Comparison of ion-exchange high-performance liquid chromatography columns for purification of Sendai virus integral membrane proteins.
The recovery and separation of the integral membrane proteins, the haemagglutinin-neuraminidase (HN) and the fusion protein (F), from a Sendai virus detergent extract were compared on three different ion-exchange high-performance liquid chromatography (IE-HPLC) columns: Mono Q, TSK DEAE-NPR and Zorbax BioSeries SAX. The detergent, either 1-O-n-octyl-beta-glucopyranoside (octylglucoside) or decyl polyethylene glycol-300 (decyl PEG-300), used for extraction of HN and F proteins from the virions, was also present in the elution buffers at a concentration of 0.1%. Recovery of HN and F proteins was primarily dependent on the detergent present in the eluent, resulting in yields of HN varying from 18 to 28 and 56 to 67%, when octylglucoside and decyl PEG-300, respectively, were used. The highest yield for HN protein was obtained by separation on either a Mono Q or a TSK DEAE-NPR column with decyl PEG-300 as the additive. Yields of F protein were lower, and the highest recovery of 46% was found in the presence of decyl PEG-300 by separation on the Mono Q column. Analysis of the fractions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by size-exclusion HPLC indicated that the HN protein eluted in the presence of decyl PEG-300 from the Mono Q and the TSK DEAE-NPR columns was obtained in pure form, while the F protein was slightly contaminated with HN. Analysis of the fractions with monoclonal antibodies directed against conformational epitopes of HN and F proteins indicated that after IE-HPLC the conformation of the proteins is largely retained.
Welling-Wester
S
S
Laboratorium voor Medische Microbiologie, Groningen, The Netherlands.
Haring
R M
RM
Laurens
H
H
Orvell
C
C
Welling
G W
GW
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006388:Hemagglutinins / Q000302:isolation & purification; D008565:Membrane Proteins / Q000302:isolation & purification*; D009439:Neuraminidase / Q000302:isolation & purification; D010222:Parainfluenza Virus 1, Human / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D014760:Viral Fusion Proteins / Q000302:isolation & purification; D014764:Viral Proteins / Q000302:isolation & purification*
D006388:Hemagglutinins; D008565:Membrane Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D009439:Neuraminidase