STRUCTURAL AND BIOLOGICAL CHARACTERIZATION OF A RHIFN ALPHA RECEPTOR

进行研究以表达和纯化胞外结构域， 人IFN-α受体（HuIFN-R）的亚结构域并产生抗体 用于研究信号转导机制。表达及 HuIFN-R的纯化：编码HuIFN-R胞外结构域的基因 HuIFN-R以与谷胱甘肽-S-转移酶（GST）融合的形式表达。 GST-受体是从包涵体中分离的， （可溶形式）和存在（不溶性形式）8 M尿素。 整体 回收率约为1毫克/升细胞培养物。 均不溶 可溶性形式抑制抗病毒和抗增殖 活性为5 - 10 U的IFN α，并显示Kd值约为 10-9 M在受体-配体结合试验中。 芘标记的 用荧光法测定IFN-α B对纯化受体蛋白的作用 极化 结果表明，荧光的饱和增加 各向异性随着受体蛋白浓度的增加而增加。 作为 预期，通过预孵育标记的IFN-γ， aB与10倍过量的未标记IFN。 GST-受体的可溶性形式： 在30 ° C下用0.1 mM IPTG诱导蛋白质表达，连续 在含有DTT，EDTA， PMSF和CHAPS（或Triton）以可溶形式从PMSF释放HuIFN-R。 包涵体 约20%的HuIFNR是在实验室中获得的。 细胞裂解物的上清液。 表达的HUIFNR的80% 仍然保留在包涵体中，并且仅溶解在 8 M尿素。 HuIFN-R亚结构域的表达和分离： 三个亚结构域，命名为M1、M2和M3（残基1-103、93-260和224- 225）。 401)在PRSET载体中表达，并用于鉴定单克隆抗体 与每个片段中发现的特异性表位反应的抗体。 在这三个子域中，只有两个，M2和M3似乎表现出 抗病毒和抗增殖活性。此外，只有M3显示 与芘标记的IFN-aB的可饱和结合，Kd与 测定纯化的受体融合蛋白的Kd值。 抗体产生：产生针对GST受体的多克隆抗体 25 - 30%抑制IFN α与受体的结合和IFN 生物活性 几种单克隆抗体免疫沉淀 天然细胞受体，并特异性地识别细胞 受体蛋白质印迹。 所有项目都已完成。 这些调查的结果将大大丰富我们的 了解干扰素和其他细胞因子 调节免疫反应并控制病毒和肿瘤生长， 他们的受体。

Studies were conducted to express and purify the extracellular domain and subdomains of human IFN-a receptor (HuIFN-R) and to generate antibodies for the investigation of signal transduction mechanisms. Expression and Purification of HuIFN-R: The gene encoding the extracellular domain of HuIFN-R was expressed as fusion with glutathione-S-transferase (GST). The GST-Receptor was isolated from inclusion bodies in the absence (soluble form) and presence (insoluble form) of 8 M urea. Overall recovery was approximately 1 mg/liter of cell culture. Both insoluble and soluble forms inhibited the antiviral and antiproliferative activities of 5 -10 U of IFN alpha and showed a Kd value of approximately 10-9 M in receptor-ligand binding assays. The binding of pyrene-labeled IFN-aB to the purified receptor proteins was measured using fluorescence polarization. Results indicated a saturable increase in the fluorescence anisotropy with increasing concentrations of receptor proteins. As expected, reduced binding was obtained by preincubating the labeled IFN- aB with a 10-fold excess of unlabeled IFN. Soluble Form of GST-Receptor: Induction of protein expression with 0.1 mM IPTG at 30oC, sequential disruption with french press in Tris pH 9.0 buffer containing DTT, EDTA, PMSF and CHAPS (or Triton) released HuIFN-R in soluble form from inclusion bodies. Approximately 20% of HuIFNR was obtained in the supernatant of cell lysate. Eighty per cent of the expressed HUIFNR still remained in the inclusion bodies and were solubilized only in the presence of 8M urea. Expression and Isolation of HuIFN-R Subdomains: Three subdomains, named M1, M2 and M3 (residues 1-103, 93-260 and 224- 401) were expressed in PRSET vector and served to identify monoclonal antibodies which reacted with specific epitopes found in each fragment. Of the three subdomains, only two, M2 and M3 appeared to exhibit antiviral and antiproloferative activities. Further, only M3 exhibited saturable binding to pyrene-labeled IFN-aB with a Kd comparable to the Kd which was determined for the purified receptor fusion-proteins. Antibody Production: Polyclonal antibodies against GST-receptor produced 25 - 30% inhiition of both IFN alpha binding to receptor and IFN biological activity. Several monoclonal antibodies immunoprecipitated the natural cellular receptor and specifically recognized the cellular receptor on western blots. All the projects have been completed. Findings from these investigations will significantly enrich our understanding of the mechanisms by which interferons and other cytokines modulate immune responses and control viral and neoplastic growth through their receptors.