Date of Graduation

5-2026

Document Type

Thesis

Degree Name

Bachelor of Science in Chemistry

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Suresh Kumar Thallapuranam

Committee Member

Andrew Dowdle

Second Committee Member

Nan Zheng

Third Committee Member

Timothy Kral

Abstract

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that is necessary for maintaining optimal phosphate levels and regulating vitamin D metabolism in the body by decreasing the cell surface expression of sodium-phosphate co-transporters and inhibiting the transcription of key enzymes involved in vitamin D biosynthesis. However, despite its importance, not much research has been conducted solely focusing on the C-terminal end of FGF23, mainly due to its extreme instability. Hence, to increase the solubility and stability of the C-terminus of FGF23, a novel rubredoxin (Rd)-based solubility tag was utilized. The objectives of the current project included the overexpression and purification of the RdRdFGF23 C-tail fusion protein, enterokinase cleavage treatment to isolate FGF23 C-tail, and its biophysical characterization. The circular dichroism (CD) spectroscopy revealed that both FGF23 and RdRdFGF23 C-tail contained helical segments in their respective secondary structures. The fluorescence spectroscopy data confirmed that the protein was properly folded in its correct tertiary conformation. Most significantly, the differential scanning calorimetry (DSC) showed that the rubredoxin tag increased the thermal stability of the C-terminus as the melting temperature (Tm) increased from 36°C to 52°C. The guanidine thiocyanate induced denaturation assay revealed that the rubredoxin-tagged C-terminus is extremely stable against chemical denaturation. The limited time-dependent trypsin digestion revealed that the rubredoxin tag protects the C-terminus from enzymatic degradation, making it proteolytically stable. The biological and metabolic assays determined that there is mitogenic activity present, and glycolysis is the preferred mode for ATP production for the C-terminal fragment. As a whole, the rubredoxin tag contributed to the overall increased stability of the C-terminus of FGF23. Through these analyses, a greater understanding of the structural and functional properties of the FGF23 C-terminal region was achieved. Further, the findings of the current study indicate that the Rd-based fusion system may be applicable to express and extract unstable and/or insoluble proteins.

Keywords

Fibroblast growth factors; FGF23; FGF23 C-tail; Biophysical characterization; Rubredoxin; Stability tag

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Available for download on Tuesday, April 27, 2027

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