Purification of viruses and macromolecular assemblies for structural investigations using a novel ion exchange method.
We describe a novel ion exchange chromatographic technique suitable for large-scale preparation of viruses and other biomacromolecular assemblies in highly purified form. The method, which utilizes anion exchange on either of two commercially available cellulose cartridges, is applied to the Escherichia coli bacteriophage PRD1. Viral particles eluted from both QMA and DEAE cartridges retain infectivity and exhibit greater homogeneity of composition, as judged by gel electrophoresis and electron microscopy, than particles purified by rate zonal sucrose gradient centrifugation. The ion exchange protocols are rapid, requiring less than 15 min elution time, and permit retrieval of the purified viral particles at high concentration in aqueous media without centrifugal pelleting. The present method is particularly well suited to the preparation of milligram to decigram quantities of virus, sufficient for many biophysical structural analyses, including investigations by solution spectroscopic and crystal diffraction techniques. The feasibility and advantages of the ion exchange chromatographic procedure are demonstrated by application of laser Raman spectroscopy to ion exchange purified PRD1 virions and subviral assemblies.
Walin
L
L
Department of Genetics, Univesity of Helsinki, Finland.
Tuma
R
R
Thomas
G J
GJ
Bamford
D H
DH
D002501:Centrifugation, Zonal; D002848:Chromatography, DEAE-Cellulose / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D009154:Mutation; D012483:Salmonella Phages / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D013059:Spectrum Analysis, Raman; D014764:Viral Proteins / Q000302:isolation & purification; D014771:Virion / Q000737:chemistry; D014780:Viruses / Q000302:isolation & purification*
D014764:Viral Proteins