title: Expression in Escherichia coli and purification of human recombinant connexin-43, a four-pass transmembrane protein.
abstract: We have recently shown, using a well-defined in vitro model, that connexin 43 (Cx43) is directly involved in human cytotrophoblastic cell fusion into a multinucleated syncytiotrophoblast. Cx43 appears to interact with partner proteins within a fusogenic complex, in a multi factorial and dynamic process. This fusogenic complex remains to be characterized and constituent proteins need to be identified. In order to identify proteins interacting with the entire Cx43 molecule (extracellular, transmembrane and intracellular domains), we produced and purified full-length recombinant Cx43 fused to glutathione S-transferase (GST-Cx43) and used it as "bait" in GST pull-down experiments. Cx43 cDNA was first cloned into the pDEST15 vector in order to construct a GST-fusion protein, using the Gateway system. The fusion protein GST-Cx43 was then expressed in Escherichia coli strain BL21-AI™ and purified by glutathione-affinity chromatography. The purified fusion protein exhibited the expected size of 70 kDa on SDS-PAGE, western blot and GST activity. A GST pull-down assay was used to show the capacity of the full-length recombinant protein to interact with known partners. Our results suggest that this method has the capacity to produce sufficient full-length recombinant protein for investigations aimed at identifying Cx43 partner proteins.
authors.affiliation: INSERM, U767, Paris F-75006, France. gcarine@yahoo.com
authors.forename: Sédami
authors.initials: S
authors.lastname: Gnidehou
authors.forename: Pascale
authors.initials: P
authors.lastname: Gerbaud
authors.forename: Guillaume
authors.initials: G
authors.lastname: Ducarme
authors.forename: Fatima
authors.initials: F
authors.lastname: Ferreira
authors.forename: Josette
authors.initials: J
authors.lastname: Badet
authors.forename: André
authors.initials: A
authors.lastname: Malassiné
authors.forename: Danièle
authors.initials: D
authors.lastname: Evain-Brion
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Frendo
mesh_terms: D000704:Analysis of Variance; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018031:Connexin 43 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D018031:Connexin 43; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase