Iron oxide magnetic particles (MPs) affinity fishing is a proven method for target protein separation from crude extract . Given the virtual infinite surface modifications that can be made on magnetic particles surface, we investigated the MPs influence in protein crystal growth as nucleation points. Functionalized and non-functionalized MPs were used as additives in lysozyme and trypsin crystallization in the presence and absence of an external magnetic field. A rational design for MPs functionalization was achieved, having MPs functionalized with chitin for lysozyme crystallization, and MP functionalized with casein for trypsin. The physico-chemical properties of the MPs were studied by Fourier transform infrared spectroscopy, dynamic light scattering, zeta potential and transmission electron microscopy. The assay was developed to overcome some crystallization drawbacks as crystal growth kinetics, yield and morphology. Improvement of some of these factors were observed, notably in the presence of functionalized MP. The presence of functionalized MP led to a faster crystal growth kinetics, still improving crystal yield and morphology without hampering crystal diffraction. The new magnetic crystallization method enables the possibility to overcome some protein crystallization difficulties, but also, due to the MP functionalization system, has the potential to be integrated in protein purification methods involving crystallization/precipitation steps. For this purpose, a high throughput screen in the presence of MP functionalized with an affinity ligand towards antibodies was designed showing protein crystal growth in different crystallization conditions.
Acknowledgments: R. dos Santos acknowledges FCT-MCTES for the research fellowship PD/BD/105753/2014 within the scope of the PhD program Molecular Biosciences PD/00133/2012. This work is supported by UCIBIO, financed by FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01- 0145-FEDER- 007728).