Sisk W P, Bradley J D, Leipold R J, Stoltzfus A M, Ponce de Leon M, Hilf M, Peng C, Cohen G H, Eisenberg R J
DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0400.
J Virol. 1994 Feb;68(2):766-75. doi: 10.1128/JVI.68.2.766-775.1994.
Two forms of herpes simplex virus glycoprotein gD were recombined into Autographa californica nuclear polyhedrosis virus (baculovirus) and expressed in infected Spodoptera frugiperda (Sf9) cells. Each protein was truncated at residue 306 of mature gD. One form, gD-1(306t), contains the coding sequence of Patton strain herpes simplex virus type 1 gD; the other, gD-1(QAAt), contains three mutations which eliminate all signals for addition of N-linked oligosaccharides. Prior to recombination, each gene was cloned into the baculovirus transfer vector pVT-Bac, which permits insertion of the gene minus its natural signal peptide in frame with the signal peptide of honeybee melittin. As in the case with many other baculovirus transfer vectors, pVT-Bac also contains the promoter for the baculovirus polyhedrin gene and flanking sequences to permit recombination into the polyhedrin site of baculovirus. Each gD gene was engineered to contain codons for five additional histidine residues following histidine at residue 306, to facilitate purification of the secreted protein on nickel-containing resins. Both forms of gD-1 were abundantly expressed and secreted from infected Sf9 cells, reaching a maximum at 96 h postinfection for gD-1(306t) and 72 h postinfection for gD-1(QAAt). Secretion of the latter protein was less efficient than gD-1(306t), possibly because of the absence of N-linked oligosaccharides from gD-1(QAAt). Purification of the two proteins by a combination of immunoaffinity chromatography, nickel-agarose chromatography, and gel filtration yielded products that were > 99% pure, with excellent recovery. We are able to obtain 20 mg of purified gD-1(306t) and 1 to 5 mg of purified gD-1(QAAt) per liter of infected insect cells grown in suspension. Both proteins reacted with monoclonal antibodies to discontinuous epitopes, indicating that they retain native structure. Use of this system for gD expression makes crystallization trials feasible.
将两种形式的单纯疱疹病毒糖蛋白gD重组到苜蓿银纹夜蛾核型多角体病毒（杆状病毒）中，并在感染的草地贪夜蛾（Sf9）细胞中表达。每种蛋白质在成熟gD的第306位残基处被截断。一种形式是gD-1(306t)，包含帕顿株单纯疱疹病毒1型gD的编码序列；另一种是gD-1(QAAt)，包含三个突变，消除了所有添加N-连接寡糖的信号。在重组之前，每个基因都被克隆到杆状病毒转移载体pVT-Bac中，该载体允许将基因减去其天然信号肽后与蜜蜂蜂毒肽的信号肽框内插入。与许多其他杆状病毒转移载体一样，pVT-Bac还包含杆状病毒多角体蛋白基因的启动子和侧翼序列，以允许重组到杆状病毒的多角体蛋白位点。每个gD基因都被设计为在第306位残基的组氨酸之后包含另外五个组氨酸残基的密码子，以便于在含镍树脂上纯化分泌的蛋白质。两种形式 的gD-1都在感染的Sf9细胞中大量表达并分泌，gD-1(306t)在感染后96小时达到最大值，gD-1(QAAt)在感染后72小时达到最大值。后一种蛋白质的分泌效率低于gD-1(306t)，可能是因为gD-1(QAAt)缺乏N-连接寡糖。通过免疫亲和色谱、镍琼脂糖色谱和凝胶过滤相结合的方法纯化这两种蛋白质，得到的产物纯度>99%，回收率极佳。每升悬浮培养的感染昆虫细胞，我们能够获得20毫克纯化的gD-1(306t)和1至5毫克纯化的gD-1(QAAt)。两种蛋白质都与针对不连续表位的单克隆抗体发生反应，表明它们保留了天然结构。使用该系统表达gD使结晶试验成为可能。
