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

Area of Science:

• Materials Science
• Nanotechnology
• Biotechnology

Background:

• Stable water suspensions of novel surface active maghemite nanoparticles (SAMNs) with a diameter of 11 ± 2 nm were developed.
• These nanoparticles exhibit unique colloidal properties and surface interactions, influenced by surface under-coordinated iron atoms.
• A facile, inexpensive, one-step, and green synthesis procedure was employed for SAMN production.

Purpose of the Study:

• To characterize and functionalize SAMNs with high-biotechnological interest ligands, specifically biotin and avidin.
• To evaluate the binding capacity and biological activity of the functionalized nanoparticles.
• To apply the functionalized SAMNs for the large-scale purification of a recombinant protein.

Main Methods:

• Surface characterization using titration curves and aqueous UV-vis spectra.
• Functionalization with biotin and avidin via aqueous incubation.
• Quantification of bound avidin and biotin binding sites using spectroscopy and fluorescent probes.
• Adsorption isotherm analysis using the Langmuir model.
• Characterization of the SAMN@avidin complex via UV-vis spectroscopy, quartz crystal microbalance, FTIR, and TEM.
• Application in magnetic purification of recombinant biotinylated human sarco/endoplasmic reticulum Ca(2+)-ATPase (hSERCA-2a).

Main Results:

• SAMNs demonstrated peculiar colloidal properties and surface interactions, with surface under-coordinated iron atoms playing a role.
• Avidin functionalization showed 10 ± 3 avidin molecules per nanoparticle, with each retaining significant biotin binding capacity (2.8 ± 0.8 biotin molecules per avidin).
• The Langmuir isotherm model described monolayer adsorption, with a maximum avidin adsorption capacity of 100 ± 27 μg/mg and a binding constant of 45.18 μL/μg.
• Successful large-scale purification of hSERCA-2a yielded approximately 500 μg of 70% pure protein from 4 L of yeast culture, with a 64% purification yield.

Conclusions:

• Novel surface active maghemite nanoparticles (SAMNs) can be easily synthesized via a green route and effectively functionalized with biomolecules like avidin.
• The SAMN@avidin complex maintains high biological activity and binding capacity, suitable for biomolecule immobilization.
• SAMNs offer a promising platform for efficient, large-scale magnetic purification of recombinant proteins, demonstrating significant potential in biotechnology and bioprocessing.