One-step separation of the components of Semliki Forest virus by cation exchange chromatography.
Although several procedures for isolating viral proteins have been described, the simultaneous separation of all the viral macromolecules in a single step has not yet been reported. We now describe TUA (Triton X-100, urea, acetic acid, pH 4.2)-SP (Sulphopropyl)-Trisacryl cation exchange chromatography, which proved to be ideal for this purpose. Optimal conditions for chromatography were established by screening on TUA-PAGE (polyacrylamide gel electrophoresis) using a horizontal linear 0-8.5 M urea gradient followed by identification of the proteins by SDS-PAGE in the second dimension. Segregation of the constituents of Semliki Forest virus cultivated in two cell lines (chicken embryo fibroblasts and Aedes albopictus cells) was studied, considering that the proteins have identical primary sequences but diverse post-translational modifications, thereby allowing the efficacy of the procedure and its applicability to different viruses to be tested. The results show that the RNA and lipids did not bind to the cation exchange in TUA and were eluted in the flow-through fractions. The proteins were fractionated using 3 linear NaCl gradients in TUA. SDS-PAGE revealed that all the proteins could be purified by this procedure. Furthermore, a direct correlation was obtained between the distance migrated by the proteins in the TUA-PAGE and their order of elution from the TUA-cation exchange column.
D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000302:isolation & purification; D012367:RNA, Viral / Q000302:isolation & purification*; D012672:Semliki forest virus / Q000032:analysis*; D014764:Viral Proteins / Q000302:isolation & purification*
D006023:Glycoproteins; D008565:Membrane Proteins; D012367:RNA, Viral; D014764:Viral Proteins