Hifi gene assembly

Spodoptera frugiperda Sf9 cells (Millipore) were maintained in Ex-Cell 420 medium (Sigma), and recombinant baculoviruses were generated with the Bac-to-Bac baculovirus expression system as previously described (Izumiya et al., 2009 (link); Kim et al., 2013 (link)). Transfer plasmid, pFAST-BAC1 vector was modified by inserting a Flag tag at the N-terminus, and CHD4, ORF57, p65, and Luciferase cDNAs were cloned into the CpoI (RsrII) site. The cDNA of ADNP, which also include C-terminal His tag was synthesized and cloned into BamHI and PstI restriction enzyme sites by HiFi Gene assembly (NEB). Recombinant baculovirus bacmid DNA was transfected into Sf9 cells by using polyethylenimine (Sigma), and recombinant viruses were subsequently amplified twice. Expression of recombinant proteins was confirmed by immunoblotting with anti-Flag monoclonal antibody (Sigma) or anti-His (BioRad). Largescale cultures of Sf9 cells (50 mL) were infected with recombinant baculovirus at a multiplicity of infection (MOI) of 0.1–1.0, and cells were harvested 48 hrs after infection. Recombinant proteins were purified as described previously (Izumiya et al., 2005 (link)). The purity and amount of protein were measured by SDS-PAGE and Coomassie blue staining, using bovine serum albumin (BSA) as a standard.