Overproduction of rat 1,25-dihydroxyvitamin D3 receptor in insect cells using the baculovirus expression system.

The rat 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor has been expressed at elevated levels in Spodoptera frugiperda cells using the baculovirus expression vector system. The recombinant 1,25-(OH)2D3 receptor is full-length, binds 1,25-(OH)2D3, and is recognized by a monoclonal antibody specific for 1,25-(OH)2D3 receptor. Densitometric scanning of Coomassie brilliant blue-stained SDS/polyacrylamide gels indicated a recombinant receptor protein level comprising 5% of the total soluble protein from the insect cells. The hydroxylapatite binding assay revealed average levels of 2 nmol of unoccupied 1,25-(OH)2D3 receptor per mg of protein in insect cells at 72 hr after infection with recombinant baculovirus. A measure of total 1,25-(OH)2D3 receptor using a ligand-independent, immunoradiometric assay disclosed average levels of 2.3 nmol of receptor per mg of protein produced by these same cells. A monoclonal antibody directed against the 1,25-(OH)2D3 receptor, and reported to cross-react with this receptor derived from several species, recognized the recombinant rat 1,25-(OH)2D3 receptor upon Western analysis. A monoclonal antibody directed specifically against the porcine receptor failed to recognize the recombinant rat 1,25-(OH)2D3 receptor protein. The cytosolic preparation of insect cells infected with recombinant baculovirus exhibited an equilibrium dissociation constant of 1 x 10(-11) M as determined by a 1,25-(OH)2D3 saturation analysis plotted by the method of Scatchard. This expression system provides an adequate source from which abundant quantities of 1,25-(OH)2D3 receptor can be purified for subsequent x-ray crystallographic analyses. IMAGES:

本研究采用杆状病毒表达载体系统（baculovirus expression vector system），在草地贪夜蛾细胞（Spodoptera frugiperda cells）中高水平表达了大鼠1,25-二羟基维生素D3[1,25-(OH)2D3]受体。所得重组1,25-(OH)2D3受体为全长形式，可特异性结合1,25-(OH)2D3，且能被靶向该受体的单克隆抗体识别。对考马斯亮蓝染色的十二烷基硫酸钠-聚丙烯酰胺凝胶（SDS/polyacrylamide gels）进行光密度扫描分析后发现，重组受体蛋白占昆虫细胞总可溶性蛋白的5%。羟基磷灰石结合实验（hydroxylapatite binding assay）结果显示，在重组杆状病毒感染72小时的昆虫细胞中，每毫克蛋白对应的未结合配体的1,25-(OH)2D3受体平均水平为2纳摩尔。采用不依赖配体的免疫放射分析法（immunoradiometric assay）检测总1,25-(OH)2D3受体水平，结果表明上述细胞每毫克蛋白产生的受体平均水平为2.3纳摩尔。一种靶向1,25-(OH)2D3受体的单克隆抗体（据报道可与多种物种来源的该受体发生交叉反应），在蛋白质免疫印迹分析（Western analysis）实验中可识别重组大鼠1,25-(OH)2D3受体；而另一种特异性针对猪源受体的单克隆抗体则无法识别该重组大鼠受体蛋白。通过1,25-(OH)2D3饱和结合实验并采用Scatchard作图法分析，测得重组杆状病毒感染的昆虫细胞胞浆制备物的平衡解离常数为1×10^-11 mol/L。该表达系统可提供充足的原材料，用于纯化获取大量1,25-(OH)2D3受体，以支持后续的X射线晶体学分析（X-ray crystallographic analyses）。图片：