Comparison of non-ionic detergents for extraction and ion-exchange high-performance liquid chromatography of Sendai virus integral membrane proteins.

The integral membrane proteins of Sendai virus haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with 2% of a non-ionic detergent, i.e., polyoxyethylene alkyl ethers varying by 8-14 hydrocarbon units in the alkyl chain and by 4-8 ethylene glycol units in the oxyethylene chain. Triton X-100 and octyl glucoside were included as reference detergents. The hydrophile-lipophile balance (HLB) and the critical micelle concentration (CMC) of the detergents were determined. A decrease in length of the oxyethylate by 8-5 ethylene glycol units and an increase in the alkylate by 8-12 hydrocarbon units resulted in higher yields of extracted proteins. The highest yields were obtained for C12E5 with an HLB of 11.7. Yields of extracted protein could be correlated with the HLB values of the polyoxyethylene alkyl ethers. The structural integrity of HN and F was not affected during extraction by either detergent as measured by their reactivity with monoclonal antibodies directed against native HN and F. Extracts were subjected to anion-exchange high-performance liquid chromatography (HPLC) on a Mono Q column in the presence of 0.1% of the detergent used for extraction. Eluate fractions were analysed by sodium dodecyl polyacrylamide gel electrophoresis and recoveries of HN and F protein were determined by size-exclusion HPLC. The immunological activity of HN and F was tested in an enzyme-linked immunosorbent assay. The highest recoveries of HN and F (80%) were obtained with C10E5 in the elution buffer. HN and F were partially purified and the immunological activity was well preserved.

Isolation of detergent-extracted Sendai virus proteins by gel-filtration, ion-exchange and reversed-phase high-performance liquid chromatography and the effect on immunological activity.

Virus envelope proteins were isolated from Triton X-100 extracts of purified Sendai virions by gel-filtration, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC). The fusion protein F, the matrix protein M and the tetrameric and dimeric form of the HN protein were isolated by gel-filtration HPLC with a solvent containing 0.1% sodium dodecyl sulphate. HN and F were also isolated by ion-exchange HPLC with 0.1% Triton X-100 in the eluent. Reversed-phase HPLC was performed on a C1 column with acetonitrile as the organic solvent. Especially the F1 and F2 component of the fusion protein F were obtained in pure form. The immunological activity of the proteins after HPLC was determined with an enzyme-linked immunosorbent assay (ELISA). After gel-filtration and ion-exchange HPLC, proteins still reacted with antiserum to the intact virus while proteins purified by reversed-phase HPLC did not react.