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

作者：Cory L. Nykiforuk, Yin Shen, Elizabeth W. Murray, J. G. Boothe, David Busseuil, Éric Rhéaume, Jean‐Claude Tardif, Alexandra Reid, Maurice M. Moloney · 发表于：Plant Biotechnology Journal · 年份：2010 · DOI：10.1111/j.1467-7652.2010.00546.x · 被引用次数：59 · 研究领域：Transgenic Plants and Applications、CRISPR and Genetic Engineering、Toxin Mechanisms and Immunotoxins

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.