Xu Lihui, Liu Yi, He Xianhui
Institute of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Cell Mol Immunol. 2006 Apr;3(2):139-43.
Programmed death-1 (PD-1), a member of CD28 family, is able to negatively regulate the TCR complex-initiated signaling by interacting with its cognate ligands (PD-L1 and/or PD-L2). PD-1/PD-L1 pathway plays an important role in down-regulating the effective phase of adaptive immune responses and the blockade of this pathway has been proved to enhance antiviral and antitumoral immunity, suggesting that it might be a potential target for the development of therapies to improve T cell responses in patients with virus infections or malignancies. In present study, the extracellular domain of human PD-1 with a carboxyl terminal His-tag (designated as sPD-1) was expressed as inclusion bodies in Escherichia coli. The product was on-column refolded, purified by immobilized metal affinity chromatography, and characterized by Western blotting. Furthermore, the soluble PD-1 with high purity possessed specific binding activity with its cognate ligand PD-L1, and the dissociation constant was 0.43 nmol/L as determined by Scatchard plot analysis. These results suggest that refolded sPD-1 from prokaryotic cells may be of therapeutic interest in enhancing antivirus and antitumoral immune responses.
程序性死亡蛋白 1（PD-1）是 CD28 家族的成员，能够通过与其同源配体（PD-L1 和/或 PD-L2）相互作用来负向调节 TCR 复合物启动的信号传导。PD-1/PD-L1 通路在下调适应性免疫反应的有效阶段中起重要作用，并且已证明阻断该通路可增强抗病毒和抗肿瘤免疫力，这表明它可能是开发改善病毒感染或恶性肿瘤患者 T 细胞反应疗法的潜在靶点。在本研究中，带有羧基末端 His 标签的人 PD-1 细胞外结构域（命名为 sPD-1）在大肠杆菌中以包涵体形式表达。产物经柱上复性，通过固定化金属亲和色谱法纯化，并通过蛋白质印迹法进行表征。此外，高纯度的可溶性 PD-1 与其同源配体 PD-L1 具有特异性结合活性，通过 Scatchard 作图分析确定解离常数为 0.43 nmol/L。这些结果表明，原核细胞中复性的 sPD-1 可能在增强抗病毒和抗肿瘤免疫反应方面具有治疗意义。
