Detergent extraction of herpes simplex virus type 1 glycoprotein D by zwitterionic and non-ionic detergents and purification by ion-exchange high-performance liquid chromatography.
Detergents (surfactants) are the key reagents in the extraction and purification of integral membrane proteins. Zwitterionic and non-ionic detergents were used for the extraction of recombinant glycoprotein D (gD-1) of herpes simplex virus type 1 (HSV-1) from insect cells infected with recombinant baculovirus. The highest yield was obtained with the two alkyl carboxybetaine detergents (N-dodecyl-N,N-dimethylammonio)undecanoate [DDMAU, critical micelle concentration (CMC) = 0.13 mM] and (N-dodecyl-N,N-dimethylammonio)butyrate (DDMAB, CMC = 4.3 mM). Therefore these zwitterionic detergents were used as additives to the elution buffers in ion-exchange high-performance liquid chromatography (HPIEC) to purify gD-1 of HSV-1 from the extracts. The non-ionic detergent pentaethyleneglycol monodecyl ether (C10E5) that was used in earlier studies [R.A. Damhof, M. Feijlbrief, S. Welling-Wester, G.W. Welling, J. Chromatogr. A, 676 (1994) 43] was used for comparison. Two columns were used, Mono Q and Resource Q, at 1 and 5 ml/min flow-rates, respectively. The results show that the detergents DDMAU and C10E5 are superior to DDMAB, when the detergents were used as additives to the elution buffers at 0.2% (w/v). With 0.2% DDMAB in the eluent, purification of HSV gD-1 was not possible. Detergents with a high CMC may be less suitable as additives in elution buffers. HPIEC at flow-rates of 1 and at 5 ml/min showed satisfactory results. At 5 ml/min HSV gD-1 was mainly concentrated in two eluent fractions. The highest recovery of gD-1 was obtained either by chromatography of a C10E5 extract using a Mono Q column at a flow-rate of 1 ml/min or by chromatography of a DDMAU extract using a Resource Q column at a flow-rate of 5 ml/min.
Welling-Wester
S
S
Laboratorium voor Medische Microbiologie, Rijksuniversiteit Groningen, Netherlands.
Feijlbrief
M
M
Koedijk
D G
DG
Welling
G W
GW
D000818:Animals; D016367:Baculoviridae / Q000737:chemistry; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D007313:Insecta; D008565:Membrane Proteins / Q000302:isolation & purification; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000302:isolation & purification*
D003902:Detergents; D008565:Membrane Proteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C040570:glycoprotein D-herpes simplex virus type 2