Quick T C, Traish A M, Niles R M
Department of Biochemistry, Boston University School of Medicine, MA 02118.
Receptor. 1994 Summer;4(2):65-80.
Full-length human retinoic acid receptor alpha 1 (hRAR alpha 1) was expressed in Sf9 insect cells using the baculovirus expression vector system (BEVS). Western blot analysis using a specific anti-RAR peptide antiserum detected two major protein bands with apparent mol wts of approximately 54 and approximately 51 kDa in extracts from insect cells infected with recombinant hRAR alpha 1 Autographica californica (AcNPV) baculovirus. Analysis of recombinant extracts from Sf9 cells labeled in vivo with [32P]orthophosphate suggested that the recombinant protein was phosphorylated. A component in the recombinant nuclear extracts specifically bound [3H]all-trans-retinoic acid (RA) and sedimented in sucrose density gradient centrifugation as a single, symmetric peak with a sedimentation coefficient of approximately 3.6S, corresponding to a protein of approx 50 kDa. Scatchard analyses determined that [3H]RA was bound in recombinant extracts by a single class of binding sites with an apparent dissociation constant of approximately 0.3 nM and nuclear and cytoplasmic extracts contained approximately 1200 and approximately 200 pmoles, respectively, of unoccupied receptor per mg protein. In competitive ligand binding assays, relative binding affinities of 9-cis- and 13-cis-RA for hRAR alpha 1 in nuclear extracts were about threefold and sixfold lower than all-trans-RA, whereas all-trans-retinol, -retinaldehyde, and -retinyl acetate demonstrated relatively weak binding. In gel mobility shift assays, the electrophoretic migration of a [32P]-labeled oligonucleotide containing the retinoic acid response element of the RAR beta gene was retarded in the presence of recombinant nuclear and cytoplasmic extracts. The apparent complex formation between recombinant hRAR alpha 1 and beta RARE was greatly enhanced by the addition of nuclear extract from wild-type AcNPV-infected Sf9 cells, possibly because of heterodimer formation between recombinant hRAR alpha 1 and a metazoan RXR homolog. Thus, recombinant hRAR alpha 1 expressed at high levels in Sf9 insect cells exhibited biochemical properties of the native protein, including nuclear translocation, specific high affinity ligand and RARE binding, and possible heterodimer formation.
使用杆状病毒表达载体系统（BEVS）在Sf9昆虫细胞中表达了全长人视黄酸受体α1（hRARα1）。用特异性抗RAR肽抗血清进行的蛋白质印迹分析在感染重组hRARα1苜蓿银纹夜蛾（AcNPV）杆状病毒的昆虫细胞提取物中检测到两条主要蛋白带，其表观分子量分别约为54 kDa和约51 kDa。对用[32P]正磷酸盐在体内标记的Sf9细胞的重组提取物分析表明，重组蛋白被磷酸化。重组核提取物中的一种成分特异性结合[3H]全反式视黄酸（RA），并在蔗糖密度梯度离心中沉降为单一的对称峰，沉降系数约为3.6S，对应于约50 kDa的蛋白质。Scatchard分析确定，[3H]RA在重组提取物中通过一类结合位点结合，表观解离常数约为0.3 nM，核提取物和细胞质提取物每毫克蛋白质分别含有约1200和约200皮摩尔未占据的受体。在竞争性配体结合试验中，核提取物中9-顺式和13-顺式RA对hRARα1的相对结合亲和力分别比全反式RA低约三倍和六倍，而全反式视黄醇、视黄醛和视黄基乙酸酯的结合相对较弱。在凝胶迁移率变动分析中，在重组核提取物和细胞质提取物存在的情况下，含有RARβ基因视黄酸反应元件的[32P]标记寡核苷酸的电泳迁移受到阻滞。通过添加来自野生型AcNPV感染的Sf9细胞的核提取物，重组hRARα1与βRARE之间的表观复合物形成大大增强，这可能是由于重组hRARα1与后生动物RXR同源物之间形成了异二聚体。因此，在Sf9昆虫细胞中高水平表达的重组hRARα1表现出天然蛋白的生化特性，包括核转位、特异性高亲和力配体和RARE结合以及可能的异二聚体形成。
