Purification of recombinant LDVICp016 and rainbow trout TNFSF members RANKL, TRAIL-like 1 and BALM. A. The LCDV-C ORF16 gene was subcloned into a modified pFastBac plasmid (Invitrogen) and a recombinant baculovirus termed vBacAH52 was generated to express a carboxi-terminally 10xHis-tagged version of the protein using the Bac-to-Bac technology (Invitrogen). Extracellular media from High5 cells grown in serum-free medium infected with vBacAH52 and harvested at 72 hours post-infection were analysed by western blot using an anti His-tag antibody (Sigma). B. The predicted extracellular mature peptide of rainbow trout (Oncorhynchus mykiss) TRAIL-like (TNFSF10; [Genbank: DQ218468]; residues F33-S291), RANKL (TNFSF11; [Genbank: DQ218471]; residues T45-R259) and BALM ([Genbank: DQ218469]; residues D29-N246) were expressed in bacteria using a cold-shock induction system based on the pColdI plasmid (Takara) following the manufacturer’s guidelines. Recombinant His-tagged proteins were purified by affinity chromatography under denaturing conditions. The purified proteins were refolded by dyalisis into an arginine-containing buffer and oligomeric assemblies corresponding most probably to trimers further purified by size exclusion chromatography. The purified proteins were analyzed on 12% SDS-PAGE and stained with Coomassie blue.