β-Cyclodextrin-Modified Magnetic Nanoparticles Immobilized on Sepharose Surface Provide an Effective Matrix for Protein Refolding

Abstract

In this article, we propose an impressive and facile strategy to improve protein refolding using solid phase artificial molecular chaperones consisting of the surface-functionalized magnetic nanoparticles. Specifically, monotosyl-β-cyclodextrin connected to the surface of 3-aminopropyltriethoxysilane (APES)-modified magnetic nanoparticles is immobilized on the sepharose surface to promote interaction with exposed hydrophobic surfaces of partially folded (intermediates) and unfolded states of proteins. Their efficiencies were investigated by circular dichroism spectroscopy and photoluminescence spectroscopy of the protein. Although the mechanism of this method is based on principles of hydrophobic chromatography, this system is not only purging the native protein from inactive inclusion bodies but also improving the protein refolding process. We chose β-cyclodextrin (β-CD) considering multiple reports in the literature about its efficiency in protein refolding and its biocompatibility. To increase the surface area/volume ratio of the sepharose surface by nanoparticles, more β-CD molecules are connected to the sepharose surface to make a better interaction with proteins. We suppose that proteins are isolated in the nanospace created by bound cyclodextrins on the resin surface so intermolecular interactions are reduced. The architecture of nanoparticles was characterized by Fourier transform infrared spectra, X-ray diffraction, scanning electron microscopy images, energy dispersive X-ray spectroscopy, nuclear magnetic resonance (1H NMR and 13C NMR), and dynamic light scattering.

Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

“…The native zona matrix porous matrix and its rough surface may be important for physiological responses when sperm bind to its surface 40 . Although individual recombinant ZP-proteins do not form native matrices, conjugated with sepharose beads provide a rough surface 41 with sufficient stiffness that responds to mechanical pressure of sperm-ZP contact. According to current models, the kinetic energy of the sperm is converted into elastic deformation of the zona matrix necessary for successful penetration 42 .…”