Date of Graduation

5-2026

Document Type

Thesis

Degree Name

Bachelor of Science in Chemistry

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Suresh Thallapuranum

Committee Member

Bin Dong

Second Committee Member

Paul Adams

Third Committee Member

Ashley Purdy

Abstract

Fibroblast growth factors (FGFs) are a family of signaling proteins that regulate a wide range of physiological processes, including metabolism, growth, and tissue homeostasis. Among these, endocrine FGFs play a particularly important role in systemic metabolic regulation and are closely associated with conditions such as obesity and its related comorbidities, including diabetes, liver disease, and cardiovascular dysfunction. Endocrine FGFs signal through fibroblast growth factor receptors (FGFRs) in a Klotho-dependent manner, which determines their tissue specificity. FGF19 and FGF21 primarily act in metabolic tissues such as the liver and adipose tissue through β-Klotho, where they regulate processes including bile acid synthesis, lipid metabolism, and insulin sensitivity. In contrast, FGF23 signals through FGFRs in complex with α-Klotho, primarily in the kidney, where it plays a critical role in maintaining phosphate homeostasis. Despite its biological importance, FGF23 is limited in therapeutic and experimental applications due to its structural instability and poor solubility in vitro.

To address these limitations, an engineered chimeric protein s1F23 C AB was developed, consisting of the full sequence of a hyperstable FGF1 variant (sFGF1) fused to the C-terminal tail of FGF23. The C-terminal region was further modified through targeted substitutions derived from FGF19 and FGF21 to enable both α- and β-Klotho binding. The objective of this study was to improve the structural stability and solubility of FGF23 while preserving and potentially expanding its biological function.

The engineered protein was successfully expressed in E. coli BL21 Star cells and purified using Nickel-Sepharose affinity chromatography. Structural characterization using circular dichroism and intrinsic fluorescence spectroscopy confirmed that the chimera maintained proper secondary and tertiary structure. ANS binding assays indicated reduced hydrophobic surface exposure, suggesting increased structural compactness. Thermal stability, assessed by differential scanning calorimetry, showed a significant increase in melting temperature from ~50°C in native FGF23 to ~68°C in the engineered chimera. Urea denaturation experiments demonstrated a cooperative unfolding transition with a midpoint (Cm) of approximately 3.8 M, indicating enhanced chemical stability. Additionally, trypsin digestion assays revealed increased resistance to proteolytic degradation relative to native FGF23. Functional assays, including cell proliferation and ATP production measurements, demonstrated that the engineered protein retained biological activity comparable to native FGF23.

Together, these results demonstrate that rational protein engineering can significantly enhance the stability and solubility of FGF23 while preserving function. This work establishes a foundation for the development of multifunctional FGF-based therapeutics capable of targeting interconnected metabolic, renal, and cardiovascular disease pathways.

Keywords

Fibroblast Growth Factor; FGF23; FGF1; Endocrine FGFs; Chimeric Protein; Protein Engineering

Additional Files
Abdullah Asif Honors Thesis (Slides).pdf (3230 kB)
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