Da-Wei LI, Jian-Feng YU, Yong-Jing CHEN, Hong-Bing MA, Zheng-Fei WANG, Yi-Bei ZHU, and Xue-Guang ZHANG^*
( Biotechnology Research Institute, Soochow University, Suzhou 215007, China )

Abstract Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed on activated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibits proliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involved in antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain was amplified and cloned into expression plasmid pGEX-5x-3. The fusion protein GST-PD-1 was effectively expressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to obtain bioactive soluble GST-PD-1. Fusion protein of above 95% purity was acquired by a convenient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependent in vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-L1 and PD-1 and increase the production of IL-2 and IFN-γ of phytohemagglutinin-activated T cells.

Key words costimulatory molecule; GST-PD-1; refolding

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Received: September 8, 2003 Accepted: December 4, 2003
This work was supported by a grant from the Major State Basic Research Development Program of China (No. 2001CB51003) and the Key Medical Laboratory of Jiangsu Province
*Corresponding author: Tel, 86-512-65125011; Fax, 86-512-65104908; E-mail, smbxuegz@public1.sz.js.cn