Nta agarose column

The cloning, expression and purification of recombinant human Hsp90α have been described previously54 (link). The region encoding full-length Hsp90 was subcloned into pET28a. Protein expression in E. coli cells was induced with 0.5 mM IPTG. Cells were harvested after 20 h of growth at 16 °C and then disrupted by sonication. The soluble lysate was clarified by centrifugation and applied to a Ni²⁺-nitrilo-triacetic acid (NTA) agarose column (QIAGEN) in buffer (50 mM Tris-Cl, 300 mM NaCl, 10 mM imidazole, 10% [v/v] glycerol, 10 mM PMSF, 10 mM DTT). Hsp90 protein was eluted with a linear gradient of 20–1,000 mM imidazole. Hsp90 was identified by SDS-PAGE, and the highly concentrated fraction was dialyzed against buffer (20 mM Tris-Cl, pH 7.5; 6 mM MgCl₂; 20 mM KCl) and then aliquoted, frozen in liquid nitrogen, and stored at −80 °C.

Recombinant PvRAMA protein was expressed as described in a previous study using the wheat-germ cell-free (WGCF) technique [28 (link), 29 (link)]. Briefly, the C-terminal of pvrama was amplified by PCR and cloned to the pEU-His vector, then expressed by the WGCF system. Recombinant PvRAMA protein was purified using a nickel-charged nitrilotriacetic acid (NTA) agarose column (Qiagen, Hilden, Germany). PvRAMA proteins were denatured with reducing sample buffer, separated in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then stained with Coomassie brilliant blue. For immunoblotting, horseradish peroxide (HRP)-conjugated anti-His antibody (ABclonal, Wuhan, China) was used to detect PvRAMA (Additional file 1: Figure S1).

Histidine-tagged MHC class II I-E^K and ICAM-1 were expressed and purified as previously described^{57 (link)}. MHC II with C-terminal hexahistidine tags on both α and β chains were expressed using a baculovirus expression system in S2 cells and purified using a Ni–nitrilotriacetic acid (NTA) agarose column (Qiagen). The histidine-tagged MHC bacmid was a gift of L. Teyton (Scripps Research Institute) and M. Davis (Stanford University). The bacmid for ICAM-1 with a C-terminal decahistidine was synthesized, and it was similarly expressed and purified in High Five cells (Invitrogen).