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Journal of Lipid Research, Vol 37, 1519-1528, Copyright © 1996 by Lipid Research, Inc.
KA McGuire, WS Davidson and A Jonas
Human apolipoprotein A-I (apoA-I) is the major protein component of high
density lipoproteins (HDL) where it defines the particle structure and
stability and functions as the main activator of the enzyme
lecithin:cholesterol acyltransferase (LCAT). ApoA-I is expressed in the
liver as a preproprotein that is targeted to the endoplasmic reticulum for
secretion; in plasma, an unknown protease removes the six amino acid long
propeptide. In this study, the cDNA coding the human proapoA- I was cloned
into an Escherichia coli vector; the overexpressed protein was purified to
99% homogeneity and was extensively characterized together with mature
apoA-I purified from plasma. SDS-PAGE, mass spectrometry, and Edman
sequence analysis showed that the initial Met residue needed for
translation in E. coli is posttranslationally removed from the N-terminal
sequence of the proapoA-I. The structural and functional analyses were
carried out on the lipid-free and the lipid-bound proteins. ProapoA-I self
associated, interacted with dimyristoyl phosphatidylcholine vesicles, and
formed secondary structures very similar to the lipid-free apoA-I.
Reconstituted HDL particles made with two initial molar ratios of
palmitoyloleoyl phosphatidylcholine/cholesterol/apolipoprotein/Na-cholate
had identical particle sizes and distributions when apoA-I or proapoA-I
were used. Particles having diameters of 79 A and 98 A, containing two
apoA-I or proapoA-I molecules per particle, were isolated and
characterized. The particles contained the same amounts of alpha-helical
structure, had very similar fluorescence properties, and activated LCAT
equally well. We conclude that proapoA-I expressed and purified from E.
coli is functionally and structurally indistinguishable from mature apoA-I
purified from plasma when analyzed in vitro. Therefore, this recombinant
proapoA-I and mutants derived from it will be important sources of protein
for analyzing apoA-I structure and function, as well as for studies of
proapoA-I processing.
ARTICLES
High yield overexpression and characterization of human recombinant proapolipoprotein A-I
Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.
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