Date of Award

4-21-2009

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Jenny J. Yang - Chair

Second Advisor

Zhi-Ren Liu

Third Advisor

Gangli Wang

Fourth Advisor

Giovanni Gadda

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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