Secretion of biologically active human proapolipoprotein A-I in a baculovirus-insect cell system: protection from degradation by protease inhibitors.
Studies of the structure and function of apolipoprotein A-I (apoA-I) often require its purification by delipidation of high density lipoprotein isolated from large quantities of human plasma and separation of apoA-I from other plasma apolipoproteins. To reduce the need for extensive purification procedures, we have developed an insect cell/baculovirus expression system for the production and secretion of human proapoA-I. The recombinant baculovirus containing full-length human apoA-I cDNA, when introduced into Spodoptera frugiperda, directs the synthesis of preproapoA-I, which is subsequently secreted into the growth medium as proapoA-I, indicating correct processing of the signal peptide during secretion. To prevent the extensive degradation of secreted proapoA-I, leupeptin and pepstatin A were added to the serum free cell culture medium. The protein was simply purified by filtration of the medium, which contained up to 80 mg/l proapoA-I, followed by chromatography on phenyl-sepharose CL-4B. The resultant proapoA-I was found to bind lipid and to activate lecithin:cholesterol acyltransferase as effectively as apoA-I from human plasma. The advantage of this expression system is the ease of purification of intact, biologically active apoA-I in high yield.
Pyle
L E
LE
Baker Medical Research Institute, Lipoprotein and Atherosclerosis Unit, Prahran, Australia.
Barton
P
P
Fujiwara
Y
Y
Mitchell
A
A
Fidge
N
N
D000818:Animals; D001054:Apolipoproteins A / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D003470:Culture Media; D018076:DNA, Complementary / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D018411:Spodoptera
D001054:Apolipoproteins A; D003470:Culture Media; D018076:DNA, Complementary; D011480:Protease Inhibitors; D011498:Protein Precursors; C035250:preproapolipoprotein A-I