Designing of artificial chaperones based on nanoparticles to assist protein refolding

In this manuscript we propose an impressive and facile strategy to improve protein refolding by using a solid phase artificial molecular chaperon consisting of a surface-functionalized magnetic nanoparticles. Mono-tosyl-β-cyclodextrin (Ts-β-CD) connected to the surface of APTES-Fe3O4 nanoparticles, which particularly immobilized on resin to interact with exposed hydrophobic surfaces of partially folding intermediates and unfolded states of proteins. We chose β-cyclodextrin regarding many reports in literature about its efficiency in the protein refolding, its biocompatibility and also it’s hydrophobically binding to critical site(s) of denatured proteins, thus avoiding protein aggregation and assisting to renaturation of proteins in native forms. The denatured α-amylase was injected directly into a column under denaturing conditions and then buffer was changed to renaturation condition to refold α-amylase. An impressive strategy to suppress α-amylase aggregation is to diminish the possibility of intermolecular interaction and increasing intramolecular interaction by adsorbing the unfolded α-amylase through flexible spacer arms that are hanging on resin surface. We compared this column with another column containing just immobilized β-cyclodextrin which a few β-cyclodextrins located on it and had larger pores. Their efficiencies were investigated by Circular Dichroism (CD) spectroscopy and photoluminescence spectroscopy (PL) of the protein. Although the functional mechanism of this method is based on hydrophobic chromatography, but this system is not only purify the native protein sections from inactive inclusion bodies but also improve the protein refolding process. Our results illustrate a maximum of 90% α-amylase renaturation yield that is more than liquid-phase artificial chaperone-assisted refolding. Moreover, we used nanoparticles to increase surface area/volume ratio in order to add more β-CD on the resin surface to make better interaction with proteins. The architecture has been characterized by Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (FESEM) and energy dispersive X-ray spectroscopy (EDX).