Addition of magnesium chloride to enhance mono-dispersityof a coiled-coil recombinant mouse macrophage protein
Parveen Pahuja • Alagiri Srinivasan •
Munish Puri
Received: 17 July 2013 / Accepted: 18 December 2013
� Springer Science+Business Media New York 2014
Abstract X-ray crystallography for the determination of
three-dimensional structures of protein macromolecules
represents an important tool in function assignment of
uncharacterized proteins. However, crystallisation is often
difficult to achieve. A protein sample fully characterized in
terms of dispersity may increase the likelihood of suc-
cessful crystallisation by improving the predictability of
the crystallisation process. To maximize the probability of
crystallisation of a novel mouse macrophage protein
(rMMP), target molecule was characterized and refined to
improve monodispersity. Addition of MgCl2 at low con-
centrations resolves the rMMP into a monodisperse solu-
tion, and finally successful crystallization of rMMP was
achieved. The effect of MgCl2 was studied using gel fil-
tration chromatography and dynamic light scattering.
Keywords Dispersity � Gel filtration chromatography �Dynamic light scattering � Crystallisation
Introduction
Macrophages are cells of myeloid origin that play signifi-
cant role in primary immune system and also in adaptive
immunity. They are also known as phagocytic cells as they
engulf the invading pathogens and present their fragments
to T cells thus acting as antigen presenting cells also. They
are primarily known to be involved in inflammation;
however, their role can be extended to many serious dis-
eases like cancer, HIV/AIDS and neurological disorders
[1–4]. They are present in myriad of activation states
producing different components in different amounts and
sometimes novel products [5, 6]. These types of differential
and novel expressions have become ubiquitous as is evi-
denced from whole genome sequencing projects that have
produced millions of coding sequences and are deposited
on public domains and databases [7]. A large number of
sequences remain functionally uncharacterized and may be
referred to as ‘hypothetical’, indicating that they are
sequences of unknown functions. There thus presents itself
an urgent need to characterize these proteins in order to aid
in the development of effective therapeutic agents and
drugs.
Structural biology is one of the most effective approa-
ches for defining the functions of proteins through solving
three-dimensional (3D) structures by X-ray and NMR
technologies [8]. X-ray crystallography, which accounts for
about 86 % of known protein structures in the Protein Data
Bank (PDB; http://www.rcsb.org/pdb/), is the most suc-
cessful method for solving high resolution structures of
proteins [9]. To implement this method, crystallisation of a
given protein is an essential step to solve its 3D-structure
using X-ray crystallography. Crystallisation involves
extensive screening for various conditions such as type of
precipitant, buffers, additives, temperature and solvent
P. Pahuja � M. Puri
Fermentation and Protein Biotechnology Laboratory,
Department of Biotechnology, Punjabi University, Patiala, India
A. Srinivasan
Department of Biophysics, All India Institute of Medical
Sciences, New Delhi, India
M. Puri (&)
Centre for Chemistry and Biotechnology, Deakin University,
Geelong Technology Precinct, Geelong, VIC 3217, Australia
e-mail: [email protected]
123
Mol Cell Biochem
DOI 10.1007/s11010-013-1934-x
concentration to achieve high quality protein crystals
suitable for high resolution X-ray diffraction. Successful
crystallisation is often followed by optimization of the
unique conditions to enhance size and diffraction-quality of
protein crystals [10]. To ensure crystallisation, the protein
sample must be highly pure, stable and homogeneous; a
pre-requisite that is met after biophysical characterisation
[11]. Such techniques are furthermore helpful in reducing
batch-to- batch variations during crystallisation and result
in more accurate screening and optimization.
Protein quality is expressed in terms of homogeneity of
molecular species present in the solution and represents a
crucial optimization parameter prior to initiating crystallisa-
tion. Purity refers to the absence of other contaminating
proteins and varying forms of the same protein. This param-
eter is also termed as ‘monodispersity’ and is a measure of
fractional density of a single type of molecular species present
in the solution. The presence of different oligomeric states (in
the form of dimers, trimers etc.) or alternatively folded states
of a protein molecule hinder the probability of successful
protein crystallisation [12, 13]. Dynamic light scattering
(DLS) is a technique often used to characterize proteins for
monodispersity and makes an indispensable tool.
GFC is used to separate protein molecules based on their
molecular size or hydrodynamic radii. It is often regarded as
an ideal method to separate different polymeric states of the
same protein. The quality of protein solutions can be ana-
lysed and confirmed using DLS. DLS is a technique which
analyses size and distribution of molecules in liquid diffused
state; commonly used to study dispersity. This technique is
also known by other names such as ‘Photon Correlation
Spectroscopy (PCS)’ and ‘Quasi Elastic Light scattering
(QELS)’. DLS uses a laser beam to act on molecules
undergoing Brownian motions in a solution. Light gets
scattered through components present in solution which
results in change in light intensity which is recorded on a
detector. The intensity change is translated into diffusional
coefficients which can give information about radial distri-
bution or particle sizes [14]. DLS is a technique that is easy
to perform and is non-invasive which means valuable sam-
ple can be recovered after DLS experiment.
The present study involves characterization of a recom-
binant mouse macrophage protein (rMMP) which is required
to achieve its crystallisation using GFC and DLS. rMMP is a
hypothetical protein derived from cDNA (RIKEN ID:
1700029K01) from mouse genome database Fantom [15].
The preliminary bioinformatics analysis revealed that rMMP
possesses a ‘coiled-coil’ structure. Coiled-coils are known to
undergo oligomeric organization at varying levels and such
polydisperse behaviour may have adverse effect on crys-
tallization. Addition of metal ions in the elution buffer may
enhance its monodispersity which is a prerequisite for
achieving crystallisation [16]. Thus, the effect of MgCl2 in
stabilising rMMP to a homogenous state was studied with
the help of GFC and DLS.
Materials and methods
Production of recombinant protein
The gene (PCR product; pool of cDNA species generated
using mRNA extracted from LPS-stimulated mouse macro-
phage cells over a time course up to 24 h was used as a
template for PCR amplification) was transferred into
pDEST17 expression vector and expressed in chemically
competent E. coli strain BL21 (DE3)pLysS (Invitrogen,
USA) as an N-terminal His-tag fusion protein [17]. A fresh
colony of bacteria from overnight incubated plates was
inoculated into 10 mL Luria–Bertani medium (Tryptone:
1 %; Yeast extract: 0.5 % and NaCl 0.5 %) containing
ampicillin at 100 lg/mL concentration. The starter culture
was grown at 37 �C, 200 rpm overnight in an orbital shaking
incubator. Proteins were expressed for 18 h at 37 �C using
auto-induction medium (1 L) [18]. Cells were harvested by
centrifugation at 10,000g, 4 �C for 20 min. A sample of this
whole cell extract was taken for Sodium dodecyl sulphate–
polyacrylamide gel electrophoresis (SDS-PAGE).
Cell pellet (5 g) was dissolved in lysis buffer (40 mL;
25 mM Hepes pH 7.4, 300 mM NaCl, 10 mM Imidazole),
and cells were disrupted using an ultra-sonicator (Vibra-
Cell VCX 500, Sonics, USA). Sonication was performed
for 15 min at 30 % amplitude using a 3 mm tapered mi-
crotip probe. All cell disruptions were carried on ice. After
sonication, insoluble material was separated from soluble
material by centrifugation (20,000g, 4 �C for 45 min) for
the recovery of recombinant protein. A sample of the sol-
uble extract was taken for SDS-PAGE analysis.
Purification
The soluble fraction obtained as a result of cell lysis was
supplemented with 10 mM imidazole and incubated with Ni-
Sepharose (3 mL) metal affinity resin (1 mL of resin has
binding affinity for 40 mg protein) for 1 h at room temper-
ature for occasional shaking. The resin was washed with
buffer A (25 mM Hepes, pH 7.4, 300 mM NaCl, 25 mM
imidazole). Finally, bound protein was eluted by 15 mL of
buffer B (25 mM HEPES pH 7.4, 300 mM NaCl and
250 mM imidazole). A sample of the eluted fractions was
removed for SDS-PAGE analysis. All liquid injections in
affinity chromatography were carried out using flow-adaptor
(Bio-Rad Inc., USA).The fractions containing target protein
were pooled and concentrated by ultrafiltration using Amicon
Ultra-15 10 kDa membrane. The resultant concentrated
protein (1 mL) was loaded onto a pre-packed Superdex-75
Mol Cell Biochem
123
PG column (HiLoad 16/60 with a bed size of 120 mL), prior
equilibrated with buffer C (25 mM Hepes, pH 7.4, 300 mM
NaCl) and attached with FPLC (BioLogic DuoFlow, Bio-Rad
Inc., USA). The protein was eluted at a flow rate of
1 mL min-1, and O.D. was measured at 280 nm. The eluted
fractions were pooled, concentrated and stored at -80 �C.
All experiments were conducted three times.
As it was predicted from in silico analysis (using Mul-
ticoil2; secondary structure prediction server) that rMMP
has a ‘coiled-coil’ structure [19], magnesium chloride was
added into all buffers to stabilize protein elution. Magne-
sium chloride (MgCl2) was added at a final concentration
of 5 mM in the elution buffer. Finally, to ascertain effect of
Mg2? on secondary structure of rMMP, gel filtration
chromatography was performed using buffer A with and
without containing MgCl2. A sample of the eluted protein
was taken for analysis by SDS-PAGE. The samples of
whole cell extract, soluble protein and protein eluted from
Ni-resin resin and Gel filtration column were run side by
side on 12 % SDS-PAGE gels. Protein concentrations of
crude extract and fractionated sample at each step were
calculated using the Bradford method [20].
Gel electrophoresis
The molecular weight analysis of collected peaks of rMMP
was determined by SDS-PAGE performed according to the
procedure of Laemmli [21], using a 12.5 % resolving gel.
Following electrophoresis, the gel was stained with Coo-
massie brilliant blue R-250 (CBB). The molecular mass of the
macrophage protein was determined by comparison with the
protein markers; phosphorylase b (97 kDa), bovine serum
albumin (66 kDa), ovalbumin (44 kDa), carbonic anhydrase
(29), soyabean trypsin inhibitor (20) and lysozyme (14).
Dynamic light scattering measurements
Dynamic light scattering was used to study in solution dis-
persity of rMMP. The experiments were performed in
duplicate at 830 nm using DynaPro-TC-04 light scattering
system (Protein Solutions, Wyatt Technology, CA). Protein
solution was used at a concentration of 5 mg mL-1 and was
centrifuged at 14,000g for 30 min at 4 �C to remove par-
ticulate matter before being placed in the 12 lL Quartz
cuvette. Mean hydrodynamic radius and peak area were
analysed using Dynamics 6.10 software. All solutions
including protein sample were clarified using 0.22 lm syr-
inge filters.
Crystallization of rMMP
The crystallization of the purified protein rMMP was
investigated following hanging drop vapour diffusion
method in a 24-well plate set up. A crystallization drop was
set up by mixing 2 lL of protein sample (6–20 mg/mL in
25 mM Hepes buffer pH 7.0) with 2 lL of reservoir buffer
on a 18 mm siliconized cover slip (procured from Hampton
Research, US). The drop was equilibrated against reservoir
buffer (1 mL) by placing the cover slip in an inverted
position over the well. The plates were observed under a
microscope and then placed in an incubator maintained at
room temperature. The plates were regularly inspected for
change in behaviour of drop and searched for conditions
that favour crystalline phase.
Results and discussion
The 6X His-tagged protein was purified by employing a
two-step purification strategy to achieve high purity. The
presence of His tag was exploited using immobilized metal
affinity chromatography (IMAC) [22]. The rMMP was
significantly expressed in the host cells and was adsorbed
to Ni?2-NTA support and eluted by increasing the imid-
azole concentration to 250 mM. Approximately 80 % of
the rec-protein could be recovered by elution from the
column at pH 7.4. All purification steps were completed
within 120 min. A pre-equilibration buffer was utilized to
eliminate adsorption of weakly bound proteins. Significant
decrease in the purification time was observed by com-
pleting the purification in one step as compared to non-
tagged proteins. This affinity tag has previously been used
to facilitate purification of many fusion proteins expressed
in bacteria by employing a Ni 2? nitrilotriacetic acid (Ni2?-
NTA) affinity column [23].
GFC was performed immediately after the first step of
IMAC purification. Two main peaks: one major and one
small peak were obtained. The major peak obtained at
72 mL shows a split at tip of the peak (Fig. 1). Two dif-
ferent molecular species were eluted (between 64 and
0
100
200
300
400
500
600
700
0 20 40 60 80 100 120 140 160 180
mA
U
Fraction Volume (mL)
3
21
Fig. 1 Analysis of rMMP by gel filtration. The concentrated protein
sample previously purified by Ni2?-affinity chromatography was
loaded onto a Superdex 75 PG column. Chromatogram exhibits a split
peak indicating towards co-elution of different types of molecular
species as a mixture
Mol Cell Biochem
123
77 mL, represented by two split peaks at 72 and 74 mL, as
a mixture) at same intensity. Splitting of peak in Fig. 1
corroborates to elution of either different proteins as a
mixture or existence of different forms of same protein. To
understand composition of eluents, the fractions corre-
sponding to the peaks 1, 2 and 3 (as shown in Fig. 1) were
collected separately and analysed on SDS-PAGE for
difference in molecular weight. It was observed that peaks
1 & 2 correspond to rMMP (Fig. 2); however, peak 3 did
not show any bands in SDS-PAGE. The GF fractions were
further investigated for DLS. The radius distribution plot
reflected a largely homogeneous solution with a peak
around 10 nm, although a slight broadening was observed
(Fig. 3).
It was hypothesized based on observations such as the
presence of split peak on chromatogram, the presence of
protein bands (representing peaks 1 & 2) at same position
in SDS-PAGE and dynamic light scattering measurements
that the protein existed in dynamic equilibrium between
two different forms. Similar co-elution of mixtures of
coiled-coil proteins containing tyrosine kinases through a
GF column was reported by Cheng and colleagues [24]. In
97 kDa
66 kDa
44 kDa
29 kDa
20 kDa
14 kDa
1 2 3 4
Fig. 2 SDS-PAGE of the purified rMMP. Lane 4 contains molecular
weight standards (molecular weight in kDa given on the right). Lane 1
contains peak 1 obtained from gel filtration chromatography; lane 2
contains peak 2 obtained from gel filtration chromatography (Fig. 1);
lane 3 peak 3 (Fig. 1) and lane 4 molecular weight marker
Fig. 3 Radius distribution
curve obtained by performing
dynamic light scattering on
rMMP eluted from gel filtration
chromatography
-100
0
100
200
300
400
500
600
700
800
0 20 40 60 80 100 120 140 160 180
mA
U
Elution Volume (mL)
Fig. 4 Analysis of rMMP by gel filtration chromatography. Magne-
sium chloride was successfully added in buffer A to resolve elution of
mixture (as shown in Fig 1) and a sharp peak was obtained
Mol Cell Biochem
123
the present study, it was concluded following DLS exper-
iments, that rMMP existed in dynamic equilibrium between
two different forms. A dynamic equilibrium was also
reported between oligomeric forms by Bitan et al. [25],
where the authors observed broadening of curves in a
radius distribution plot from DLS.
Crystallisation requires protein sample to be in a mono-
disperse state. Previous efforts to crystallize rMMP weren’t
successful. Thus, it was attempted to resolve poly-disperse
system to a mono-disperse system. It is reported in literature
that metal ions stabilize native proteins and help them stay in
a single state restricting their dynamicity [26, 27].
To resolve unstable protein states, purification was per-
formed using buffers supplemented with MgCl2 (5 mM)
(Fig. 4). It was observed during GFC that MgCl2 resulted in
shift from a split peak towards a distinct sharp peak at 74 mL
of column volume. A calibration curve for the gel filtration
column was prepared using gel filtration standards (procured
from Bio-rad Inc.; 151-1901). The semi log curve prepared
from their elution data is presented in Fig. 5. As per the
calibration curve, the elution volume of the target protein
(74 mL) corresponds to log value of 1.585 giving a molec-
ular weight of approximately 38.5 kDa. This suggests that
rMMP may be eluting in the form of dimers. The peak
observed at 72 mL corresponds approximately to 44 kDa,
which is further away from theoretical monomeric weight of
20 kDa. Therefore, the presence of different oligomeric
formation may be ruled out and the presence of different
folded states may be suggested to be playing role. However,
addition of MgCl2 suggests stabilization of rMMP to a
uniform/single state. This was further confirmed by DLS
experiments which displayed resolution of radius of distri-
bution. No other curve was present in the radius distribution
plot, further indicating mono-dispersity of the purified pro-
tein (Fig. 6). The polydispersity index was below 0.15
indicating monodispersity of the sample preparation.
It was, therefore, concluded that MgCl2 stabilised the
target protein resulting in the sharp peak with a single
molecular species further confirmed by DLS studies
showing mono-dispersity. The protein sample prepared by
0
0.5
1
1.5
2
2.5
3
3.5
4
40 50 60 70 80 90 100 110 120
log
[Mr ]
Elution Volume (mL)
Fig. 5 Calibration curve of gel filtration column. The gel filtration
standards were run on GF column to prepare standard curve. The
following five standard proteins such as Thyroglobulin (bovi-
ne) = 670 kDa; c-globulin (bovine) = 158 kDa; Ovalalbumin
(chicken) = 44 kDa; Myoglobulin (horse) = 17 kDa and Vit
B12 = 1.35 kDa were run on the column
Fig. 6 Radius distribution
curve obtained by performing
dynamic light scattering on
rMMP eluted from gel filtration
chromatography. The
smoothening of curve is caused
by use of magnesium chloride in
elution buffer
Mol Cell Biochem
123
following above mentioned strategy was successfully used
to achieve crystallization and the crystals thus obtained
were diffracted up to 5 A resolution (data not shown). The
crystallization of the rMMP was obtained following
hanging drop using poly ethylene glycol 8000 as pre-
cipitant, 2-Methyl-2,4-pentanediol as co-precipitant and
magnesium chloride as additive at pH 7.0 (Fig. 7).
Such behaviour by rMMP can be understood by evalu-
ating the intramolecular ionic interactions. The sequence
contains 17.3 % of basic and 16.1 % of acidic amino acids
[28]. The spatial distribution of these residues in the
sequence allows i ? 3 and i ? 4 positioning in the helices
and that these ionic interactions can stabilize the helix. The
high content of charged residues can also result in the
intermolecular ionic interactions leading to oligomeric
forms. The molecular weight of the split peaks indicates
that they may be dimers of rMMP. The different elution
profiles may reflect the differences in the associations of
the monomeric form. These interactions are probably
charge mediated. The presence of MgCl2 can prevent this
by counter–ion interaction. This reduces the polydispersity
by reducing the intermolecular interactions. The helix
formation per se is not dependent on the side chain-side
chain interactions. The effect of MgCl2 on the stability of
helical coils has been observed earlier [29].
Finally, the purified protein exhibited a single band with
a molecular weight corresponding to *23 kDa estimated
by 12.5 % SDS-PAGE under denaturing conditions
(Fig. 8). The molecular weight of the purified protein was
further investigated by bioinformatics analysis (ProtoPa-
ram tool; www.web.expasy.org/protparam) which indi-
cated a theoretical molecular mass of 20.318 kDa.
Bioinformatics analysis also resulted in the discovery that,
rMMP exhibits homology to a coiled-coil ferritin-like
protein that protects DNA from oxidative damage [30].
Conclusion
The present study demonstrates that rMMP protein exists
transiently between its differently folded states. Addition of
MgCl2 to the elution buffer stabilizes the protein and helps it
remain in a mono-disperse state. The effect of MgCl2 was also
confirmed by DLS investigations. The purified sample is
subsequently suitable for crystallisation screening. The pres-
ent study establishes the significance of gel filtration chro-
matography as well as dynamic light scattering as important
techniques in characterization of dispersity of proteins.
Acknowledgments Authors thank all members of the University of
Queensland, Australia structural genomics group who inspired us to
work on this protein. MP thanks Deakin University for encouraging
collaboration with participating institutions. PP thanks DU for pro-
viding trainee scholarship to pursue partial work at bioprocessing
laboratory.
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