Recombinant Protein Expression in Drosophila S2 Cells

The gene of AaHig was amplified and inserted into the pMT/BiP/V5-His A vector (Invitrogen), and then the recombinant plasmids were transfected into Drosophila S2 cells in combination with a Hygromycin selection vector pCoHygro for stable cell construction. The primers for PCR and gene cloning are shown in the S1 Table. The stable cell screening and purification were described in our previous study [2 (link),3 (link)]. Briefly, the transfected cells were selected using 300 μg/mL Hygromycin-B (Invitrogen, Cat. No# 10687–010) for 4 weeks. The resistant cells were grown in spinner flasks, switched to Express Five serum-free medium (GIBCO, Invitrogen, Cat. No# 10486025) for 3 days, and induced with copper sulfate at a final concentration of 500 μM for 4 days. The culture medium was collected for protein purification with a metal affinity resin (Clontech, Cat. No# PT1320-1). The protein was eluted with 150 mM imidazole, extensively dialyzed against PBS (pH 7.8), and concentrated via centrifugal filtration through a 5-kDa filter (Millipore Corp., Cat. No# pLCC07610). The protein concentration was measured using a Protein Assay Dye (Bio-Rad, Cat. No#500–0006) and a Nanodrop 2000c spectrophotometer (Thermo Scientific). The protein purity was checked with sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the specificity of purification was confirmed by western blotting.