Expression and recovery of biologically active recombinant Apolipoprotein AI Milano from transgenic safflower ( Carthamus tinctorius ) seeds

Summary Apolipoprotein AI Milano (ApoAI Milano ) was expressed as a fusion protein in transgenic safflower seeds. High levels of expression corresponding to 7 g of ApoAI Milano per kilogram of seed have been identified in a line selected for commercialization. The ApoAI Milano fusion protein was extracted from seed using an oilbody‐based process and matured in vitro prior to final purification. This yielded a Des‐1,2‐ApoAI Milano product which was confirmed by biochemical characterization including immunoreactivity against ApoAI antibodies, isoelectric point, N‐terminal sequencing and electrospray mass spectrometry. Purified Des‐1,2‐ApoAI Milano readily associated with dimyristoylphosphatidylcholine in clearance assays comparable to Human ApoAI. Its biological activity was assessed by cholesterol efflux assays using Des‐1,2‐ApoAI Milano :1‐palmitoyl‐2‐oleoyl phosphatidylcholine complexes in vitro and in vivo . This study has established that high levels of biologically functional ApoAI Milano can be produced using a plant‐based expression system.