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

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.