Cloning, Expression, Purification and Characterization of Heparin-binding Pocket of Recombinant FGF1

Fibroblast growth factors are polypeptide members of the FGF family, which to date comprises of at least 22 members. They belong to a group of growth factors and are involved in a variety of cellular processes including wound healing, angiogenesis, differentiation and development (organogenesis). Amongst FGF members, human acidic FGF-1 and basic FGF-2 are the most characterized. FGF-1 and FGF-2 are known to share more than 80% sequence similarity and have an identical structural fold. However, their biological roles are quite different. FGFs bind to heparin and heparan sulfate ligands through their heparin-binding pockets. The interactions are primarily electrostatic in nature. The heparin-binding pocket of the protein is significant in the interaction of protein with heparin. Therefore, it is important to characterize the heparin-binding pocket. This research project focuses on the characterization of heparin-binding pocket peptide of FGF1, located at the C-terminal of FGF1 (25 amino acids). To achieve this objective a fusion protein was initially created with the FGF1 C-terminal peptide fused to Rubredoxin (Rub) protein. The fused protein was expressed in BL21 (DE3) cells and purified using affinity chromatography. The FGF1 C-terminal heparin binding peptide (FGF1) was then generated by thrombin cleavage of the fused peptide and characterized by Circular Dichroism (CD), Fluorescence and Mass Spectroscopy. Characterizing the C-terminal heparinbinding region of FGF1 will aid in the understanding of the interactions involved between FGF1 and Fibroblast Growth Factor Receptors and subsequent signal transduction cascades. It will also assist in the development of agonists and antagonists of FGF1 that could potentially be used to regulate various cellular processes, both physiological and pathological. In addition, it could help in understanding the interaction of heparin with other proteins that contain the heparin-binding pocket.