Date Published: April , 2009
Publisher: A.I. Gordeyev
Author(s): O. Nekrasova, A. Tagway, A. Ignatova, A. Feofanov, M. Kirpichnikov.
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 membrane-bound 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. Using the KcsA-Kv1.3 hybrid protein as a model, we developed an approach for the study of the membrane localization, within E.coli cells, of recombinant channels containing functionally active sites composed of eukaryotic, voltage-gated potassium channels. The technical scheme is based on commonly used biochemical techniques, as well as on fluorescent methods of analysis. It includes:
– fractioning of E.coli cells, producing recombinant membrane proteins by means of the differential centrifugation of the cellular lysate;
– analysis of the pellet fractions with the help of extraction using different detergents;
– determination of membrane proteins within the cellular lysate through a “carbonate wash” method;
– determination of KcsA-Kv1.3 tetramer formation by means of SDS-PAGE electrophoresis;
– detection of the insertion and proper folding of a target protein within the cellular membrane by means of fluorescent methods of analysis using whole E.coli cells.