Characterization and purification of bacteriophages using chromatofocusing.
The technique of chromatofocusing was applied to the characterization and purification of three bacteriophages that are routinely used for testing virus filters: phiX174, PR772, and PP7. Chemically well-defined eluent buffers were used, instead of the more commonly used chromatofocusing polyampholyte buffers. Chromatographic column packings were selected to minimize band broadening by confining bacteriophage adsorption solely to the exterior particle surface. Under the conditions used it was determined that bacteriophages could be made to focus into narrow bands in a retained pH gradient with recoveries of live phage that ranged from 15 to nearly 100% as determined by a plaque-forming assay. Retention times and apparent isoelectric point data were obtained for samples consisting either of purified bacteriophage, or samples consisting of crude preparations of bacteriophages containing host cell impurities. Isoelectric point estimates were obtained using modified, previously described models. The results obtained suggest that chromatofocusing is a simple and rapid method for obtaining approximate isoelectric points for bacteriophages and probably other types of viruses. It is also likely a useful method for purifying these materials.
Brorson
Kurt
K
Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA.
Shen
Hong
H
Lute
Scott
S
Pérez
Jessica Soto
JS
Frey
Douglas D
DD
D001435:Bacteriophages / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point