Synthesis and characterization of β-cyclodextrin-conjugated magnetic nanoparticles and their uses as solid-phase artificial chaperones in refolding of carbonic anhydrase bovine

Surface-functionalized magnetic nanoparticles are widely used in various fields of biotechnology. In this study, beta-cyclodextrin-conjugated magnetic nanoparticles (CD-APES-MNPs) are synthesized and the use of CD-APES-MNPs as a solid-phase artificial chaperone to assist protein refolding in vitro is demonstrated using carbonic anhydrase bovine (CA) as model protein. CD-APES-MNPs are fabricated by grafting monotosyl-beta-cyclodextrin (Ts-beta-CD) onto 3-aminopropyltriethoxysilane (APES)-modified magnetic nanoparticles (APES-MNPs). Results obtained from transmission electron microscopy (TEM) and vibrating sample magnetometery (VSM) show that the synthesized magnetic nanoparticles are superparamagnetic with a mean diameter of 11.5 nm. The beta-CD grafting is confirmed by Fourier transform infrared spectroscopy (FTIR) and elemental analysis. The amount of beta-CD grafted on the APES-MNPs is found to be 0.042 mmol g(-1) from elemental analysis. Our refolding results show that a maximum of 85% CA refolding yield can be achieved using these beta-CD-conjugated magnetic nanoparticles which is at the same level as that using liquid-phase artificial chaperone-assisted refolding. In addition, the secondary and tertiary structures of the refolded CA are the same as those of native protein under optimal conditions. These results indicate that CD-APES-MNPs are suitable and efficient stripping agents for solid-phase artificial chaperone-assisted refolding due to easier and faster separation of these nanoparticles from the refolded samples and also due to recycling of the stripping agents. (C) 2010 Elsevier Inc. All rights reserved.