Expression and recovery of biologically active recombinant Apolipoprotein AI(Milano) 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.
Nykiforuk
Cory L
CL
SemBioSys Genetics Inc., Calgary, Alberta, Canada. nykiforukc@sembiosys.com
Shen
Yin
Y
Murray
Elizabeth W
EW
Boothe
Joseph G
JG
Busseuil
David
D
Rhéaume
Eric
E
Tardif
Jean-Claude
JC
Reid
Alexandra
A
Moloney
Maurice M
MM
D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D031187:Carthamus tinctorius / Q000235:genetics* / Q000378:metabolism; D002784:Cholesterol / Q000097:blood; D050356:Lipid Metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010713:Phosphatidylcholines / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D030821:Plants, Genetically Modified / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012639:Seeds / Q000235:genetics* / Q000378:metabolism
D016632:Apolipoprotein A-I; D010713:Phosphatidylcholines; D011993:Recombinant Fusion Proteins; D002784:Cholesterol; C028694:1-palmitoyl-2-oleoylphosphatidylcholine