Refoiding and Characterization of Recombinant Human GST-PD-1 Fusion Protein Expressed in Escherichia coli

Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-Sx-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was per-formed to obtain bioactive soluble GST-PD-I. Fusion protein of above 95% purity was acquired by a conve-nient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin 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-Ll and PD-l and increase the production of IL-2 and IFN-γ of phytohemagglutinin-activated T cells.