimenko Z.A., Ovchinnikova T.V., Kirpichnikov M.P., Arseniev A.S. (2009). Lipid-protein nanodiscs: possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides. Biochemistry Mosc. 74 (7), 756–65 [+]

    High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). (15)N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and (31)P-NMR spectroscopy. The 2D 1H-(15)N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased (1)H(N) line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. (15)N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (approximately 40 nsec at 45 degrees C) is indicative of additional peptide motions within the Aam-I/LPN complex.

    ID:353
  16. Nekrasova O.V., Ignatova A.A., Nazarova A.I., Feofanov A.V., Korolkova Y.V., Boldyreva E.F., Tagvei A.I., Grishin E.V., Arseniev A.S., Kirpichnikov M.P. (2009). Recombinant Kv channels at the membrane of Escherichia coli bind specifically agitoxin2. J Neuroimmune Pharmacol 4 (1), 83–91 [+]

    Potassium voltage-gated channels (Kv) are considered as molecular targets in a number of serious neuronal, immune, and cardiac disorders. Search for efficient low-molecular weight modulators of Kv channel function provides a basis for the development of an appropriate therapy for various Kv-mediated diseases. We report here on a new bacterial cell-based system, which is suitable for study of interactions between ligands and ligand-binding sites of eukaryotic Kv1.3 and Kv1.1 channels. To create this system, high-level expression of KcsA-Kv1.3 and KcsA-Kv1.1 hybrid proteins (ligand-binding sites of Kv1.3 or Kv1.1 fused with prokaryotic KcsA potassium channel) was achieved in the plasma membrane of Escherichia coli. An efficient procedure of E. coli conversion to intact spheroplasts was developed. We demonstrate that fluorescently labeled agitoxin 2 binds specifically to high-affinity and lower-affinity sites of KcsA-Kv1.3 and KcsA-Kv1.1, respectively, at the membrane of spheroplasts. Number of binding sites per cell is estimated to be (1.0 +/- 0.6) x 10(5) and (0.3 +/- 0.2) x 10(5) for KcsA-Kv1.3- and KcsA-Kv1.1-presenting cells, respectively, that allows reliable detection of ligand-receptor interactions by confocal laser scanning microscopy. This bacterial cell-based system is intended for screening of ligands to membrane-embedded pharmaceutical targets.

    ID:786
  17. Birikh K.R., Bernad P.L. Jr, Shmanai V.V., Malakhov A.D., Shchepinov M.S., Korshun V.A. (2009). SNP detection using trityl mass tags. Methods Mol. Biol. 578, 345–61 [+]

    A new method suitable for single nucleotide polymorphism (SNP) detection using differential oligonucleotide probe extension has been developed. Sulfur-linked laser-cleavable trityl labels are implemented in this protocol. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption-ionization of the cleavable labels. The usefulness of this method for SNP genotyping is demonstrated.

    ID:758
  18. Birikh K.R., Korshun V.A., Bernad P.L. Jr, Malakhov A.D., Milner N., Khan S., Southern E.M., Shchepinov M.S. (2008). Novel mass tags for single nucleotide polymorphism detection. Anal. Chem. 80 (7), 2342–50 [+]

    A new method suitable for single nucleotide polymorphism detection and other applications based on oligonucleotide probe extension has been developed. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption/ionization of the cleavable labels. A new family of sulfur-linked laser-cleavable trityl labels with vastly improved flying abilities is implemented in this study. Corresponding reagents compatible with automated oligonucleotide synthesis are presented. Utility of this method for SNP genotyping is demonstrated.

    ID:765

Oksana Nekrasova

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 52, office. 264
  • Phone: +7(495)3306638
  • E-mail: okatja@yandex.ru

An efficient method for production of recombinant α-КТх peptides – the blockers of potassium channels (2017-11-28)

A bioengineering method for production of peptide blockers of potassium Kv1 channels has been developed that provides: high yield of the target peptides (12-22 mg/l culture); retaining the native amino acid sequence of α-КТх peptides; high yield of the renatured form of the peptides with correctly formed three and four disulfide bonds; simple and reliable procedure of peptide isolation and purification. The recombinant peptides of the α-KTx family obtained by this method have the activity of the natural blockers. High affinity potassium channel blockers from scorpion venom are widely used to study the structure and function of the channels and have a promising medical value.

Publications

  1. Nekrasova O., Yakimov S., Kirpichnikov M., Feofanov A. (2017). Recombinant scorpion toxins: Focus on four-disulfide peptide blockers of Kv1-channels. Bioengineered , 1–5 [+]

    We have recently developed a simple and effective bioengineering approach to large-scale production of alpha-KTx, peptide toxins from scorpion venoms, that block voltage-gated potassium channels with high affinity and specificity. This approach was successfully approved for different peptides containing three disulfide bonds. To extend this method to production of peptide toxins with four disulfide bridges, in particular, maurotoxin and hetlaxin, appropriate conditions of a cleavage reaction with tobacco etch virus (TEV) protease need to be found. For this, the interplay between efficiency of TEV hydrolysis and sensitivity of the target peptides to disulfide reducing agents was studied, and optimized protocols of TEV cleavage reaction were worked out. Maurotoxin and hetlaxin were produced in a folded form avoiding in vitro renaturation step with yields of 14 and 12 mg/liter of culture, respectively.

    ID:1978
  2. Nekrasova O., Kudryashova K., Fradkov A., Yakimov S., Savelieva M., Kirpichnikov M., Feofanov A. (2016). Straightforward approach to produce recombinant scorpion toxins-Pore blockers of potassium channels. J. Biotechnol. 241, 127–135 [+]

    Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEV can be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.

    ID:1690

Development of integrated transcriptomic and proteomic approach to search for blockers of potassium channels in animal venoms (2016-03-27)

Authors: 

Kuzmenkov A.I. , Vassilevski A.A., Grishin Eu.V.

Department of molecular neurobiology

Kudryashova K.S., Nekrasova O.V., Kirpichnikov M.P. 

Bioengineering Department  

Feofanov A.V. 

Laboratory of optical microscopy and spectroscopy of biomolecules

Annotation: 

An original approach was developed to search for new ligands of potassium channels. It combines the bioengineering cellular test system and transcriptomic and proteomic analysis of animal venoms. Using this approach eight high-affinity peptide blockers of voltage-gated potassium channel Kv1.1 (including five new peptides) were found in the venom of the scorpion Mesobuthus eupeus. The proposed approach is a versatile and effective tool for directed search for  blockers of potassium channels in natural venoms.

Publications

  1. Kuzmenkov A.I., Vassilevski A.A., Kudryashova K.S., Nekrasova O.V., Peigneur S., Tytgat J., Feofanov A.V., Kirpichnikov M.P., Grishin E.V. (2015). Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1: AN INTEGRATED TRANSCRIPTOMIC AND PROTEOMIC STUDY. J. Biol. Chem. 290 (19), 12195–209 [+]

    The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs.

    ID:1310