Asymmetrical flow field-flow fractionation in purification of an enveloped bacteriophage ϕ6

Abstract

Basic and applied virus research requires specimens that are purified to high homogeneity. Thus, there is much interest in the efficient production and purification of viruses and their subassemblies. Advances in the production steps have shifted the bottle neck of the process to the purification. Nonetheless, the development of purification techniques for different viruses is challenging due to the complex biological nature of the infected cell cultures as well as the biophysical and -chemical differences in the virus particles. We used bacteriophage ϕ6 as a model virus in our attempts to provide a new purification method for enveloped viruses. We compared asymmetrical flow field-flow fractionation (AF4)-based virus purification method to the well-established ultracentrifugation-based purification of ϕ6. In addition, binding of ϕ6 virions to monolithic anion exchange columns was tested to evaluate their applicability in concentrating the AF4 purified specimens. Our results show that AF4 enables one-hour purification of infectious enveloped viruses with specific infectivity of ~1 × 10 PFU/mg of protein and ~65-95% yields. Obtained purity was comparable with that obtained using ultracentrifugation, but the yields from AF4 purification were 2-3-fold higher. Importantly, high quality virus preparations could be obtained directly from crude cell lysates. Furthermore, when used in combination with in-line light scattering detectors, AF4 purification could be coupled to simultaneous quality control of obtained virus specimen.

Controlled Disassembly and Purification of Functional Viral Subassemblies Using Asymmetrical Flow Field-Flow Fractionation (AF4)

“…The retention times at the peak maxima varied from ~28 min to 29.5 min between experiments that were performed with different virus batches and on different dates ( Figure S3A, Supplementary Materials ). Our recent AF4-multi-angle light-scattering measurement (MALS) study showed that ultracentrifugation-based purification of φ6 yields virus specimen that has a relatively homogenous size distribution and little aggregates [ 5 ]. Virus-sized particles are generally well retained in the AF4 channel and elute at low cross-flow rates [ 5 , 38 , 39 , 45 ].…”

“…Our recent AF4-multi-angle light-scattering measurement (MALS) study showed that ultracentrifugation-based purification of φ6 yields virus specimen that has a relatively homogenous size distribution and little aggregates [ 5 ]. Virus-sized particles are generally well retained in the AF4 channel and elute at low cross-flow rates [ 5 , 38 , 39 , 45 ]. When compared to our previous study on φ6 that was performed with a thicker 350 μm spacer and resulted in virus elution at the end of the cross-flow gradient [ 5 ], here the thinner spacer promoted virus elution at higher cross-flow rates (peak maxima at a cross-flow rate of ~0.25 mL/min), providing improved resolution between monomeric viruses and putative larger sample components that are present if purification is performed with less purified inputs such as lysates or virus precipitates [ 5 ].…”

“…φ6 was produced as previously described [ 5 , 36 ]. Purified virions were resuspended in a 10 mM potassium phosphate buffer (pH 7.2) supplemented with 1 mM MgCl 2 .…”

“…The AF4 experiments and data collection were performed using an AF2000 MT instrument and Postnova AF2000 software (Postnova Analytics, Landsberg, Germany) as previously described [ 5 , 38 , 39 ], except that a teflon spacer of 250 μm and a regenerated cellulose (RC) membrane with a molecular weight cut-off (MWCO) value of 10 kDa (Postnova) were used (Lots 1551623 and 1653893). Channel flow was monitored in volts (V) at 260 nm (Shimadzu SPD-20A; Shimadzu, Kyoto, Japan).…”

“…φ6 virions are composed of protein (~70% of weight), lipid (~20% of weight), and double-stranded (ds)RNA (~10% of weight) [ 3 , 4 ]. The total mass of virions is ~99 MDa [ 4 ] and the reported diameter is ~70–86 nm, depending on the method used [ 4 , 5 , 6 , 7 ]. The viral genome is segmented into three dsRNAs molecules that are designated as small (S, 2949 bp), medium (M, 4065 bp), and large (L, 6374 bp), based on their different lengths.…”