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

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Abstract


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.

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].

O Nekrasova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: genchem@ibch.ru
ul. MiklukhoMaklaya 16/10, 117997, Moscow, Russia

A Tagway

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

ul. MiklukhoMaklaya 16/10, 117997, Moscow, Russia

A Ignatova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Biological Faculty, Lomonosov Moscow State University

ul. MiklukhoMaklaya 16/10, 117997, Moscow, Russia; Vorobyevi Gori 1, Moscow, 119992, Russia

A Feofanov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Biological Faculty, Lomonosov Moscow State University

ul. MiklukhoMaklaya 16/10, 117997, Moscow, Russia; Vorobyevi Gori 1, Moscow, 119992, Russia

M Kirpichnikov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Biological Faculty, Lomonosov Moscow State University

ul. MiklukhoMaklaya 16/10, 117997, Moscow, Russia; Vorobyevi Gori 1, Moscow, 119992, Russia

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Copyright (c) 2009 Nekrasova O., Tagway A., Ignatova A., Feofanov A., Kirpichnikov M.

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