کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020888 1069215 2011 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression in Escherichia coli and purification of human recombinant connexin-43, a four-pass transmembrane protein
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Expression in Escherichia coli and purification of human recombinant connexin-43, a four-pass transmembrane protein
چکیده انگلیسی

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 78, Issue 2, August 2011, Pages 174–180
نویسندگان
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