1708Journal of Lipid Research Volume 53, 2012
Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
incidence of cardiovascular disease (CVD) ( 1–3 ). Apolipo-
protein (apo)A-I, the main protein component of HDL,
plays a direct role in HDL’s cardioprotective effects ( 4–6 ).
However, much less is known about apoA-II, the second
most abundant protein in HDL ( ≈ 20% in mass). Synthe-
sized primarily in the liver, apoA-II is a 77 amino acid pro-
tein with a single cysteine at position 6. In healthy humans,
apoA-II exists almost entirely as a disulfi de-linked homodi-
mer (molecular mass, 17.4 kDa), though it can be occa-
sionally found as a heterodimer with other Cys-containing
proteins ( 7 ). It is primarily found in mature, spherical
HDL particles with a small fraction associated with VLDLs
and chylomicrons ( 8 ). ApoA-II has been associated with
numerous aspects of HDL metabolism ( 9–15 ), but, despite
many years of study, its overall physiological function is
still enigmatic ( 16 ).
The impact of apoA-II on cardiovascular disease also re-
mains unclear. In humans, recent case-control studies
have pointed to an inverse correlation between plasma
apoA-II levels and CVD ( 17–19 ). However, the only known
case of human apoA-II defi ciency has shown no evidence
of CVD ( 20, 21 ). In mice, overexpression of murine
apoA-II consistently increased aortic fatty streak lesions
( 22–24 ). In this animal model, HDL particles with high
apoA-II content have reduced binding and selective lipid
uptake by the class B scavenger receptors ( 15, 25 ) and are
poor activators of hepatic lipase ( 11, 14, 26 ). Furthermore,
they are proinfl ammatory and ineffi cient antioxidants rel-
ative to those containing primarily apoA-I ( 14, 23 ). The
combination of these properties may partly explain the
proatherogenic phenotype produced by apoA-II overexpres-
sion. This effect may be mediated directly or, as suggested by
recent studies by Julve et al. (27), apoA-II may alter the
proteome of discrete HDL particle subfractions to indi-
rectly modulate factors such as lipoprotein lipase.
Abstract Recombinant expression systems have become
powerful tools for understanding the structure and func-
tion of proteins, including the apolipoproteins that com-
prise human HDL. However, human apolipoprotein (apo)
A-II has proven diffi cult to produce by recombinant tech-
niques, likely contributing to our lack of knowledge about
its structure, specifi c biological function, and role in cardio-
vascular disease. Here we present a novel Escherichia coli -
based recombinant expression system that produces highly
pure mature human apoA-II at substantial yields. A Mxe
GyrA intein containing a chitin binding domain was fused at
the C terminus of apoA-II. A 6× histidine-tag was also added
at the fusion protein’s C terminus. After rapid purifi cation
on a chitin column, intein auto-cleavage was induced under
reducing conditions, releasing a peptide with only one extra
N-terminal Met compared with the sequence of human ma-
ture apoA-II. A pass through a nickel chelating column re-
moved any histidine-tagged residual fusion protein, leaving
highly pure apoA-II. A variety of electrophoretic, mass spec-
trometric, and spectrophotometric analyses demonstrated
that the recombinant form is comparable in structure to hu-
man plasma apoA-II. Similarly, recombinant apoA-II is com-
parable to the plasma form in its ability to bind and
reorganize lipid and promote cholesterol effl ux from mac-
rophages via the ATP binding cassette transporter A1.
This system is ideal for producing large quantities of recom-
binant wild-type or mutant apoA-II for structural or func-
tional studies. —Smith, L. E., J. Yang, L. Goodman, X.
Huang, R. Huang, J. Dressman, J. Morris, R. A. G. D. Silva,
W. S. Davidson, and G. Cavigiolio. High yield expression
and purifi cation of recombinant human apolipoprotein A-II
in Escherichia coli. J. Lipid Res . 2012. 53: 1708–1715.
Supplementary key words intein • fusion protein • affi nity tag • bacte-
ria • inexpensive • low temperature cleavage • high density lipoprotein.
Numerous human and animal studies have established
an inverse correlation between HDL plasma levels and
This work was supported by New Investigator Award no. 8KT-0021 from the
Tobacco-Related Disease Research Program of California and grant HL113059
from the National Heart Lung and Blood Institute (NHLBI) (G.C.); by grants
HL62542, HL67093, and HL082734 from NHLBI (W.S.D.); and by K99/
R00 grant HL087561 from NHLBI (R.A.G.D.S.).
Manuscript received XXX and in revised form 24 May 2012.
Published, JLR Papers in Press, May 25, 2012
High yield expression and purifi cation of recombinant
human apolipoprotein A-II in Escherichia coli
Loren E. Smith , 1, * Jun Yang , 1, * Leah Goodman , † Xinqi Huang , † Rong Huang , * James Dressman , *
Jamie Morris , * R. A. Gangani D. Silva , * W. Sean Davidson , 2, * and Giorgio Cavigiolio 2,†
Department of Pathology and Laboratory Medicine,* University of Cincinnati , Cincinnati, OH 45273 and
the Children’s Hospital Oakland Research Institute , † Oakland, CA 94609
Abbreviations: CBD, chitin binding domain; CD, circular dichro-
ism; CVD, cardiovascular disease; DMPC, dimyristoyl phospatidylcho-
line; His-tag, histidine-tag; IPTG, isopropyl ? -D-1-thiogalactopyranoside;
OD, optical density; STB, standard tris buffer.
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed.
e-mail: firstname.lastname@example.org, parafi email@example.com, or davidswm@
by guest, on January 9, 2016
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