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Abstract
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.
Highlights
Studies of the structure and function of apolipoprotein A-Ioften 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
ApoE had been expressed in and secreted from insect cells [4], a previous report on the expression of apoA-I in Spodopteru frugiperdu described the production of intracellular apoA-I with a consequent requirement for more extensive purification [5].ApoA-I and proapoA-I possessing a modified amino-terminus have been expressed in Escherichia coli [6,7,8] which has led to complicated strategies for removal of the additional methionine [9]
To determine optimum conditions for harvesting apoA-I secreted from cells, 5 x lo6 infected cells were incubated as monolayers in 25-cm2 flasks in serum free (SF)-medium for up to 7 days
Summary
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. Secretion of biologically active human proapolipoprotein A-I in a baculovirus-insect cell system: protection from degradation by protease inhibitors.J. Lipid. ApoE had been expressed in and secreted from insect cells [4], a previous report on the expression of apoA-I in Spodopteru frugiperdu described the production of intracellular apoA-I (possessing a modified amino-terminus) with a consequent requirement for more extensive purification [5].ApoA-I and proapoA-I possessing a modified amino-terminus have been expressed in Escherichia coli [6,7,8] which has led to complicated strategies for removal of the additional methionine [9].
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