Recombinant proteins were produced and purified using Drosophila Expression System (DES®, Life technologies). Briefly, pMT-BIP-CD83 scFv-V5-His, pMT-BIP-rH9HA-V5-His, or pMT-BIP-rH9HA-CD83 scFv-V5-His plasmids were co-transfected into Drosophila melanogaster S2 cells together with a hygromycin B resistance plasmid (pCoHYGRO, Life technologies). Antibiotic selection was carried out for four weeks using hygromycin B at a concentration of 250 µg/ml and a single cell clone was obtained via limiting dilution43 (link). Recombinant proteins were secreted into culture supernatant after CuSO4 (500 µM) induction and then purified by Profinity™IMAC uncharged column (Bio-Rad). Protein expression was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 10−12% gradient polyacrylamide gels (Bio-Rad) followed by Coomassie staining (Life Technologies). Furthermore, proteins were also transferred onto nitrocellulose membranes (Amersham) for western blotting using anti-H9HA monoclonal antibodies44 (link). The concentration of purified recombinant proteins was determined by Pierce™ BCA Protein Assay Kit (Life Technologies) according to the manufacturer’s protocol.
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