Helfrich W, Van Geel M, The T H, De Leij L
Department of Clinical Immunology, University Hospital Groningen, The Netherlands.
Int J Cancer Suppl. 1994;8:70-5. doi: 10.1002/ijc.2910570715.
The cluster-2 antigen, also called EGP-2, is a 38-kDa transmembrane glycoprotein with a distribution that is largely confined to human epithelial cells and their derived carcinomas. Monoclonal antibodies (MAbs) directed against EGP-2 have been extensively studied as anti-tumor agents, yet the function of the antigen is not known. In the present study we used a biotinylated recombinant soluble derivative of the EGP-2 (sEGPbio) as a probe to detect a possible EGP-2 ligand, using various carcinoma cell lines as a substrate. The recombinant soluble EGP-2 was expressed in the Autographa californica nuclear polyhedrosis virus (baculovirus) expression system. The sEGP-2, to which we engineered a poly-histidine affinity tag, was purified from infected Spodoptera frugiperda insect cells using immobilized metal-ion-affinity chromatography (IMAC). In Western blot analysis the sEGPbio probe bound to a 30-kDa protein band in 2 out of 5 of the assessed carcinoma cell lines, suggesting that this band may be an EGP-2 ligand. Interestingly, binding only occurred when, prior to SDS-PAGE, cell lysates had been subjected to a reducing agent (2-mercapto-ethanol). The physiological significance of this phenomenon and nature of the detected 30-kDa protein band remains to be determined.
集群2抗原，也称为EGP-2，是一种38 kDa的跨膜糖蛋白，其分布主要局限于人类上皮细胞及其衍生的癌组织。针对EGP-2的单克隆抗体（MAb）作为抗肿瘤药物已得到广泛研究，但该抗原的功能尚不清楚。在本研究中，我们使用生物素化的重组可溶性EGP-2衍生物（sEGPbio）作为探针，以各种癌细胞系为底物来检测可能的EGP-2配体。重组可溶性EGP-2在苜蓿银纹夜蛾核多角体病毒（杆状病毒）表达系统中表达。我们在sEGP-2上设计了一个多组氨酸亲和标签，使用固定化金属离子亲和色谱法（IMAC）从感染的草地贪夜蛾昆虫细胞中纯化该蛋白。在蛋白质免疫印迹分析中，sEGPbio探针在5个评估的癌细胞系中的2个中与一条30 kDa的蛋白条带结合，表明这条带可能是EGP-2配体。有趣的是，只有在SDS-PAGE之前，细胞裂解物经过还原剂（2-巯基乙醇）处理后才会发生结合。这一现象的生理意义以及检测到的30 kDa蛋白条带的性质仍有待确定。
