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Molecular Cloning of the Nucleotide Binding Domain of Sulphonylurea Receptor 1, a Component of the ATP-sensitive K-channel

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Author(s): C.N. Fokunang | K.A. Watson | L. Skipper | A. Purvis | C. Browning

Journal: Journal of Medical Sciences
ISSN 1682-4474

Volume: 5;
Issue: 3;
Start page: 141;
Date: 2005;
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Keywords: Nucleotide binding domain | cloning sulphonylurea receptor1 | atp-sensitive k- channel | hamster

ABSTRACT
The aim of this study was to amplify and clone the Nucleotide Binding Domain (NBD1) of Sulphonylurea Receptor 1 (SUR1) from hamster. SURI is a component of the ATP-sensitive K-channel (KATP channels) which play a central role in the control of insulin secretion from the β-pancreatic cells. The KOD HiFi DNA polymerase was used to amplify the gene encoding the NBD1 region of SUR1. This was then digested with restriction enzyme EcoRI and cloned into pETBlue-1 Blunt vector and subsequently into a larger pPIC9 (8023 base pair) vector. The cloned insert was transformed using NovaBlue Competent Cells. The analysis of transformants by double restriction digest using restriction enzymes SnaBI and XbaI confirmed that cloning was successful. The correct orientation of cloned NBD1 inserts were selected for the recombinant protein expression and purification into yeast Pichia pastoris and subsequently for X-ray crystallographic studies.

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