Title
Biophysical analysis of BMV virions purified using a novel method
Author
Aleksander Strugała, Monika Kręcisz, Jakub Dalibor Rybka, Anna Urbanowicz, Kamil Szpotkowski, Paulina Bierwagen, Marek Figlerowicz, Maciej Kozak, Christoph Böttcher, Michał Giersig
Year
2017
Journal
Journal of Chromatography B
Abstract
Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2 mg of virus from 1 g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Chemical stability, Biochemistry