2008 Amgen Scholars Program Final Presentation - Rational Design of Phosphorylation Sites into the Erbin-PDZ Domain
P.I.: Tanja Kortemme, Ph.D.
Lead GRA: Colin A. Smith
10. METHODS Acrylamide gel electrophoresis of protein elutions ladder E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 E1 E2 Batch 1 Batch 2 Grew transformed E. Coli cells to an optical density of ~0.5 Induced with IPTG Spun down and lysed cells two hours after induction Purified with the Ni + beads for 6xHis
11. METHODS Acrylamide gel electrophoresis of protein elution Batch 3 Serial dilutions from 1/5000 of sample to 1/32000 of sample Grew transformed E. Coli cells to an optical density of ~0.5 Induced with IPTG Spun down and lysed cells two hours after induction Purified with the Ni + beads for 6xHis
12. METHODS PHASE II Computational redesign of the Erbin-PDZ protein QuickChange to produce mutant sequences Express and purify mutant Erbin-PDZ proteins Check with pkaPS phosphorylation motif predictor Fluorescence anisotropy binding assay Circular dichroism analysis Phosphorylation assay Visual inspection of mutant sequences Mutation Strategy Cloning Structural Analysis