Date of Graduation

8-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Ivey, D. Mack

Committee Member

McNabb, David

Second Committee Member

Sakon, Joshua

Third Committee Member

Kral, Timothy

Keywords

C.difficile; SpoIIE

Abstract

The SpoIIE protein plays a crucial role in the sporulation process of Clostridioides difficile, a pathogen responsible for severe gastrointestinal diseases. Understanding the structural and functional aspects of SpoIIE is pivotal for deciphering its role in sporulation and exploring its potential as a therapeutic target. This study investigates the impact of varying concentrations of L-arabinose on the overexpression of SpoIIE in C. difficile, aiming to optimize protein yield and purity for subsequent crystallization and structural determination via X-ray crystallography. Using a recombinant expression system in Escherichia coli, different concentrations of L-arabinose were employed to induce SpoIIE overexpression. Protein purification was performed using affinity chromatography, followed by an assessment of yield and purity. Our findings demonstrate that varying L-arabinose concentrations significantly influence SpoIIE protein expression levels and purity. Using the data from this research will guide successful crystallization trials that yield crystals suitable for structural analysis, revealing critical insights into SpoIIE’s architecture and potential regulatory mechanisms. This structural data will enhance our understanding of SpoIIE’s role in C. difficile sporulation, laying a foundation for future studies targeting this protein for therapeutic interventions. This research underscores the importance of optimizing inducer concentrations for efficient protein production and structural studies, offering new avenues for advancing our knowledge of C. difficile pathogenesis and treatment strategies.

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