Ross T K, Prahl J M, Herzberg I M, DeLuca H F
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10282-6. doi: 10.1073/pnas.89.21.10282.
The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.
杆状病毒基因表达系统已被用于在草地贪夜蛾昆虫细胞和烟草天蛾昆虫幼虫中生产小鼠视黄酸受体（RAR）γ型。羟基磷灰石结合试验显示，昆虫细胞中每毫克蛋白质的未占据受体产量为300皮摩尔，而感染昆虫幼虫中的水平平均为每毫克蛋白质100皮摩尔RARγ。通过Scatchard方法绘制的视黄酸饱和分析确定，感染昆虫细胞的胞质制剂表现出2.1 nM的平衡解离常数。一种针对RARγ的多克隆抗体将重组受体蛋白识别为一种54000道尔顿的物种。电泳迁移率变动分析表明，来自产生RARγ的昆虫细胞或幼虫的蛋白质提取物能够结合视黄酸反应元件。这与昆虫细胞产生的维生素D受体的特异性DNA结合行为形成对比，后者需要存在哺乳动物来源的核辅助蛋白。RARγ和维生素D受体之间的这种区别表明，这两种受体对其各自DNA反应元件特异性结合的分子要求存在差异。
