We report on the surface characterization, functionalization and application of stable water suspensions of novel surface active maghemite nanoparticles (SAMNs), characterized by a diameter of 11 ± 2 nm and possessing peculiar colloidal properties and surface interactions. These features permitted the acquisition of titration curves and aqueous UV-Vis spectrum and suggested a role played by surface under-coordinated iron atoms. This new class of nanoparticles was obtained through an easy, inexpensive, one-step, green procedure and functionalized with ligands of high biotechnological interest, such as biotin and avidin, by simple incubation in aqueous solution. Bound avidin was determined by measuring the disappearance of free avidin absorbance at 280 nm, as a function of nanoparticle increasing concentration, showing the presence of 10 ± 3 avidin molecules per nanoparticle. The biological activity of SAMN@avidin complex was evaluated and the number of available biotin binding sites was determined, using biotinyl-fluorescein as a probe, showing that each bound avidin molecule is able to bind 2.8 ± 0.8 biotin molecules, confirming the maintenance of biological activity and excellent binding capacity of SAMN@avidin complex. Furthermore Langmuir isotherm model was used to describe the biomolecule specific monolayer adsorption onto the particle surface, and, in the case of avidin, the maximum adsorption capacity was 100 ± 27 µg avidin/mg SAMN, while the binding constant is 45.18 µL µg-1. The SAMN@avidin complex was characterized by UV-Vis spectroscopy, quartz crystal microbalance, FTIR spectroscopy and transmission electron microscopy. Finally, SAMN@avidin was applied for the large scale purification of recombinant biotinylated human sarco/endoplasmic reticulum Ca2+-ATPase (hSERCA-2a), expressed by Saccharomyces. cerevisiae. The protein was magnetically purified and about 500 µg of a 70% pure hSERCA-2a were recovered from 4 liters of yeast culture, with a purification yield of 64%.

Avidin functionalized maghemite nanoparticles and their application for recombinant human biotinyl-SERCA purification

MAGRO, MASSIMILIANO;BARATELLA, DAVIDE;SALVIULO, GABRIELLA;VIANELLO, FABIO
2012

Abstract

We report on the surface characterization, functionalization and application of stable water suspensions of novel surface active maghemite nanoparticles (SAMNs), characterized by a diameter of 11 ± 2 nm and possessing peculiar colloidal properties and surface interactions. These features permitted the acquisition of titration curves and aqueous UV-Vis spectrum and suggested a role played by surface under-coordinated iron atoms. This new class of nanoparticles was obtained through an easy, inexpensive, one-step, green procedure and functionalized with ligands of high biotechnological interest, such as biotin and avidin, by simple incubation in aqueous solution. Bound avidin was determined by measuring the disappearance of free avidin absorbance at 280 nm, as a function of nanoparticle increasing concentration, showing the presence of 10 ± 3 avidin molecules per nanoparticle. The biological activity of SAMN@avidin complex was evaluated and the number of available biotin binding sites was determined, using biotinyl-fluorescein as a probe, showing that each bound avidin molecule is able to bind 2.8 ± 0.8 biotin molecules, confirming the maintenance of biological activity and excellent binding capacity of SAMN@avidin complex. Furthermore Langmuir isotherm model was used to describe the biomolecule specific monolayer adsorption onto the particle surface, and, in the case of avidin, the maximum adsorption capacity was 100 ± 27 µg avidin/mg SAMN, while the binding constant is 45.18 µL µg-1. The SAMN@avidin complex was characterized by UV-Vis spectroscopy, quartz crystal microbalance, FTIR spectroscopy and transmission electron microscopy. Finally, SAMN@avidin was applied for the large scale purification of recombinant biotinylated human sarco/endoplasmic reticulum Ca2+-ATPase (hSERCA-2a), expressed by Saccharomyces. cerevisiae. The protein was magnetically purified and about 500 µg of a 70% pure hSERCA-2a were recovered from 4 liters of yeast culture, with a purification yield of 64%.
2012
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2527531
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