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.
Van Ede
J
J
Laboratorium voor Medische Microbiologie, Groningen, The Netherlands.
Nijmeijer
J R
JR
Welling-Wester
S
S
Orvell
C
C
Welling
G W
GW
D000818:Animals; D000911:Antibodies, Monoclonal; D002642:Chick Embryo; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006388:Hemagglutinins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D009439:Neuraminidase / Q000032:analysis; D010222:Parainfluenza Virus 1, Human / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013501:Surface-Active Agents; D014760:Viral Fusion Proteins / Q000032:analysis; D014764:Viral Proteins / Q000032:analysis*
D000911:Antibodies, Monoclonal; D003902:Detergents; D006388:Hemagglutinins; D008565:Membrane Proteins; D013501:Surface-Active Agents; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D009439:Neuraminidase