Plasmid construction of FcRnECD for expression in Sf9 insect cells

The parent construct for α-chain/β2m expresses two Open Reading Frames (ORFs) within one baculovirus multiple-target expression plasmid, pBacugs4X-1. Protein targets are based on the following amino acid sequences (both α-chain and β2m each contain their native leaders): α-chain 1–297 based on NCBI reference sequence {"type":"entrez-protein","attrs":{"text":"NP_004098","term_id":"4758346","term_text":"NP_004098"}}NP_004098, β2m 1–119 based on NCBI reference sequence {"type":"entrez-protein","attrs":{"text":"NP_004039","term_id":"4757826","term_text":"NP_004039"}}NP_004039. Codons for both ORFs were engineered by GeneComposer for highly expressed baculovirus genes, such that BamHI, HindIII, BglII, and EcoRI restriction sites were eliminated from the inserts to facilitate cloning [65]. Both genes, including flanking restriction sites, were synthesized at GeneArt. The ORF for α-chain was cloned behind the polyhedrin promoter via unique BamHI and HindIII sites; in this case, the BamHI site preceded the signal sequence while HindIII followed the sequence “TGAT” such that two stops are introduced after the C-terminal residue. The ORF for β2m was cloned behind the p10 promoter via unique BglII and EcoRI sites; the BglII site preceded the signal sequence, while EcoRI followed the sequence “TGATAA” such that two stops are introduced after the C-terminal residue. Using this cloning scheme, polyhedrin and p10 promoters are arranged in divergent/opposing orientations within the baculovirus transfer vector. ORFs were sequence verified prior to the commencement of expression studies.