Protein expression and purification.

Eukaryotic expression was conducted by transfection of GP5 expression vector into HEK-293T cells for 48 h using Lipofectamine 2000 reagent. The transfected cells were lysed in RIPA lysis buffer supplemented with protease inhibitors and clarified by centrifugation at 12,000 rpm at 4°C for 15 min to collect supernatants. Protein A/G beads were incubated with mouse anti-GP5 antibody and WCLs at 4°C, eluted by 0.05 M glycine-HCl buffer (pH 2.2; 0.2 M glycine, 0.2 M HCl), and neutralized by 1 M Tris buffer (pH 10.4).