Preparation of a Flavonol Specific Glucosyltransferase found in Grapefruit and Site-Directed Mutants for Protein Crystallization

This research was designed to determine the conditions necessary to remove c-myc and 6x-His tags from a flavonol specific glucosyltransferase found in grapefruit (CP3GT) using thrombin in preparation for crystallization. X-ray crystallography of CP3GT crystals may elucidate structural features that account for flavonol specificity in some glucosyltransferase enzymes. A thrombin cleavage site was inserted into WT CP3GT and one mutant. Recombinant CP3GT was expressed in yeast and purified. Optimal conditions for thrombin digestion were explored. Digestion with 100U of thrombin for 2 hours at 4o C was optimal for removing tags from CP3GT. Storage at 4o C for 2 hours resulted in approximately 70% retention of activity. The effect of thrombin treatment on CP3GT activity was tested. Purified CP3GT protein with and without tags was tested for activity with the flavonol quercetin. Data showed no significant difference in overall activity between tagged and native protein