M. Rupar, M. Ravnikar, M. Tušek-Žnidarič, P. Kramberger, L. Glais, I. Gutiérrez-Aguirre
Journal of Chromatography A, 1272 (2013) 33-40(2013) 33-40
Obtaining pure virus suspensions is an essential step in many applications, such as vaccine production, antibody production, sample preparation for procedures requiring enrichment in viruses and other in vitro characterizations. Purification procedures usually consist of complex, long lasting and tedious protocols involving several ultracentrifugation steps. Such complexity is particularly evident in the case of plant viruses, where the virus needs to be isolated from the complex plant tissue matrix. Convective Interaction Media (CIM) monoliths are chromatographic supports that have been successfully utilized for the purification of large bio-molecules such as viruses, virus like particles and plasmids from various matrixes. In this study a CIM monolith based procedure was developed for the fast purification from plant tissue of the filamentous Potato virus Y (PVY) (virion size, 740 nm × 11 nm), which is one of the most important plant viruses causing great economical losses in potato production. Different mobile phases, chemistries and sample preparation strategies were tested. The presence of the virus in the chromatographic fraction was monitored with viral RNA quantification (RT-qPCR), viral protein purity estimation (SDS-PAGE) and viral particle integrity observation (transmission electron microscopy). The optimized procedure involves initial clarification steps, followed by chromatography using CIM quaternary amine (QA) monolithic disk column. In comparison to classical purification procedure involving ultracentrifugation through sucrose and caesium chloride, the developed CIM-QA purification achieved comparable yield, concentration and purity. Plant nucleic acids were successfully removed. Purification showed good reproducibility and moreover it reduced the purification time from four working days required for classic purification to a day and a half. This is the first study where a filamentous virus was purified using CIM monolithic supports. The advantages of this new purification procedure make it an attractive method in serological diagnostic tool production, which requires purified viruses for the immunization step. Moreover, the outcome of this study could serve as starting point for the improvement of the purification methods of other important filamentous viruses.
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