Expression and Characterization of the β 1 Isoform of Glucokinase Regulatory Protein (GKRP) from <i>Homo sapiens</i> β‐Islet Cells

TL;DRAbstract

Glucokinase (GK) is a member of a superfamily of hexokinases that phosphorylates glucose to glucose‐6‐phosphate and is modulated by a regulatory protein (GKRP), with association of the inhibitory complex occurring in the presence of fructose‐6‐phosphate (F6P) and dissociation promoted in the presence of fructose‐1‐phosphate (F1P). Using complementary single‐stranded synthetic oligonucleotides to construct duplex DNA corresponding to the unique 3′‐terminus of β‐islet mRNA splicing variant, the chimeric gene was subcloned and overexpressed in E. coli yielding the putative β 1‐GKRP isoform produced in pancreatic β‐islet cells as described by Alvarez, et al. After purification of β 1‐GKRP by metal chelation affinity chromatography, the variant protein was tested for its ability to bind and inhibit human liver glucokinase. A qualitative differential binding assay was developed using a chitin binding domain/glucokinase (CBD/GK) fusion protein immobilized on a chitin column as bait against β

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