Date of Graduation

5-2026

Document Type

Thesis

Degree Name

Bachelor of Science in Chemistry

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Thallapuranam, Suresh

Committee Member

Adams, Paul

Second Committee Member

Ferguson, Alishia

Abstract

Fibroblast Growth Factor 1 (FGF1) plays a crucial role in cell proliferation, angiogenesis and wound healing; however, its therapeutic application is limited by instability and susceptibility to proteolytic degradation at the wound site. Engineering more stable FGF1 variants with enhanced biological activity is therefore essential for improving its clinical potential. Thrombin also plays a very important role in the blood clotting mechanism and consequently in the wound healing process. This study aims to reverse the amino acid sequence of FGF1 so that thrombin will not recognize its cleavage site, thereby achieving maximal thrombin cleavage resistance for effective therapeutic potential. A new variant of FGF1, termed retroFGF1, was designed in which the primary sequence of the FGF1 was reversed, essentially challenging the central dogma of biology. BL21* E. coli host cells were transformed to express retroFGF1, overexpressed to optimize growth conditions, and the retroFGF1 protein was purified for characterization experiments. After completing Circular dichroism (CD) to evaluate secondary structure, retroFGF1 showed an alpha helix structure in contrast to the expected beta-trefoil structure of native FGF1. Fluorescence spectroscopy of retroFGF1 showed a red shift of emission maxima to a longer wavelength than FGF1; this suggests increased solvent exposure of tryptophan residues and subtle conformational rearrangements that may contribute to the enhanced functional properties of the variant. Differential Scanning Calorimetry (DSC) showed a higher Tm for retroFGF1 compared to native FGF1, indicating an increased thermal stability. Trypsin digestion also showed increased resistance for retroFGF1 compared to FGF1. In addition to exhibiting superior enzymatic and thermal stability compared to the FGF1 protein, retroFGF1 also shows surprisingly increased resistance to thrombin-mediated cleavage, which is a trait achieved due to reversal of its sequence. The retro FGF1 protein also showed improved chemical stability to urea in addition to enzymatic and thermal stability; this supports its improved functional robustness compared to native FGF1. The newly engineered FGF1 variant demonstrates enhanced biological activity with comparable cell proliferation rate along with improved stability under physiological conditions showing its potential for mitogenic applications. These combined properties highlight its potential as a promising candidate for FGF-based wound healing.

Keywords

Wound healing; Fibroblast Growth Factor; FGF1; thrombin resistant; mitogenic; metabolic

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