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

Abstract

Summary: Apolipoprotein AI Milano (ApoAIMilano) was expressed as a fusion protein in transgenic safflower seeds. High levels of expression corresponding to 7 g of ApoAIMilano per kilogram of seed have been identified in a line selected for commercialization. The ApoAIMilano 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-ApoAIMilano 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-ApoAIMilano readily associated with dimyristoylphosphatidylcholine in clearance assays comparable to Human ApoAI. Its biological activity was assessed by cholesterol efflux assays using Des-1,2-ApoAIMilano:1-palmitoyl-2-oleoyl phosphatidylcholine complexes in vitro and in vivo. This study has established that high levels of biologically functional ApoAIMilano can be produced using a plant-based expression system. © 2010 SemBioSys Genetics Inc. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.