Type of Document Master's Thesis Author Castiblanco, Adriana P Author's Email Address adripaka@yahoo.com URN etd-04212009-135436 Title Expression and Purification of Engineered Calcium Binding Proteins Degree Master of Science Department Chemistry Advisory Committee
Advisor Name Title Jenny J. Yang Committee Chair Gangli Wang Committee Member Giovanni Gadda Committee Member Zhi-Ren Liu Committee Member Keywords
- Ion exchange chromatography
- Hydrophobic interaction chromatography
- Protein refolding
- Calcium sensing receptor
- Affinity chromatography
- Calmodulin
- STIM1
Date of Defense 2009-03-25 Availability unrestricted Abstract Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding.The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.
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