Damhof R A, Feijlbrief M, Welling-Wester S, Welling G W
Laboratorium voor Medische Microbiologie, Rijksuniversiteit Groningen, Netherlands.
J Chromatogr A. 1994 Jul 29;676(1):43-9. doi: 10.1016/0021-9673(94)80454-0.
Selective elution of Sendai virus integral membrane proteins by ion-exchange high-performance liquid chromatography (HPIEC) using different detergent concentrations was reported before [S. Welling-Wester, M. Freijlbrief, D.G.A.M. Koedijk, M.A. Braaksma, B.R.K. Douma and G.W. Welling, J. Chromatogr., 646 (1993) 37]. In the present study this novel approach was applied to the purification of the integral membrane glycoprotein D of Herpes simplex virus type 1 and 2. The glycoproteins D of types 1 (gD-1) and 2 (gD-2) were cloned into the baculovirus expression system and produced in protein-free cultured insect cells. Detergent extracts of recombinant baculovirus-infected insect cells containing gD-1 or gD-2 were prepared using pentaethyleneglycol monodecyl ether, for extraction (final concentration 2%, w/v). The same detergent was used as additive in the elution buffers for HPIEC on a Mono Q HR 5/5 column. At low (0.005%) detergent concentration, most of the proteins present in the extract including part of gD were eluted with the sodium chloride gradient whereas a subsequent blank run using the same gradient at higher detergent concentration (0.1%) resulted in selective elution of pure gD.
之前已有报道称，使用不同去污剂浓度，通过离子交换高效液相色谱法（HPIEC）对仙台病毒整合膜蛋白进行选择性洗脱[S. Welling-Wester、M. Freijlbrief、D.G.A.M. Koedijk、M.A. Braaksma、B.R.K. Douma和G.W. Welling，《色谱杂志》，646（1993）37]。在本研究中，这种新方法被应用于单纯疱疹病毒1型和2型整合膜糖蛋白D的纯化。将1型（gD-1）和2型（gD-2）的糖蛋白D克隆到杆状病毒表达系统中，并在无蛋白培养的昆虫细胞中产生。使用五乙二醇单癸醚制备含有gD-1或gD-2的重组杆状病毒感染昆虫细胞的去污剂提取物用于提取（终浓度2%，w/v）。在Mono Q HR 5/5柱上进行HPIEC时，相同的去污剂用作洗脱缓冲液中的添加剂。在低去污剂浓度（0.005%）下，提取物中存在的大多数蛋白质（包括部分gD）随氯化钠梯度洗脱，而随后在较高去污剂浓度（0.1%）下使用相同梯度进行的空白运行导致纯gD的选择性洗脱。
