Whitehorn E A, Tate E, Yanofsky S D, Kochersperger L, Davis A, Mortensen R B, Yonkovich S, Bell K, Dower W J, Barrett R W
Affymax Research Institute, Palo Alto, CA 94304, USA.
Biotechnology (N Y). 1995 Nov;13(11):1215-9. doi: 10.1038/nbt1195-1215.
A general method for expression, purification, immobilization, detection and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subunit of interleukin-2 receptor (IL-2R alpha) and E-selectin are used as illustrative examples of cell surface receptors. DNA encoding the ECD of the proteins are fused at their 3' end to a chimeric DNA which serves to generically "tag" the recombinant ECD. The resulting fusion protein contains a substrate sequence for protein kinase-A (PKA) adjacent to the signal sequence from human placental alkaline phosphatase (HPAP), The HPAP signal sequence directs the formation of the phosphatidylinositol-glycan (PI-G) anchorage of the protein at the cell surface. When these chimeric genes are expressed in CHO cells, the ECDs are detected on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase-C (PI-PLC). Based on protein processing known to occur for native HPAP, twenty amino acids from the HPAP signal sequence remain at the C-terminus of the ECD. A high affinity monoclonal antibody was generated against this common epitope. This antibody can be used to detect, purify and immobilize the ECDs. In addition, the ECDs can be radiolabeled with 32P by treatment with PKA and maintain the ability to bind their natural ligands. This "tagging" method has been successfully applied to many other type I proteins which serve as cell surface receptors.
一种表达、纯化、固定、检测和放射性标记I型膜蛋白细胞外结构域（ECD）的通用方法。I型白细胞介素-1受体（IL-1RtI）、白细胞介素-2受体α亚基（IL-2Rα）和E-选择素用作细胞表面受体的示例。编码这些蛋白ECD的DNA在其3'端与一种嵌合DNA融合，该嵌合DNA用于对重组ECD进行通用“标记”。所得融合蛋白在来自人胎盘碱性磷酸酶（HPAP）的信号序列附近含有蛋白激酶A（PKA）的底物序列，HPAP信号序列指导该蛋白在细胞表面形成磷脂酰肌醇聚糖（PI-G）锚定。当这些嵌合基因在CHO细胞中表达时，ECD可在细胞表面检测到，并且可用磷脂酰肌醇特异性磷脂酶C（PI-PLC）处理将其释放。基于已知天然HPAP发生的蛋白质加工过程，HPAP信号序列中的20个氨基酸保留在ECD的C末端。针对该共同表位产生了一种高亲和力单克隆抗体。该抗体可用于检测、纯化和固定ECD。此外，通过PKA处理，ECD可用32P进行放射性标记，并保持结合其天然配体的能力。这种“标记”方法已成功应用于许多其他作为细胞表面受体的I型蛋白。
