Journal of Biomaterials and Nanobiotechnology, 2011, 2, 258-266
doi:10.4236/jbnb.2011.23033 Published Online July 2011 (
Copyright © 2011 SciRes. JBNB
Rapid and Facile Purification of Apolipoprotein
A-I from Human Plasma Using Thermoresponsive
Martin Lundqvist1, Tord Berggård2, Erik Hellstrand3, Iseult Lynch1, Kenneth A. Dawson1,
Sara Linse3, Tommy Cedervall1,3#
1Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Dublin, Ireland; 2Department
of Protein Technology, Lund University, Lund, Sweden; 3Biophysical Chemistry, Lund University Chemical Centre, Lund, Sweden.
Received February 26th, 2011; revised March 30th 2011; accepted April 18th, 2011.
Nanoparticles can be used to purify proteins from plasma. We report here the purification of apolipoprotein A-I (apoA-I)
with high specificity from human plasma using copolymeric nanoparticles. We present an optimized protocol using
50:50 NiPAM:BAM copolymer nanoparticles with thermo-responsive properties as an affinity resin. Repeated pelleting
and washing of nanoparticle-captured apoA-I is achieved through temperature cycling. The protein is then eluted using
urea followed by an ion exchange step for protein concentration and depletion of nanoparticles.
Keywords: Apolipoprotein A-I, Nanoparticles, Selective Purification
1. Introduction
The isolation and purification of proteins from complex
bodily fluids is a challenging task, especially for low
abundance proteins. The presence of a very high number
(often several thousands) of other proteins means that
classical methods like ion exchange chromatography and
gel filtration yield only partial purification and a large
number of steps may be needed. Affinity purification pro-
vides a shorter purification route and can lead to much
simplified purification schemes if an entity with high af-
finity and high specificity for the desired protein is at
hand. Commonly used entities are peptides, proteins, spe-
cific antibodies or low Mw ligands coupled to chroma-
tography resins. For example, factor IX can be purified
with vey high specificity from human plasma using con-
formation-specific antibodies that bind solely to the
metal-stabilized conformer [1]. An example of a low Mw
ligand is p-aminomethyl-benzene-sulfonamide for affin-
ity purification of carbonic anhydrase [2]. Examples of
peptides and proteins used successfully in single-step
purification are the streptococcal IgA-binding peptide for
human IgA [3], and an affibody, derived from staphylo-
coccal protein A and selected by phage display, for Taq
DNA polymerase [4]. While antibodies and other protein
ligands offer very high specificity with excellent purity
of the isolated protein, the scale-up costs are high be-
cause the required amount of ligand scales linearly with
batch size. Development of less expensive and more
versatile tools for affinity purification is therefore still a
tractable goal.
Protein purification methods based on nanoparticles
have recently been developed, especially nickel-ni-tri- lo-
triacetate modified magnetic nanoparticles for purifying
poly-His-tagged proteins [5-7]. The magnetism of the
particles makes the purification extremely easy. However,
this approach requires engineering of the protein to be
purified and is not feasible for purification of proteins
from bodily fluids. Non-tagged proteins may be purified
using magnetic nanoparticles modified with specific high
affinity ligands as described above for chromatographic
Here we report the use of thermo-responsive copoly-
mer nanoparticles for protein purification. The special
properties of NiPAM:BAM copolymer particles allow
*This work was funded by an Irish Research Council for Science, En-
gineering and Technology Postdoctoral Fellowship (M.L.), the
Marianne and Marcus Wallenberg Foundation (M.L.), the EU FP6
roject NanoInteract (NMP4-CT-2006-033231), and the SFI SRC
BioNanoInteract (07 SRC B1155), Centre for Nano-Vaccine, Copen-
hagen, Denmark, and the Swedish Research Council (VR).
Rapid and Facile Purification of Apolipoprotein A-I from Human Plasma Using Thermoresponsive Nanoparticles
Copyright © 2011 SciRes. JBNB
for repeated precipitation and re-suspension through tem-
perature cycling, and specific interaction makes it po-
ssible to purify with high efficiency one specific protein
from human plasma, apolipoprotein A-I (apoA-I). ApoA-I
is the major protein constituent of high-density lipopro-
tein (HDL). HDL is involved in reverse cholesterol
transport and is thought to play an important role in low-
ering the risk of coronary artery disease [8-10]. The tra-
ditional purification of apolipoproteins from plasma is a
time consuming process [11-15]. The lipoproteins are
normally isolated from plasma by one or several prepara-
tive ultracentrifugation steps. The purified lipoproteins
contain a high degree of lipids and several proteins. The
apolipoproteins are delipified using organic solvents and
single apolipoproteins are separated from the mixture of
proteins by biophysical and biochemical methods. This
process is not protein friendly and takes days if not
weeks. In contrast, the method presented here can be
completed in less than 12 hours yielding apoA-I of high
quality and purity.
2. Material and Methods
2.1. 70 nm NIPAM:BAM 50:50 Polymer
N-isopropylacrylamide-N-tert-butylacrylamide (NIPAM:
BAM) copolymer particles of 70 nm diameter with 50:50
ratio of the monomers were synthesized in SDS micelles
by free radical polymerization as described previously
[16], although higher SDS concentration was used in the
present work. The procedure for the synthesis was as fo-
llows: 2.8 g monomers (in the appropriate wt/wt ratio),
and 0.28 g crosslinker (N,N-methylenebisacrylamide)
were dissolved in 190 mL MilliQ water with 0.8 g SDS
and degassed by bubbling with N2 for 30 min. Polymeri-
sation was induced by adding 0.095 g ammonium persul-
fate initiator in 10 mL degassed MilliQ water and heating
at 70˚C for 4 hours [17]. Particles were extensively dial-
ysed against MilliQ water for several weeks, changing
the water daily. Particles were lyophilized and stored in
the fridge until used.
2.2. Plasma
Human blood was withdrawn from healthy humans into
vessels pre-treated with EDTA-solution. The vessels
where centrifuged for 5 min at 800 Relative Centrifuga-
tion Force (RCF). The supernatants (the plasma) were
transferred to new vessels and stored in –80˚C freezer
until the time of use. Before use the plasma vessel was
thawed and centrifuged for 3 min at 16.1 kRCF and the
supernatant was transferred to a new vessel, or in the
case where more than one plasma vessel was needed the
supernatants were pooled.
2.3. Particle and Plasma Mixtures
Stock solutions of particles were made by dispersing
lyophilized copolymer particles, to a concentration of 10
mg/mL, in 10 mM Tris/HCl, pH 7.5 with 0.15 mM NaCl
and 1 mM EDTA or in 10 mM phosphate, pH 7.5 with
0.15 mM NaCl and 1 mM EDTA. The mixture was kept
on ice until all the particles were dispersed (1-3 hours)
before mixing with plasma. After mixing the particles
and plasma the sample was kept on ice except during the
centrifugation steps, which were performed at room tem-
In one experiment, lyophilized copolymer particles
were dissolved directly in plasma by adding plasma to
the particles and keeping the mixtures on ice for 1 hour.
2.4. Ion-Exchange Chromatography
Ion exchange chromatography was performed on a Hi-
Trap DEAE FF 1 mL column from GE Healthcare at
room temperature in 10 mM Tris/HCl, pH 7.5 with 1 mM
EDTA (buffer A). Urea fractions (eluates from the nano-
particles) were pooled and passed through a 0.45 µm
syringe filter. After filtration the urea and salt concentra-
tions were lowered by ten-fold dilution with buffer A.
(Alternatively, the salt and urea concentration may be
lowered by dialysis against buffer A). The sample was
thereafter loaded onto the column. ApoA-I was eluted by
stepwise increasing the NaCl concentration: the first step
was elution with 50 mM NaCl in buffer A to remove the
nanoparticles; the second step (the elution of apoA-I) is
performed with 10 0.15 M NaCl in buffer A.
In this study two different SDS-PAGE gels have been
used: PIERCE, PreciseTM Protein Gels 4% - 20% pre-
made gel, which are commercially available; and 12%
gels which were cast immediately prior to use.
2.6. Commercial ApoA-I
ApoA-I (A-0722) was obtained from Sigma-Aldrich and
used as obtained.
2.7. Biophysical Analysis by UV-, CD- and
Fluorescence Spectrometry
The commercial apoA-I (Sigma-Aldrich, A-0722) was
diluted 10 times with 10 mM phosphate, 0.15 M NaCl, 1
mM EDTA, pH 7.5, to approximately 0.15 mg/mL which
corresponds approximately to the concentration of the
purified apoA-I as estimated from the absorbance at 280
The CD signal between 200 and 260 nm was recorded
on a Jasco J-720 spectropolarimeter at 25˚C using a 1
mm quartz cuvette. The fluorescence emission spectra,
Rapid and Facile Purification of Apolipoprotein A-I from Human Plasma Using Thermoresponsive Nanoparticles
Copyright © 2011 SciRes. JBNB
after excitation at 295 nm, were recorded between 310
and 400 nm on a Perkin-Elmer luminescence spectrome-
ter LS 50B in a 1 cm quartz cuvette and the UV absorb-
ance spectra were recorded between 250 and 300 in a 1
cm cuvette.
2.8. Reconstitution of Phospholipids:ApoA-I
Phospholipids vesicles with apoA-I were reconstituted as
previously described [18]. Briefly, apoA-I was mixed
with phosphatidyl choline (PC) and minute amounts of
radio-labelled 14C-POPC PC (260:1 PC/apoA-I molar
ratio) and dialyzed in order to generate rHDL. The sam-
ple was separated by gel filtration on a Superose 6 col-
umn, and eluted fractions were analyzed by scintillation
counting and apoA-I content was determined by apoA-I
3. Results and Discussion
The purification scheme is based on the high affinity and
specificity of 50:50 NiPAM:BAM copolymer nanoparti-
cles for apoA-I [19,20] and the temperature responsive
behaviour of the nanoparticles. The copolymer particles
are expanded and mainly dispersed as individual particles
at temperatures below 10˚C, but collapsed and aggre-
gated (at least if the particle concentration is high enough)
at temperatures over 10˚C. These properties make the
50:50 NiPAM:BAM copolymer nanoparticles ideal for
use in an affinity based purification scheme for apoA-I
that can be completed within a day, as shown in Scheme
3.1. Purification Scheme
The purification scheme consists of the following steps;
1) Mixing of copolymer particles with plasma and incu-
bation on ice for 60 minutes (the mixture contains 10 mg
of 50:50 NiPAM:BAM nanoparticles of 70 nm diameter
per mL human plasma); 2) Pelleting the particles by
heating to room temperature and centrifugation for 3
minutes at 16 kRCF followed by aspiration of the super-
natant (which is discarded); 3) Washing of the pellet by
addition of 10 mM Tris/HCl pH 7.5, 1 mM EDTA, 150
mM NaCl, 100 µL per mg of nanoparticles 4) Repeat
step 2; 5) Repeat step 3; 6) Repeat step 2; 7) Repeat step
3; 8) Repeat step 2; 9) Elution of bound protein from the
nanoparticles by addition of 6 M urea, 10 mM Tris/HCl
pH 7.5, 1 mM EDTA, 100 µL per mg of nanoparticles,
and incubation on ice for 30 minutes; 10) Pelleting of the
particles by heating to room temperature and centrifuga-
tion for 3 minutes at 16 kRCF, followed by collection of
the supernatant (saved for step 15); 11) Repeat step 9; 12)
Repeat step 10; 13) Repeat step 9; 14) Repeat step 10; 15)
Scheme 1. The approximate experimental run time is indi-
cated at the right-hand side of the chart.
Ten-fold dilution of the eluate (supernatant from steps 10,
12 and 14) with 10 mM Tris/HCl pH 7.5, 1 mM EDTA,
and loading onto an DEAE (HiTrap FF) ion-exchange
column; 16) Washing of the column with 10 mM
Tris/HCl pH 7.5, 1 mM EDTA, (10 mL per 1 mL resin);
17) 2nd washing step of the DEAE column using 10 mM
Tris/HCl pH 7.5, 1 mM EDTA, 50 mM NaCl, (10 mL
per 1 mL resin); 18) Elution of apoA-I from the DEAE
column in 10 mM Tris/HCl pH 7.5, 1 mM EDTA, 150
mM NaCl, (10 mL per 1 mL resin, collection of 1 mL
fractions). Samples from all steps were analysed by SDS
PAGE and the samples after steps 5, 7, 9, 11, 13, and 18
are shown in Figure 1.
The purification scheme yields pure protein free from
nanoparticles, and each step has been optimized to pro-
vide the highest yield and the least disruption to the pro-
tein native state. Buffer composition, incubation time, and
washing conditions were varied during this optimization
and the amount of apoA-I adsorbed to the co-polymer
particles was investigated using SDS PAGE.
Rapid and Facile Purification of Apolipoprotein A-I from Human Plasma Using Thermoresponsive Nanoparticles
Copyright © 2011 SciRes. JBNB
3.2. Role of Salt and Buffer Composition in the
Nanoparticle-Binding Step
The results from different incubation conditions for co-
polymer particles with human plasma are shown in Fig-
ure 2. The effect of buffer and salt was evaluated by
comparing incubation in phosphate- or trizma-based
buffers at 10 mM, pH 7.5 with 1 mM EDTA, with or
without 0.15 M NaCl, and all solutions were incubated
for 1, 3, and 6 hours. The nanoparticle bound apoA-I was
eluted from the particles with SDS-loading buffer after
three washing steps (using the same buffer and salt con-
centration as during the respective incubation). The re-
sults (Figure 2) clearly show that the buffer composition
is important, as more apoA-I is retrieved from particles
in Tris/HCl buffers than from particles in phosphate
buffer (Figure 1). The salt concentration is also impor-
Figure 1. SDS-PAGE of samples from the different stages of
the purification of apoA1. Lane 1 = supernatant after the
2nd wash (step 5), lane 2 = supernatant after the 3rd wash
(step 7). Lanes 3, 4, and 5 show the supernatants after the
urea elution steps (9, 11, and 13, respectively). Lane 6 shows
the eluate of the DEAE ion-exchange column (step 17). The
left lane contains molecular weight standards with the Mw
of the proteins given to the left.
Figure 2. SDS PAGE investigation of the effects of buffer
composition and incubation time. In order to study the
amount of apoA-I bound to the nanoparticles proteins were
eluted from the nanoparticles into SDS loading buffer after
three washes using the same buffer as in the incubation step.
NaP = 10 mM sodium phosphate buffer, pH 7.5, 1 mM
EDTA; Tris = 10 mM trizma base/HCl, pH 7.5, 1 mM
EDTA; NaCl = 0.15 M NaCl; Time = incubation time in
hours. The left lane contains molecular weight standards
with the Mw of the proteins given to the left.
tant, and the best buffer condition for the initial incuba-
tion step is 10 mM Tris /HCl, 1 mM EDTA, 0.15 M
NaCl. We also find that one hour incubation is sufficient
to maximize the amount of apoA-I bound to the copoly-
mer particles. Longer incubation does not increase the
amount of apoA-I retrieved.
After incubation, pelleting and aspiration of the su-
pernatant, apoA-I is the dominant protein bound to the
nanoparticles, but serum albumin is often a contaminant.
Cedervall et al. showed [20] that serum albumin dissoci-
ates much faster from the copolymer particle surface than
apoA-I. This difference allows for serum albumin to be
washed away while the major fraction of apoA-I remains
attached to the copolymer particles.
3.3. Role of Salt and Buffer Composition in the
Nanoparticle-Washing Step
To optimize the washing procedures, buffers with dif-
ferent NaCl concentrations, 0.15, 0.5, 1.0, and 2.0 M,
were tested. As shown in Figure 3 there are no dramatic
differences after the first wash, while after the second
wash 1 and 2 M NaCl leads to more efficient removal of
serum albumin than 0.15 and 0.5 M NaCl. In the third
wash, there is too little protein to allow for any quantita-
tive comparison, and there is no obvious difference in the
amount of apoA-I retrieved from the nanoparticles. Fi-
gure 3 also shows that the amount of apoA-I that is lost
in the three wash steps is virtually the same for all four
NaCl-concentrations. For simplicity 10 mM Tris/HCl pH
7.5, 1 mM EDTA, 0.15 M NaCl was selected for both the
sample incubation and washing steps. This buffer condi-
tion is also favorable for the subsequent purification
steps (see below).
3.4. Optimization of the Elution Step
In order to optimize the elution of apoA-I from the
Figure 3. Optimization of the NaCl concentration during
the washing steps. SDS PAGE of the protein eluted from the
nanoparticle pellet after three washes (lanes 2-5), and of the
supernatant from the 1st (lanes 7-10), 2nd (lanes 11-14) and
3rd (lanes 15-18) washing steps. The NaCl concentration
was 0.15, 0.5, 1.0 or 2.0 M from left to right among the four
samples at each step.
Rapid and Facile Purification of Apolipoprotein A-I from Human Plasma Using Thermoresponsive Nanoparticles
Copyright © 2011 SciRes. JBNB
nanoparticles, at least two factors are necessary to con-
sider in order to maximize the protein yield. The effi-
ciency of the elution is, of course, important for the final
protein yield. However, the buffer composition after the
elution is also important for the protein yield as it will
affect the subsequent purification steps. Buffers with
high salt concentration, high and low pH, different de-
tergents, or urea were tested for their elution efficiency
(Figure S3 in supplementary material), determined as the
highest amount of protein in the gel bands. The highest
amount of apoA-I was eluted using detergents, followed
by 3 M NaCl, followed by 6 M urea. At lower NaCl
concentration, or at pH 4 or 9.2, little or no elution could
be observed. Urea was chosen as eluant for the purifica-
tion scheme as it allows the eluted proteins to be loaded
directly onto an ion exchange column without changing
the buffer. The detergents, although they are the best
eluants, were not chosen as it could be difficult to remove
them from the protein, resulting in a lower purity of the
final product.
Figure 1 shows the different stages in the purification
of apoA-I schematically. The amount of eluted apoA-I
decreases in each step of elution with 6 M urea (Figure 1,
Lanes 3-5). The small amount of apoA-I remaining in the
third urea eluate does not motivate a fourth urea step.
3.5. Ion Exchange Chromatography
After elution of apoA-I from the copolymer particles,
ion-exchange (DEAE) chromatography is used to sepa-
rate apoA-I from impurities, mainly the proteins apoli-
poprotein E and serum albumin [19] and copolymer par-
ticle fragments and to remove urea and change the buffer.
Before loading onto the DEAE-column the pooled sam-
ple is diluted 10 times with 10 mM phosphate buffer pH
7.5, 1 mM EDTA, to reduce the NaCl concentration.
Bound molecules are eluted from the DEAE-column in a
stepwise manner. Non-protein substances are eluted in
buffer with 50 mM NaCl, apoA-I is eluted in buffer with
0.15 M NaCl, and serum albumin elutes in 3 M NaCl,
which regenerates the column (serum albumin would
elute much earlier if a gradient elution profile were
If the first step (elution in 50 mM NaCl) is not in-
cluded, something that scatters light under 270 nm is co-
eluted with apoA-I (data not shown). The fraction from
the first elution step does not contain protein (no visible
bands in a coomassie stained gel). Analysis by NMR
spectroscopy (data not shown) indicates an interaction
between buffer molecules and very large molecules and/
or a slight increase in the solution viscosity evidenced as
a slower tumbling of the molecules and a broader peak
[21]. The most probable explanation for the observed phe-
nomena is that the eluted fraction consists of fragments
of the copolymer particles. However, this has to be in-
vestigated further.
3.6. Maximizing ApoA-I Yield
Figure 3 shows that even though apoA-I has a high af-
finity for the copolymer particle surface, a large fraction
of the apoA-I is washed away in the first washing step.
Two experiments were conducted to maximize the yield
of purified apoA-I. The effect of the copolymer particle
concentration was checked by keeping the total sample
volume and the total plasma concentration constant while
the concentration of particles was varied. After incuba-
tion, the same amount (by weight) of copolymer particles
was removed from each sample and the amount of bound
apoA-I analyzed by SDS PAGE after elution in SDS
loading buffer. The results show that the total binding of
apoA-I increases with the particle concentration (Figure
S1, supplementary material), hence, one limiting factor
in the yield is the available particle surface area. To con-
firm this result a second experiment was conducted in
which different amounts of lyophilized copolymer parti-
cles were dispersed in the same volume of plasma. The
trend of increasing yield of apoA-I with increasing
amount of available surface area was confirmed (Figure
S2, supplementary material). Table 1 shows the amounts
of purified apoA-I for two experiments with different
particle concentrations. The purification efficiency, in per-
cent, is calculated assuming that the concentration of
apoA-I in the original plasma is ~1 mg/ml. These results
show that it is not the apoA-I concentration in blood that
is the limiting factor for the yield of purified apoA-I, but
the solubility (dispersability) of the copolymer particles
Table 1. Comparison of the efficiency of the copolymer
nanoparticles at purifying apoA-1 from plasma with two
different dispersion methods—with TBS as an intermediate
step, or direct dispersion in plasma.
a 2b
Theoretical max apoA-I (mg)c 3.6 2.2
Particles (mg)d 45 22
Plasma/particles (ml/mg)e 0.08 0.1
Amount purified apoA-I (μg)f 224 277
Purification efficiency (%)g 6 13
Purified apoA-I/amount particles (μg/mg)h 5 13
aCopolymer particles first dispersed in TBS before mixing with plasma;
bCopolymer particles directly dispersed in plasma; cCalculated at a nominal
apoA-I concentration of 1 mg/ml plasma; dTotal amount of copolymer parti-
cles used in the experiment; eRatio between plasma and copolymer particles
in the experiment; fTotal amount of purified apoA-I in the experiment;
gPurification efficiency in percent purified apoA-I compared to the nominal
apoA-I concentration; hAmount purified apoA-I per mg copolymer particles.
Rapid and Facile Purification of Apolipoprotein A-I from Human Plasma Using Thermoresponsive Nanoparticles
Copyright © 2011 SciRes. JBNB
Figure S1. ApoA1 retrieved with different amounts of
polymer particles added to 210 μl plasma in a total volume
of 910 μl. Lanes 1, 2, 3, 4, 5, 6, 7, and 8 represent 0.007, 0.07,
0.14, 0.35, 0.7, 1.4, 2.1, and 7.0 mg co-polymer particles,
respectively. The 0.35, 0.7, 1.4, 2.1 and 7.0 mg samples con-
tain protein from an identical amount of polymer particles.
Figure S2. Lanes 1, 2, 3, and 4 represent 10, 5, 2.5 and 1 mg
co-polymer nanoparticles, respectively, that were dissolved
in 1 ml plasma. Washed with 3 x 1000, 500, 250 and 100 μL,
respectively, of 10 mM trizma/HCl pH 7.5, 1 mM EDTA,
0.15 M NaCl. The pellets were dissolved in 100, 50, 25 and
10 μL SDS-loading buffer, respectively, and 10 μL of each
sample was loaded on the electrophoresis gel.
Figure S3. Elution of apoA1 from the co-polymer particles
using different agents. Lanes 1, 2, and 3 are eluates in 1, 2,
and 3 M NaCl, respectively, lane 4 the eluate with 2% triton,
lane 5 with 2% tween-20, lane 6 with 6 M urea, lane 7 10
mM sodium acetate buffer, pH 4, lane 8 10 mM Tris/HCl
pH 9.2, and lane 9 buffer with 2% SDS. All eluates except in
lanes 7 and 8 contain 10 mM Tris/HCl pH 7.5.
in plasma and thus the amount of available surface area.
The highest concentration of particles that can be dis-
persed in plasma is ~10 mg per ml, as above at this con-
centration particle aggregation occurs, and thus the over-
all surface area available for bind decreases again.
3.7. Lipid-Free ApoA-I
Preliminary results indicate that the particles bind apoA-I
together with lipids in the first step of the purification
scheme. As the goal was to purify lipid free apoA-I, the
final purified sample was checked for cholesterol and
triglycerides with commercial enzyme-based detection
kits (see SI). No lipids were detected in the purified
apoA-I samples suggesting that the purified apoA-I is
lipid free.
3.8. Comparison with Commercial ApoA-I
Integrity, Structure and Function
The purified apoA-I was compared to commercially
available apoA-I. On a coomassie stained 1D SDS-PAGE
gel, the commercial protein gives rise to three bands,
while the protein purified by the method described here
gives rise to one band on the same gel with similar
amounts of sample loaded, as shown in Figure 4(a). The
identity of the purified apoA-I has been confirmed re-
peatedly by mass spectrometry [21,22]. The apparent
differences in size between the three bands in the com-
mercial sample of apolipoprotein A-1 may be due to
modifications or proteolysis. Another explanation may be
that the copolymer particles specifically target a sub-
population of apoA-I, for example, the fraction with oxi-
dized side chains. This is less likely as the apoA-I found
in the wash fractions has the same mobility as the puri-
fied protein. Thus, the three bands in commercial apoA-I
are most likely due to the harsh conditions used during
the existing purification process, whereas the protocol
presented here leads to a homogeneous protein prepara-
Three different spectroscopic methods, UV absorbance,
intrinsic tryptophan fluorescence and far UV circular
dichroism spectroscopy, are used to evaluate and com-
pare the structure of apoA-I purified via the method pre-
sented here with commercial apoA-I. The UV absorb-
ance spectra, Figure 4(b), gives information on local
structural changes around aromatic amino acids; the in-
trinsic tryptophan fluorescence spectra, Figure 4(c), gives
information on changes in the tertiary structure; and the
far UV circular dichroism spectra, Figure 4(d), reports
on changes in the secondary structure. In all of the spec-
tra there are no or very small deviations between the
commercial and purified apoA-I, indicating that the terti-
ary and secondary structure of apoA-I are essentially the
same in the two samples.
The phospholipids binding function of the purified
apoA-I was examined by reconstitution of phospholip-
ids:apoA-I vesicles. Figure S4 shows that the copolymer
particle purified apoA-I interacts with and forms vesicles
together with phospholipids in a similar way to that
shown previously for apoA-I purified with a traditional
purification protocol [18].
Rapid and Facile Purification of Apolipoprotein A-I from Human Plasma Using Thermoresponsive Nanoparticles
Copyright © 2011 SciRes. JBNB
(a) (b)
(c) (d)
Figure 4. Comparison between commercial apoA-I and the apoA-I purified by the method described here. (a) Lane 1 = mo-
lecular weight standards with the Mw of the proteins given to the left, lane 2 = commercial apoA-I, lane 3 = apoA-I purified
in this work. In (b), (c) and (d), dark grey = commercial apoA-I and light grey = purified apoA-I. (b) absorbance, (c) fluores-
cence and (d): circular dichroism spectra.
Figure S4. ApoA1 was mixed with labelled PL in order to
generate rHDL. The sample was analyzed by gelfiltration.
Eluted fractions were analyzed by scintillation counting and
apoA1 ELISA.
4. Conclusions
Copolymer nanoparticles offer efficient and facile puri-
fication of apoA-I from complex protein mixtures, such
as plasma. The purification scheme presented here dra-
matically reduces the time needed for purification. The
final yield of pure apoA-I is up to 13 mg/g nanoparticles,
which is comparable to other affinity chromatography
systems. The particles also bind apolipoprotein E, A-IV,
and A-II with high specificity. These apolipoproteins are
in much lower concentrations in plasma and are therefore
a challenge to purify. However, in a scaled up purifica-
tion protocol it should be possible to purify also these
proteins using the copolymer particles. Additionally,
slight modification of the comonomer ratio could be used
to optimize the particles for selective binding of these
The copolymer particles are easy and inexpensive to
produce and their physical and chemical characteristics
(including surface charge and/or hydrophobicity or sur-
face energy) are easy to vary. Additionally, the particles
can be modified with ligands with high specificity for
selected target proteins. These features make the co-
polymer particles an attractive choice for purification of
proteins, not limited to apolipoproteins.
5. Acknowledgments
We thank Professor Björn Dahlbäck and Dr Cecilia
Oslakovic for help with the reconstitution of phosphol-
ipid:apoA-I vesicles and for valuable comments on the
manuscript. Access to and use of UCD Conway Mass
Spectrometry Resource instrumentation is gratefully ac-
Rapid and Facile Purification of Apolipoprotein A-I from Human Plasma Using Thermoresponsive Nanoparticles
Copyright © 2011 SciRes. JBNB
6. Supporting Information Available
Supplementary information regarding the optimization of
the purification protocol is presented. Figures showing
the effects of particle concentration on apoA-I purity
(Figure S1), and yield (Figure S2) and the effectiveness
of various agents to elute the purified apoA-I from the
particles (Figure S3), and the reconstituted phospholip-
ids:apoA-I vesicles (Figure S4).
[1] H. A. Liebman, S. A. Limentani, B. C. Furie and B. Furie,
“Immunoaffinity Purification of Factor-Ix (Christmas
Factor) by Using Conformation-Specific Antibodies Di-
rected against the Factor-Ix - Metal-Complex,” Proceed-
ings of the National Academy of Sciences of the United
States of America, Vol. 82, No. 11, 1985, pp. 3879-3883.
[2] S. O. Falkbring, P. O. Göthe, P. O. Nyman, L. Sundberg
and J. Porath, “Affinity Chromatography of Carbonic An-
hydrase,” FEBS Letters, Vol. 24, No. 2, 1972, pp. 229-
235. doi:10.1016/0014-5793(72)80773-7
[3] C. Sandin, S. Linse, T. Areschoug, J. M. Woof, J. Rein-
holdt and G. Lindahl, “Isolation and Detection of Human
IgA Using a Streptococcal IgA-binding Peptide,” Journal
of Immunology, Vol. 169, No. 3, 2002, pp. 1357-1364.
[4] K. Nord, E. Gunneriusson, J. Ringdahl, S. Ståhl, M.
Uhlen and P.-A. Nygren, “Binding Proteins Selected from
Combinatorial Libraries of an [alpha]-Helical Bacterial
Receptor Domain,” Nature Biotechnology, Vol. 15, No. 8,
1997, pp. 772-777. doi:10.1038/nbt0897-772
[5] H. W. Gu, K. M. Xu, C. J. Xu and B. Xu, “Biofunctional
Magnetic Nanoparticles for Protein Separation and Patho-
gen Detection,” Chemical Communications, Vol. 9, 2006,
pp. 941-949. doi:10.1039/b514130c
[6] D. B. Shieh, C. H. Su, F. Y. Chang, Y. N. Wu, W. C. Su,
J. R. Hwu, J. H. Chen and C. S. Yeh, “Aqueous Nickel-
nitrilotriacetate Modified Fe3O4-NH3+ Nanoparticles for
Protein Purification and Cell Targeting,” Nanotechnology,
Vol. 17, No. 16, 2006, pp. 4174-4182.
[7] C. C. You, A. Verma and V. M. Rotello, “Engineering the
Nanoparticle-Biomacromolecule Interface,” Soft Matter,
Vol. 2, No. 3, 2006, pp. 190-204.
[8] N. E. Miller, “Associations of High-Density-Lipoprotein
Subclasses and Apolipoproteins with Ischemic-Heart- Di-
sease and Coronary Atherosclerosis,” American Heart
Journal, Vol. 113, No. 2, 1987, pp. 589-597.
[9] W. P. Castelli, R. D. Abbott and P. M. McNamara, “Sum-
mary Estimates of Cholesterol Used to Predict Coronary
Heart-Disease,” Circulation, Vol. 67, No. 4, 1983, pp.
730-734. doi:10.1161/01.CIR.67.4.730
[10] G. G. Rhoads, C. L. Gulbrandsen and A. Kagan, “Serum-
Lipoproteins and Coronary Heart-Disease in a Population
Study of Hawaii Japanese Men,” New England Journal of
Medicine, Vol. 294, No. 6, 1976, pp. 293-298.
[11] C. Edelstein, D. Pfaffinger and A. M. Scanu, “Advantages
and Limitations of Density Gradient Ultracentrifugation
in the Fractionation of Human-Serum Lipoproteins—Role
of Salts and Sucrose,” Journal of Lipid Research, Vol. 25,
No. 6, 1984, pp. 630-637.
[12] R. J. Havel, H. A. Eder and J. H. Bragdon, “The Distribu-
tion and Chemical Composition of Ultracentrifugally Se-
parated Lipoproteins in Human Serum,” The Journal of
Clinical Investigation, Vol. 34, No. 9, 1955, pp. 1345-
[13] D. W. Swinkels, H. L. M. Haklemmers and P. N. M. De-
macker, “Single Spin-Density Gradient Ultracentrifuga-
tion Method for the Detection and Isolation of Light and
Heavy Low-Density-Lipoprotein Subfractions,” Journal
of Lipid Research, Vol. 28, No. 10, 1987, pp. 1233-1239.
[14] T. Tadey and W. C. Purdy, “Chromatographic Tech-
niques for the Isolation and Purification of Lipoproteins,”
Journal of Chromatography B: Biomedical Sciences and
Applications, Vol. 671, No. 1-2, 1995, pp. 237-253.
[15] K. H. Weisgraber, T. P. Bersot, R. W. Mahley, G.
Franceschini and C. R. Sirtori, “A-I Milano Apoprotein
Isolation and Characterization of a Cysteine-Containing
Variant of the A-I Apoprotein from Human High Density
Lipoproteins,” The Journal of Clinical Investigation, Vol.
66, No. 5, 1980, pp. 901-907. doi:10.1172/JCI109957
[16] X. Wu, R. H. Pelton, A. E. Hamielec, D. R. Woods and
W. McPhee, “The Kinetics of Poly(N-Isopropylacryla-
mide) Microgel Latex Formation,” Colloid and Polymer
Science, Vol. 272, No. 4, 1994, pp. 467-477.
[17] I. Lynch, I. Miller, W. M. Gallagher and K. A. Dawson,
“Novel Method to Prepare Morphologically Rich Poly-
meric Surfaces for Biomedical Applications via Phase
Separation and Arrest of Microgel Particles,” Journal of
Physical Chemistry B, Vol. 110, No. 30, 2006, pp. 14581-
[18] C. Oslakovic, M. J. Krisinger, A. Andersson, M. Jauhiai-
nen, C. Ehnholm and B. Dahlback, “Anionic Phospholip-
ids Lose Their Procoagulant Properties When Incorpo-
rated into High Density Lipoproteins,” Journal of Bio-
logical Chemistry, Vol. 284, No. 9, 2009, pp. 5896-5904.
[19] T. Berggård, et al., “140 Mouse Brain Proteins Identified
by Ca2+-Calmodulin Affinity Chromatography and Tan-
dem Mass Spectrometry,” Journal of Proteome Research,
Vol. 5, No. 3, 2006, pp. 669-687.
[20] T. Cedervall, I. Lynch, M. Foy, T. Berggård, S. C. Don-
nelly, G. Cagney, S. Linse and K. A. Dawson, “Detailed
Identification of Plasma Proteins Adsorbed on Copolymer
Nanoparticles,” Angewandte Chemie International Edi-
tion, Vol. 46, No. 30, 2007, pp. 5754-5756.
[21] T. Cedervall, I. Lynch, S. Lindman, T. Berggard, E. Thu-
lin, H. Nilsson, K. A. Dawson and S. Linse, “Understand-
Rapid and Facile Purification of Apolipoprotein A-I from Human Plasma Using Thermoresponsive Nanoparticles
Copyright © 2011 SciRes. JBNB
ing the Nanoparticle-Protein Corona Using Methods to
Quantify Exchange Rates and Affinities of Proteins for
Nanoparticles,” Proceedings of the National Academy of
Sciences of the United States of America, Vol. 104, No. 7,
2007, pp. 2050-2055.doi:10.1073/pnas.0608582104
[22] M. Lundqvist, I. Sethson and B. H. Jonsson, “Protein
Adsorption onto Silica Nanoparticles: Conformational
Changes Depend on the Particles’ Curvature and Protein
Stability,” Langmuir, Vol. 277, No. 20, 2004, pp. 10639-
ApoA-I: Apolipoprotein A-I; RCF: Relative Centrifugal Force; PC: Phosphatidyl Choline.