Synthesis and characterization of beta-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 mono-tosyl-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.
Badruddoza
A Z M
AZ
Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore.
Hidajat
K
K
Uddin
M S
MS
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D002942:Circular Dichroism; D008280:Magnetics; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D047392:beta-Cyclodextrins / Q000138:chemical synthesis / Q000737:chemistry*
D047392:beta-Cyclodextrins; D002256:Carbonic Anhydrases; C031215:betadex