Asymmetric flow field flow fractionation methods for virus purification

Abstract

Detailed biochemical and biophysical characterization of viruses requires viral preparations of high quantity and purity. The optimization of virus production and purification is an essential, but laborious and time-consuming process. Asymmetric flow field flow fractionation (AF4) is an attractive alternative method for virus purification because it is a rapid and gentle separation method that should preserve viral infectivity. Here we optimized the AF4 conditions to be used for purification of a model virus, bacteriophage PRD1, from various types of starting materials. Our results show that AF4 is well suited for PRD1 purification as monitored by virus recovery and specific infectivity. Short analysis time and high sample loads enabled us to use AF4 for preparative scale purification of PRD1. Furthermore, we show that AF4 enables the rapid real-time analysis of progeny virus production in infected cells.

Keywords: Bacteriophage PRD1; Icosahedral virus; Membrane virus; Monolithic chromatography; Real-time analysis of progeny virus production; Ultracentrifugation.