Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7614843 | Journal of Chromatography B | 2018 | 25 Pages |
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 Ï6 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 Ï6. In addition, binding of Ï6 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 ~1â¯Ãâ¯1013â¯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.
Keywords
DLSradius of hydrationVLPAF4Quaternary aminePESCIMTCAMWCOBSAbovine serum albumintrichloroacetic acidSDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresisField flow fractionationVirus purificationAsymmetrical flow field-flow fractionationSingle strandedchannel flowDouble strandedConvective interaction mediaRegenerated celluloseRadius of gyrationCross-flowMALSplaque forming unitVirus like particleDynamic Light ScatteringMulti angle light scatteringpfupolyethersulfonepolyethylene glycolPEGMacromolecular complexmolecular weight cut-off
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Mirka Lampi, Hanna M. Oksanen, Florian Meier, Evelin Moldenhauer, Minna M. Poranen, Dennis H. Bamford, Katri Eskelin,