Studying of Membrane Localization of Recombinant Potassium Channels in E.coli

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The effective expression of recombinant membrane proteins in E.coli depends upon the targeting and insertion of proteins into the cellular membrane, as well as on those proteins adopting the correct spatial structure. A significant technological problem involves the design of approaches for detecting the location of target proteins within a host cell. Using a hybrid potassium channel KcsA-Kv1.3 as a model, we developed a technological scheme which is suitable for the study of membrane localization in E.coli cells of recombinant proteins containing voltage-gated eukaryotic potassium channels as the functional active site. The scheme involves both biochemical and fluorescent methods for detecting target proteins in the cytoplasmic membrane of E.coli, as well as the study of the ligand-binding activity of membrane-embedded proteins.

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Studies on trans-membrane proteins and membranebound proteins are one of the current trends in biology. Membrane proteins participate in most cellular processes – signal reception and intercellular communications, molecular and ionic transport- and they play a role in the pathogenesis of many diseases and, as such, are the targets for most pharmaceutical preparations [1]. Because of the low level of biosynthesis of many membrane proteins in biological tissues, the main source of these proteins for structural-functional studies is from recombinant molecules produced in various systems for heterologous expression [2]. Bacterial cells (in particular, Escherichia coli) represent the most widely used, and most productive, system for the biosynthesis of recombinant membrane proteins [3]. At the same time, the heterologous expression in E.coli of membrane proteins is associated with numerous problems involving the general toxicity of these proteins to the host cells. Besides, recombinant proteins are often produced in aggregated form (with inclusion bodies) necessitating careful preparation to refold such proteins. It would seem more practical to work out an approach for the functional expression of membrane proteins in a bacterial membrane [4].
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Список литературы

  1. Byrne V, Iwata S (2002) Membrane protein complexes. Curr Opinion in Struct Biol 12: 239-243.
  2. Grisshammer R, Tate CG (1995) Overexpression of integral membrane proteins for structural studies. Q Rev. Biophis 28: 314-422.
  3. Jana S, Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67:289-298.
  4. Drew D, Froderberg L, Baars L, de Gier JW (2003) Assembly and overexpression of membrane proteins in Escherichia coli. Biochim Biophys Acta 1610: 3-10.
  5. Legros, C, Pollmann, V, Knaus, H-G, Farrell, AM, Darbon, H, Bougis, PE, MartinEauclaire, MF, Pongs, O (2000) Generating a high-affinity scorpion toxin receptor in KcsA-Kv1.3 chimeric potassium channels. J Biol Chem 275: 16918-16924.
  6. Legros C, Schulze C, Garcia ML, Bougis PE, Martin-Eauclaire MF, Pongs O (2002) Engineering-specific pharmacological binding sites for peptidyl inhibitors of potassium channels into KcsA. Biochemistry 41: 15369–15375.
  7. Shieh CC , Coghlan M, Sullivan JP, Gopalakrishnan M (2000) Potassium channels: molecular defects, diseases, and therapeutic opportunities. Pharmacol Rev 52:557–594.
  8. Beeton C, Wulff H, Barbaria J, Clot-Faybesse O, Pennington M, Bernard D, Cahalan MD, Chandy KG, Beraud E (2001) Selective blockade of T lymphocyte K(-) channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc Natl Acad Sci U S A 98:13942-13947.
  9. Kaczorowski GJ, Garcia ML (1999) Pharmacology of voltage-gated and calciumactivated potassium channels. Curr Opin Chem Biol 3: 448–458.
  10. MacKinnon R (2003) Potassium channels. FEBS Lett 555: 62-65.
  11. Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R (1998) The structure of the potassium channel: molecular basis of Kconduction and selectivity. Science 280: 69-77.
  12. MacKinnon R, Cohen SL, Kuo A, Lee A, Chait BT (1998) Structural conservation in prokaryotic and eukaryotic potassium channels. Science 280:106-109.
  13. Sambrook, J, Fritsch, EF and Mniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  14. Laemmli, UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
  15. Irizarry SN, Kutluay E, Drews G, Hart SJ, Heginbotham L (2002) Opening the KcsA K- channel: tryptophan scanning and complementation analysis lead to mutants with altered gating. Biochemistry 41: 13653-13662.
  16. Cortes DM, Perozo E (1997) Structural dynamics of the Streptomyces lividans Kchannel (SKC1): oligomeric stoichiometry and stability. Biochemistry 36: 10343-10352.
  17. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99: 303-310.
  18. van Heerikhuizen H, Kwak E, van Bruggen EF, Witholt B (1975) Characterization of a low density cytoplasmic membrane subfraction isolated from Escherichia coli. Biochim Biophys Acta 413: 177-191.
  19. Schrempf H, Schmidt O, Kummerlen R, Hinnah S, Muller D, Betzler M, Steinkamp T, Wagner R (1995) A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans. EMBO J 14: 5170-5178
  20. Carlone GM, Thomas ML, Rumschlag HS, Sottnek FO (1986) Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 24: 330-332.
  21. Gauthier A, Puente JL, Finlay BB (2003) Secretin of the enteropathogenic Escherichia coli type III secretion system requires components of the type III apparatus for assembly and localization. Infect Immun 71:3310-3319.
  22. Fiermonte G, Walker JE, Palmieri F (1993) Abundant bacterial expression and reconstitution of an intrinsic membrane-transport protein from bovine mitochondria. Biochem J 294: 293-299
  23. Kerman A, Ananthanarayanan VS (2005) Expression and spectroscopic characterization of a large fragment of the mu-opioid receptor. Biochim Biophys Acta 1747:133-40
  24. Shimada Y, Wang ZY, Mochizuki Y, Kobayashi M, Nozawa T (2004) Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell-free synthesis systems. Biosci Biotechnol Biochem 68:19421948.
  25. Van Dalen A, Schrempf H, Killian JA, de Kruijff B (2000) Efficient membrane assembly of the KcsA potassium channel in Escherichia coli requires the protonmotive force. EMBO Rep 1:340-346.
  26. Nekrasova OV, Ignatova AA, Nazarova AI, Feofanov AV, Korolkova YV, Boldyreva EF, Tagvei AI, Grishin EV, Arseniev AS, Kirpichnikov MP (2008) Recombinant Kv Channels at the Membrane of Escherichia coli Bind Specifically Agitoxin2. J Neuroimmune Pharmacol doi: 10.1007/s11481-008-9116-4.
  27. Garcia ML, Garcia-Calvo M, Hidalgo P, Lee A, MacKinnon R (1994) Purification and characterization of three inhibitors of voltage-dependent K- channels from Leiurus quinquestriatus var. hebraeus venom. Biochemistry 33: 6834-6839.
  28. Valiyaveetil FI, MacKinnon R, Muir TW (2002) Semisynthesis and folding of the potassium channel KcsA. J Am Chem Soc 124:9113-9120.

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© Nekrasova O., Tagway A., Ignatova A., Feofanov A., Kirpichnikov M., 2009

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