Molecular-assisted refolding: Study of two different ionic forms of recombinant human fibroblast growth factors

Despite several early reports on the refolding-enhancement effect of a stimuli-responsive polymer, Eudragit, the exact mechanism by which Eudragit interacts with protein molecules to improve refolding remains not well-understood. This study provides clear elucidation of the mode of involvement of Eudragit in facilitating the refolding of two ionic forms of human fibroblast growth factor: (i) human basic fibroblast growth factor (hbFGF) and (ii) human acidic fibroblast growth factor (haFGF). Eudragit enhanced the refolding yield of hbFGF to 74% at 0.5 mg ml−1 refolding protein concentration, but had little effect on its acidic counterpart. Spectroscopic and chromatographic analyses provide clear biomolecular evidence that the oppositely charged polymer and hbFGF protein interact during refolding by forming an ionic complex. This ionic conjugation shields exposed hydrophobic residues of the hbFGF refolding intermediates, thus minimizing hydrophobic-prone aggregation of the molecules. The refolded hbFGF protein can be readily dissociated from the polymer by ion-exchange chromatography. Circular dichroism spectroscopy confirmed that the interaction between Eudragit and hbFGF during refolding did not compromise the final refolded protein structure. This study conclusively shows that Eudragit exhibits charge selectivity in its role as a refolding enhancer, which will have a significant impact on its future use as a refolding additive.