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Abstract #47720 Published in IGR 13-4
Thermal Stability Assay for Glaucoma-Causing Myocilin Mutants using Maltose Binding Fusion Protein (MBP)
Harris JL; Lieberman R; Burns JN; Orwig SFASEB Journal 2010; 24
Myocilin is a protein closely linked to inherited forms of glaucoma, a neurodegenerative disorder associated with increased intraocular pressure. In myocilin, over 90% of reported glaucoma-causing missense mutations are localized to the C-terminal olfactomedin (OLF) domain. To determine the relative stability of wild-type and mutant OLF, we developed a fluorescence thermal stability assay without removal of MBP. The fluorescence thermal shift assay can accommodate relatively low concentrations of protein (0.25-0.5 mg/ml) and uses a real time PCR (RT-PCR) instrument to conduct a slow melt while measuring fluorescence. The excitation and emission settings of the RT-PCR instrument are compatible with Sypro Orange, a dye known to bind to hydrophobic regions of proteins, which become increasingly exposed as the temperature of the protein-dye solution is raised. Given upper and lower limits of the fluorescence signal, a melting point can be determined as the midpoint of the unfolding transition. We expressed and purified the OLF (amino acids 228-504) domain of myocilin and select glaucoma mutants (D380A, I477N, I477S, K423E) as maltose-binding fusion (MBP) proteins in E. coli. In comparison to wild-type, all four MBP-OLF mutants tested were destabilized but retained a discrete melting transition expected for a folded protein. Our study appears to be a novel application of the fluorescence stability assay to a MBP fusion protein and lays the foundation for the identification of tailored therapeutic molecules to delay the onset of glaucoma.
J.L. Harris. Chemistry, Capital University, Columbus, United States.
Classification:
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
