Active respiratory syncytial virus purified by ion-exchange chromatography: characterization of binding and elution requirements.
Two viruses, respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) were used to evaluate viral purification by an affinity resin column (Matrex Cellufine Sulfate (MCS); Amicon Division, WR Grace & Co.). Viable RSV was purified significantly from crude cell lysate by a single pass through a column containing the anionic MCS resin. Most cell protein and albumin eluted from the MCS resin with phosphate buffered saline (PBS) but RSV eluted at high ionic strength, i.e., greater than or equal to 0.6 M NaCl. Further purification was possible by sucrose step gradient centrifugation. The RSV prepared by column purification or by column plus sucrose gradient separation was both intact and infective. RSV and pure samples of VSV were used to optimize ionic strength and salts for elution from the MCS column: 0.8 M NaCl removed most of the viral protein. The capacity of the MCS gel for RSV or VSV was found to be about 0.6-0.8 mg viral protein per ml of hydrated resin. Detergent-solubilized viral membrane proteins bound to the MCS resin in 0.145 M NaCl and eluted with higher salt concentrations. Thus, this resin also may be a useful aid for relatively gentle purification of these proteins.
Downing
L A
LA
Department of Physiology and Biophysics, Wright State University, Dayton, OH.
Bernstein
J M
JM
Walter
A
A
D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D006801:Humans; D008278:Magnesium Sulfate; D009994:Osmolar Concentration; D012136:Respiratory Syncytial Viruses / Q000302:isolation & purification*; D014764:Viral Proteins / Q000302:isolation & purification
D014764:Viral Proteins; D008278:Magnesium Sulfate