仙台病毒膜蛋白提取及离子交换高效液相色谱法中去污剂的比较
Comparison of detergents for extraction and ion-exchange high-performance liquid chromatography of Sendai virus membrane proteins.
作者信息
Welling G W, Hiemstra Y, Feijlbrief M, Orvell C, van Ede J, Welling-Wester S
机构信息
Laboratorium voor Medische Microbiologie, Rijksuniversiteit Groningen, Netherlands.
出版信息
J Chromatogr. 1992 May 22;599(1-2):157-62. doi: 10.1016/0021-9673(92)85468-9.
The integral membrane proteins of Sendai virus, haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with a non-ionic and two zwitterionic detergents, i.e., pentaethylene glycol monolauryl ether (C12E5), lauryldimethylamine oxide (LDAO) and dodecyldimethylammoniopropane-1-sulphonate (SB12), respectively. The extracts were subjected to ion-exchange high-performance liquid chromatography (HPIEC) using 0.1% of the detergent in the eluent on four different columns (MA7Q, Zorbax BioSeries SAX, Mono Q and PL-SAX) with a quaternary amine as interacting ligand and with different pore sizes: non-porous and 30, 80 nm and 400 nm, respectively. The relative recoveries of protein were similar for all the columns. The highest recovery of HN and F protein and the best separation were obtained with C12E5. Analysis of HPIEC fractions with monoclonal antibodies directed against conformational epitopes showed that C12E5 had less effect on the conformation than the other two detergents.
用非离子型和两种两性离子去污剂，即五乙二醇单月桂醚（C12E5）、月桂基二甲基氧化胺（LDAO）和十二烷基二甲基氨丙烷-1-磺酸盐（SB12），分别从纯化的仙台病毒颗粒中提取其整合膜蛋白血凝素神经氨酸酶（HN）和融合蛋白（F）。提取物采用离子交换高效液相色谱（HPIEC），在四种不同的柱子（MA7Q、Zorbax BioSeries SAX、Mono Q和PL-SAX）上进行，洗脱液中含有0.1%的去污剂，柱子以季胺作为相互作用配体，孔径不同：分别为无孔、30nm、80nm和400nm。所有柱子上蛋白质的相对回收率相似。使用C12E5时，HN和F蛋白的回收率最高，分离效果最佳。用针对构象表位的单克隆抗体分析HPIEC级分表明，C12E5对构象的影响比其他两种去污剂小。
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