Refolding Circulary Permutated GFP From Inclusion Bodies; Accessing the Chromophore of Green Fluorescent Protein

Abstract

The Green Fluorescent Protein(GFP) which is derived from the jellyfish Aequorea Victoria has become an invaluable tool in molecular and cellular biology research due to its ability to form a chromophore in vitro. Its amino acid components, Ser65, Tyr66, and Gly67 undergo post‐translational, autocatalytic reaction to create a five membered ring chromophore. This mature P‐hydroxybenzylidene imidazolinone chromophore is formed in exposure to molecular oxygen via an intramolecular reaction. Even though several possible mechanisms have been suggested for chromophore biosynthesis, none have been proven definitive. The chemical ligation of unnatural amino acids onto the chromophore residues will halt the chromophore formation without affecting protein refolding process. Because the chromophore is located in the center of the beta‐barrel protein, making any subtle changes to the chromophore is difficult to accomplish. Therefore, a permutated variant of GFP has been constructed where N and C termini of the protein are linked and a new N terminus closer to S65, T66, G67 is created. The truncation of N terminus results in GFP where unnatural amino acids can be ligated in place of the residues that comprise the chromophore. Subsequent refolding of this protein will help eluciadate the mechanisms of chromophore formation. The focus of current studies is to find the optimal renaturing condition for this variant of GFP from the inclusion bodies, before the chromophore has a chance to form.