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Journal of Lipid Research, Vol 37, 673-683, Copyright © 1996 by Lipid Research, Inc.
MG Sorci-Thomas, JS Parks, MW Kearns, GN Pate, C Zhang and MJ Thomas
To facilitate the investigation of apoA-I structure:function relationships
as they relate to LCAT activation and lipid binding, we have developed an
apoA-I baculoviral expression and purification system that yields milligram
quantities of wild-type or mutant proapoA-I. Baculovirus-infected Sf-9
cells, grown in suspension, were found to secrete high levels of human
wild-type (40-50 mg/l) or mutant apoA-I protein (1-38 mg/l), which was
determined to be > 95% pure following a two-step purification procedure.
In the case of wild-type apoA-I, ELISA showed that approximately 13-18% of
the total protein secreted into the culture medium was apoA-I. To isolate
pure protein from culture medium, 72 h post-infection medium was subjected
to preparative reverse phase high performance liquid chromatography (HPLC),
followed by DEAE ion- exchange chromatography. Purity and molecular size
determination of wild-type proapoA-I protein was verified by SDS
polyacrylamide gel electrophoresis, electrospray mass spectrometry, and
N-terminal sequencing. In addition, recombinant discoidal
apoA-I:phospholipid complexes prepared from wild-type or plasma apoA-I
showed similar particle size and LCAT activation properties. To fully
characterize the utility of this expression system, the expression levels
of various mutant apoA-I proteins were compared to wild-type. Despite a
lower production level seen with selected apoA-I mutants, milligram
quantities of these purified mutant proteins were also obtained. In
summary, we show that baculovirus-derived wild-type proapoA-I shows
properties similar to plasma apoA-I relative to recombinant HDL formation,
LCAT reactivity, and alpha-helical content. In addition, we show that a
variety of mutant forms of human proapoA-I can be expressed and purified in
abundant quantity from baculoviral-infected Sf-9 cells.
ARTICLES
High level secretion of wild-type and mutant forms of human proapoA-I using baculovirus-mediated Sf-9 cell expression
Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157-1040, USA.
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M. G. Sorci-Thomas, M. Thomas, L. Curtiss, and M. Landrum Single Repeat Deletion in ApoA-I Blocks Cholesterol Esterification and Results in Rapid Catabolism of Delta 6 and Wild-type ApoA-I in Transgenic Mice J. Biol. Chem., April 14, 2000; 275(16): 12156 - 12163. [Abstract] [Full Text] [PDF]
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M. G. Sorci-Thomas, L. Curtiss, J. S. Parks, M. J. Thomas, and M. W. Kearns Alteration in Apolipoprotein A-I 22-Mer Repeat Order Results in a Decrease in Lecithin:Cholesterol Acyltransferase Reactivity J. Biol. Chem., March 14, 1997; 272(11): 7278 - 7284. [Abstract] [Full Text] [PDF]
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D. Sviridov, L. E. Pyle, and N. Fidge Efflux of Cellular Cholesterol and Phospholipid to Apolipoprotein A-I Mutants J. Biol. Chem., December 27, 1996; 271(52): 33277 - 33283. [Abstract] [Full Text] [PDF]
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