Biochemical Engineering Journal 128 (2017) 1218

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Design of bio-inspired silica-encapsulated protein A for improved

immunoprecipitation assays
Ki Sung Park, Mi-Ran Ki, Ki Baek Yeo, Jung Hoon Choi, Seung Pil Pack
Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-Ro, Sejong 30019, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Staphylococcus aureus Protein A (SpA) has a high afnity to the Fc region of antibodies (Abs), and
Received 25 March 2017 SpA-immobilized matrices are widely used for Ab purication or immunoprecipitation (IP) assays.
Received in revised form 3 August 2017 Here, we employed a bio-inspired silica-encapsulation method to improve the Ab-binding ability of
Accepted 29 August 2017
an SpA-immobilized matrix. Two silica-forming peptides (SFPs), namely R5 and EctP1, were separately
Available online 1 September 2017
introduced at the C-terminus of SpA to generate two recombinant fusion proteins (SpA-SFPs) with auto-
silicifying abilities. When SpA-SFPs were employed as Ab-binders on a solid surface (96-well plate), they
Keywords:
showed an effective Ab-binding ability and a better performance than intact SpA. A high binding ability
Staphylococcus aureus Protein A (SpA)
Biosilica
was observed even when an SFP-mediated SpA silica matrix (SpA-SFP@SiO2 ) was prepared. SpA-SFP@SiO2
Encapsulation showed a higher performance than commercially obtained SpA-Agarose particles and no loss of matri-
Immunoprecipitation (IP) ces. Moreover, in IP assays, SpA-SFP@SiO2 showed an approximately 300% higher precipitation of target
Silica-forming peptide (SFP) protein than the commercial SpA-Agarose product when a small amount of cell lysate was used. These
ndings demonstrated that SpA-SFP could be useful for the development of an efcient immunoassay
system.
2017 Elsevier B.V. All rights reserved.

1. Introduction
Antibodies (Abs) have been widely used for sensing or capturing
biomolecules in biotechnology and related elds. Abs have been
used for applications including the treatment of cancers, evalua-
tion of therapeutics, drug development, and bio-sensing systems
[1]. Furthermore, monoclonal Abs (mAbs) have become established
as a gold standard reagent in the biopharmaceuticals industry [2].
However, the purication of Abs is laborious and requires a robust
process with stringent quality control. In particular, the manu-
facture of mAbs involves multiple steps including expression in
mammalian cell culture and high-quality purication with virus
clearance [3]. Ab purication is typically achieved by afnity chro-
matography with Staphylococcus aureus Protein A (SpA) or G (SpG),
which have high afnities for binding to Abs [4]. SpA and SpG have
high afnities and specicities to the Fc domain of immunoglob-
ulins, and it is possible to employ these proteins for the one-step
purication of Abs [57].
SpA was identied as a 42-kDa cell surface protein in the cell
wall of S. aureus. Its potential function is for immune evasion by
Corresponding author.
E-mail address: spack@korea.ac.kr (S.P. Pack).
neutralizing Abs of the host immune system [810]. It can inter-
rupt the formation of a complex between Abs and the Fc domain
receptors on the surface of phagocytes by capturing the Fc domain
of Abs before they can interact with the receptors [10]. Because
of its high afnity for the Fc domain of Abs, SpA is commonly
used in vitro not only for Ab purication via afnity chromatog-
raphy [6], but also for immunoprecipitation (IP) assays with mAbs
to isolate and detect specic target proteins from cell lysates. In
addition, the co-IP method is used to provide direct evidence of
proteinprotein interactions either in vitro or in vivo [11]. Afn-
ity chromatography and IP assays have been commonly performed
with binding partners of IgGs, such as SpA and/or SpG, immobi-
lized on a solid matrix [7,12]. Commercially available kits typically
use these proteins immobilized on agarose beads (or patented
variants) via chemical cross-linking, enabling the production of a
precipitated bead-Ab-target protein complex [12,13]. An optimal
matrix for conjugated SpA or SpG requires appropriate physico-
chemical characteristics and the capacity to expose the conjugated
proteins on a high portion of the matrix surface area. Therefore,
the most important consideration of immobilization is to maximize
the amount of the afnity agent that can be exposed on the matrix
surface.
Our group has established a recombinant protein system for the
production of a biosilica matrix without chemical cross-linking via

http://dx.doi.org/10.1016/j.bej.2017.08.017
1369-703X/ 2017 Elsevier B.V. All rights reserved.
 K.S. Park et al. / Biochemical Engineering Journal 128 (2017) 1218
the spontaneous immobilization of biomolecules [14,15] that fea-
tures a low process cost, high loading capacity, and easy scale-up
process. Especially, biosilica nano/micro particles have attracted
growing attention as industrial materials because biosilica matri-
ces are novel organic-inorganic composites [16]. Silica particles
have good biomolecular properties including biocompatibility, and
their surfaces can be modied with a wide variety of functional
groups. They are suitable as carriers for drug delivery, scaffolds
for tissue engineering, and platforms for bio-sensing and imaging
[1621]. Most of all, biosilica matrices are promising support mate-
rials because of their highly porous structure, easy immobilization
procedure, and relatively high stability over a broad range of pH
and temperature conditions [22,23].
Some marine organisms, such as diatoms (silafns and
polyamines) and sponges (silicateins), produce biosilica for their
external supporting structure using particular proteins or pep-
tides [22,23]. Brott and co-workers [24] reported a short (19 amino
acid) R5 peptide derived from the Cylindrotheca fusiformis silafn-1
protein that catalyzes biosilica formation. Most biomimetic biosil-
ica formation is mediated by R5 and polyamines in addition to
other amine-bearing biomolecules such as lysine-rich proteins [25].
Recently, our group found several novel silica-forming peptides
(SFPs), for example, EctP1 and EctP2, which are R5-like short pep-
tides that mediate the formation of biosilica[26]. In addition, we
tried to improve the stability and reusability of endoglucanase
(EG) via a bio-inspired silica-encapsulation strategy (EG-SFP@SiO2 )
[27] or to improve the sustained release of drugs using a biosilica-
encapsulated matrix [28]. In this study, we designed a recombinant
fusion protein, SpA-SFP, by genetically introducing SFP at the
C-terminus of SpA to develop a new SpA-immobilized matrix
with a high Ab-binding ability. We found that SpA-SFP and its
SFP-mediated SpA silica matrix (SpA-SFP@SiO2 ) showed better per-
formance than commercially available SpA-immobilized matrix
reagents in IP assays.
2. Materials and methods
2.1. Protein expression
The plasmids for SFP-fused recombinant SpA (SpA-R5 and SpA-
EcptP1) and wild-type SpA (Fig. S1 of Data in Brief) were each
transformed into Escherichia coli strain DE3 BL21 (Agilent Technolo-
gies, Santa Clara, CA). The protein expression conditions of SpA-R5,
SpA-EcptP1, and SpA were optimized by expression testing with an
incubation temperature shift and induction by isopropyl 1-thio--
d-galactopyranoside (IPTG). The E. coli cells were grown at 37 C
to an absorbance (A600 ) of 0.50.7, and then induced with IPTG
(0.1 mM) and incubated overnight at 20 C.
2.2. Protein purication
The grown cells of recombinant E. coli were harvested by cen-
trifugation at 5,000 g for 20 min at 4 C. The pellet was frozen
at 70 C for at least 2 h and was then dissolved with 50 mM
NaH2 PO4 , 300 mM NaCl, and 1% glycerol, pH 7.5 buffer contain-
ing 1 mM phenylmethanesulfonyl uoride (PMSF). The cells were
lysed by ultra-sonication with a Digital Sonier 250 (Branson Ultra-
sonics, Danbury, CT) at 40% amplitude for 10 min (1 s sonication
and 5 s intervals). The lysed cells were pelleted by centrifuga-
tion at 14,000 g for 40 min at 4 C to collect the supernatant
containing the protein of interest. The recombinant SpA proteins
were puried by HisPurTM Cobalt resin (Thermo Fisher Scientic,
Waltham, MA) for afnity chromatography with a buffer composed
of 50 mM NaH2 PO4 , 300 mM NaCl, 1% glycerol, pH 7.5, and 100 mM
imidazole. Puried proteins were analyzed by sodium dodecyl
13
Fig. 1. Protein expression analysis by western blot.
To conrm the yield and molecular weight of each puried Staphylococcus aureus
Protein A (SpA) variant, we performed SDS-PAGE and western blots. The predicted
molecular weight of each protein was SpA-R5: 35.329 kDa, SpA-EctP1: 35.610 kDa,
and SpA: 32.949 kDa. All SpAs were highly expressed and had a high solubility in
Escherichia coli DE3 cells, and the correct molecular weight was obtained for each of
the puried proteins by western blotting.
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and west-
ern blotting (Fig. 1). The concentration of protein was measured
using the Bradford assay (Thermo Fisher Scientic) by determining
the absorbance at 595 nm.
2.3. Silicication and immobilization
Bio-silicication mediated by SFPs (R5 and EctP1) was per-
formed by reaction with tetramethyl orthosilicate (TMOS),
phosphate buffer, and SpA-fused SFPs (SpA-SFPs). SpA-SFPs were
puried using a buffer comprised of 50 mM NaH2 PO4 , 300 mM
NaCl, and 1% glycerol, pH 7.5 for bio-silicication. The puried
proteins were concentrated to 1 mg/mL using an Amicon Ultra
Centrifugal Filter (0.5 mL, 10 K, Millipore, Billerica, MA) follow-
ing the manufacturers instructions. To facilitate the use of the
puried proteins in downstream applications, excessive residual
imidazole was removed from the elution buffer. Each puried SpA-
SFP (75 g/mL) was incubated with 100 mM TMOS and 100 mM
NaH2 PO4 buffer with shaking at 25 C for 1 h. To determine the
efciency of immobilization, the formed silica was pelleted by cen-
trifugation at 14,000 g for 10 min at 4 C to remove non-reacted
agents, and the concentration of non-immobilized protein in the
supernatant was measured by the Bradford assay (Thermo Fisher
Scientic).
2.4. Immunoassay in 96-well microtiter plate
The Ab-binding characteristics of the puried SpAs (SpA-R5,
SpA-EctP1, and SpA) were evaluated by an immune assay using
a 96-well microtiter plate. The microtiter plate, Nunc-ImmunoTM
MicroWellTM 96-well solid plate (Nalgen Nunc International, Pen-
eld, NY), has a strong ability to adsorb protein and is widely
used for direct enzyme-linked immunosorbent assays (ELISA). The
prepared SpAs were adsorbed to the plate at 4 C overnight and
unbound SpAs were removed by washing with 1 Tris-buffered
saline containing 0.1% Tween 20 (TBST) at least 3 times. Blocking
with 1% bovine serum albumin (BSA) in 1 TBST was performed for
1 h, followed by washing with 1 TBST at least 3 times. An near-
infrared (NIR) uorescence dye IRDye 800CW-labeled Ab (IRDye
800CW, 0.5 mg/mL; LI-COR Biosciences, Lincoln, NE) was employed
after serial dilution from 1:100 to 1:6400 and the signal of IRDye
800CW-labeled Ab (NIR-labeled Ab) was detected using an imaging
scanner (Odyssey CLx; LI-COR Biosciences).
2.5. IP assays
SpA-SFP@SiO2 was pelleted to remove non-reacted TMOS by
centrifuging at 14,000 g for 10 min at 4 C and then washed with
100 mM NaH2 PO4 buffer 3 times. Subsequently, the silica pellet
was suspended in 100 mM NaH2 PO4 buffer for downstream analy-
sis. Twenty microliters of SpA-SFP@SiO2 sample was resuspended
with 1 TBST to a total volume of 100 L for immunoprecipitation
14 K.S. Park et al. / Biochemical Engineering Journal 128 (2017) 1218
and blocked with 1% BSA in 1 TBST for 1 h, followed by washing
at least 3 times with 1 TBST. To investigate the binding ability of
SpA-SFP@SiO2 with Abs, 100 L of NIR-labeled Ab (IRDye 800CW,
0.5 mg/mL) was employed at a 1:100 ratio for 2 h. After the precip-
itation step and each wash step, the NIR uorescence signal was
detected using an IR imaging scanner (Odyssey CLx). For compari-
son with a typical commercial product, we purchased SpA-Agarose
beads (0.5 mL agarose in 2 mL phosphate-buffered saline [PBS];
Santa Cruz Biotechnology, Dallas, TX) and performed IP assays using
the same protocol described above.
2.6. IP of green uorescent protein (GFP) in cell lysates
To determine the expression of GFP in cell lysates, mouse anti-
GFP mAb (200 g IgG2a in 1 mL PBS with < 0.1% sodium azide and
0.1% gelatin) and SpA-Agarose beads (0.5 mL agarose in 2 mL PBS)
were purchased from Santa Cruz Biotechnology. Prior to IP assays,
SpA-SFP@SiO2 samples were prepared and 100 L of cell lysate
diluted 1:10 or 1:1000 was prepared from a 200-mL culture of
recombinant GFP-expressing E. coli. Each cell lysate sample was
incubated with 100 L of anti-GFP mAb (0.2 mg/mL) for 2 h at
25 C. Then, target binders (SpA-SFP@SiO2 or SpA-Agarose) were
incubated with the sample with shaking for 2 h, after which the
resultant target binder-GFP complexes were pelleted at 14,000 g
to remove unreacted agents (which remained in the supernatant)
and washed 3 times with 1 TBST buffer. The GFP uorescence sig-
nal was measured for GFP detection. To investigate the performance
of each SpA-matrix in IP assays, we employed an NIR-labeled Ab
and measured the NIR uorescence signal using an imaging scanner
(Odyssey CLx).
3. Results and discussion
3.1. SFP-mediated encapsulation of SpA-SFPs on biosilica
Two SFP-fused SpA variants (SpA-SFPs) were prepared to
construct SpA-immobilized silica matrices by genetically intro-
ducing one SFP, either R5 (SSKKSGSYSGSKGSKRRIL) or EctP1
(SSRSSSHRRHDHHDHRRGS), at the C-terminus of SpA (Fig. S1 of
Data in Brief). Each SpA-SFP was expressed through the heterolo-
gous recombinant production in E. coli and obtained by His6 x Tag
purication system (Fig. 1). Silica encapsulation (bio-silicication)
mediated by SFP (R5 or EctP1) was carried out in a buffer com-
prised of 100 mM TMOS and 100 mM phosphate, pH 8.0 for 1 h at
25 C. SpA-SFPs were successfully immobilized on biosilica to pro-
duce SpA-SFP@SiO2 with a short reaction time of silicication and
over 90% yield (Fig. S2 of Data in Brief). Wild-type recombinant
SpA, which was used as a control, also showed silicication with
over 90% yield.
However, different shapes of silica matrices were observed by
scanning electron microscopy (SEM) as shown in Fig. 2. SFP (R5
or EctP1)-mediated silica matrices are composed of sphere-shaped
nanoparticles (Fig. 2A and B). In contrast, no SFP-mediated silica
matrix, wild type SpA@SiO2 , formed a sol-gel state and showed a
large size clump-like morphology (Fig. 2C). This is not based on the
peptide-mediated or templated silicication. The SpA might be
simply attached during the sol-gel formation so that a high-amount
release of SpA was observed from the silica matrices (See details
in Section 3.3). The simply-attached SpA might be easily released
from SpA@SiO2 , while SpA-SFPs were immobilized on the biosilica
matrices. The effect of the protein isoelectric point (pI) or negative
charge of SpA may be involved. The pI values of SpA (4.5943), SpA-
R5 (5.1272), and SpA-EctP1 (5.4388) were slightly different, but
their negative charges were clearly different between the wild-type
and SpA-SFPs (SpA: 10.5, SpA-R5: 4.5, SpA-EctP1: 5.0). SpA-
SFPs may have more favorable properties for silica formations.
Since sphere-shaped structures of SpA-SFP@SiO2 can effectively
provide large surface area to the immobilized proteins, more active
sites of SpA can be displayed. Therefore, they are more likely to
facilitate proteinprotein interactions and have a high afnity for
binding to one another. In addition, SFP-mediated silicication is
involved at the C-terminus of fusion protein. In case of SpA-SFP (Fig.
S3 of Data in Brief), silica formation reactions occurred mostly dis-
tant from the binding sites of SpA, which were maintained as active
forms. Moreover, the SpA-SFP is strongly attached to biosilica at the
C-terminus, so that large portion of SpA-SFP@SiO2 might expose
their binding sites effectively. These results proposed that SFP-
mediated biosilica formed more active-SpA-immobilized matrices
that may be suitable as Ab binders for immunoprecipitation assays.
3.2. Ab-binding abilities of spA-R5, spA-EctP1, and SpA on a solid
surface
We investigated the Ab-binding abilities of the prepared SpA-
SFPs (SpA-R5 and SpA-EctP1) on a solid surface and compared
them with that of wild-type SpA. We measured the amounts of
the Abs bound by SpA-SFPs or SpA (not on SFP-mediated silica
matrix) adsorbed on the solid surface of a 96-well immunoassay
plate, which is manufactured for ELISA. This experiment is intended
to show no difference in the Ab-binding abilities between SpA
and SpA-SFPs even on a solid surface. Here, large biomolecules
( > 10 kDa) that possess ionic groups are easily adsorbed through
the ionic interactions with surface wells of the plate.
Each kind of SpA was adsorbed on the well surface of the plate
and unattached SpA was removed by washing with TBST buffer at
least 3 times. Then, the SpA-adsorbed wells were incubated with
an NIR-labeled Ab and the NIR uorescence signal was measured
to estimate the amount of the bound Ab. We investigated the limit
of detection (LOD) of each SpA by applying different concentra-
tions of Ab (Fig. 3). As shown in Fig. 3A, SpA-R5, SpA-Ectp1, and
SpA were adsorbed on the plate and then incubated with serially
diluted samples (from 1:100 to 1:6400 dilution) of NIR-labeled Ab
(0.5 mg/mL). At low concentrations (1 g/mL) of SpA and SpA-R5,
no signal was detected, which means that the adsorbed amount
of SpA is not sufcient for the meaningful binding of the Ab. How-
ever, in the case of EctP1-SpA (1 g/mL), a weak signal was detected
only at 1:100 to 1:200 dilutions of the samples (Fig. 3A-b). When
we measured the signal intensities produced by the different con-
centrations of Ab before washing the unbound Ab from the plate, a
clear NIR uorescence signal was observed, indicating that the NIR
dye signal is detectable (Fig. 3B). Since a 1 g/mL concentration was
too low for SpAs to bind the Ab, 10-fold higher concentrations of
SpAs (10 g/mL) were adsorbed on the plate, followed by incuba-
tion with the NIR-labeled Ab, and then the NIR uorescence signals
were analyzed as shown in Fig. 3C. To determine the amount of Ab
that was non-specically adsorbed on the surface of the microtiter
plate (i.e., showing no interaction with SpA), empty wells without
adsorbed SpA were used as a control and were only blocked with
BSA. No NIR uorescence signal was detected as shown in Fig. 3C-d.
The results of Fig. 3 demonstrated that all the SpAs (SpA-R5, SpA-
EctP1, and SpA) showed a sufcient afnity to bind with the Fc
region of Abs. In fact, SpA-SFPs are more likely to get adsorbed on
the solid surface than SpA. The slightly higher adsorbing amount
was observed for SpA-EctP1 than the other two.
3.3. Ab-binding ability of biosilica-encapsulated SpAs
Two kinds of SpA-SFP@SiO2 matrices (R5 or EctP1) were pre-
pared through the SFP-mediated biosilica-encapsulation method,
in which a silica matrix with SFP-fused protein is spontaneously
 K.S. Park et al. / Biochemical Engineering Journal 128 (2017) 1218 15

Fig. 2. Scanning electron microscopy (SEM) images of SpA-immobilized biosilica matrix.

A: SpA-R5@SiO2 , B: SpA-EctP1@SiO2 , C: SpA@SiO2, wild-type SpA without peptide fusion. Scale bars are 2 m.

Fig. 3. Binding activity test of SpAs by immunoassay in a microtiter plate.

Serial dilutions (1:100 to 1:6400) of an NIR-labeled antibody (Ab) were incubated in a microtiter plate containing adsorbed SpA. A: 1 g/mL of each SpA was adsorbed on the
microtiter plate surface overnight. B: Total NIR uorescent signal of labeled Ab before washing steps. C: 10 g/mL of each SpA was adsorbed on the plate surface overnight.
Lane a) SpA-SFP (R5), b) SpA-SFP (EctP1), c) SpA, d) no treatment control (NTC). After SpA adsorption, all wells were blocked with 1% bovine serum albumin (BSA) to reduce
non-specic binding before the Ab incubation step. Schematic illustration of SpAAb interactions on the microtiter plate surface was depicted. The adsorbed SpA binds to
the Fc region of the Ab and can be detected using an NIR-labeled Ab.

SpA@SiO2 (control), we carried out severe washing steps by vor-

Fig. 4. Releasing test of SpA-SFP or SpA from biosilica matrices (SpA-R5@SiO2 , SpA-
EctP1@SiO2 ,and SpA@SiO2 ).
Released SpAs from biosilica matrices were adsorbed on the solid phase immunoas-
say plate and they were incubated with a 1:1000 diluted NIR-labeled Ab to measure
the amount indirectly. SUP: Supernatant obtained by centrifugation after biosilica
preparations. The NIR uorescence signal was measured in each washing steps. All
wells of plate were blocked with 1% bovine serum albumin (BSA) to reduce non-
specic binding before the Ab incubation step. Fluorescence intensities of Ab were
analyzed by densitometry of scanned images using ImageJ.
formed. We rst checked out whether SpA-SFPs are well encapsu-
lated into the resultant biosilica matrix. The washing solutions of
SpA-immobilized biosilica were analyzed though the solid phase
immunoassay to estimate the amount of SpA or SpA-SFP released
from the biosilicas (Fig. 4). For two kinds of SpA-SFP@SiO2 and
texing and sonication. The released SpA or SpA-SFP in the washing
solution was adsorbed on the immunoassay plate and the amounts
of the adsorbed amounts were measured by NIR-labeled Ab. As
shown in Fig. 4, in each washing step, the released amount of
SpA from SpA@SiO2 is approximately 5 times higher than those
from SpA-SFP@SiO2 . The results indicated that SpA-SFPs in SpA-
SFP@SiO2 are better encapsulated than SpA in SpA@SiO2 .
We compared the performance of IP using SpA-SFP with that
of IP using a commercial product, SpA-Agarose beads (Santa Cruz
Biotechnology). The IP technique is commonly used to isolate
and purify target proteins from cell lysates with specic Abs.
We prepared two kinds of SpA-SFP@SiO2 matrices through the
SFP-mediated biosilica-encapsulation method. Each sample was
blocked with BSA to reduce non-specic binding and then incu-
bated with a 1:200 dilution of an NIR-labeled Ab (0.5 mg/mL) for
2 h at 25 C. After washing 3 times in TBST, the signal of the NIR
uorescence dye was measured using an IR imaging scanner to
estimate the efciency of SpA-SFP@SiO2 binding to the Abs. We
measured the NIR uorescence signal after each step (separation of
the supernatant, each of 3 washing steps, and the nal precipitation
of the matrices) to estimate the Ab-binding ability of the SpA-
SFP@SiO2 matrices (Fig. 5). In both types of SpA-SFP@SiO2 matrices,
a strong Ab-binding afnity was observed and the assay process
was performed without any loss of particles during washing steps.
In the case of SpA-Agarose beads (Santa Cruz Biotechnology), a
similar Ab-binding ability was observed, but some losses of beads
16 K.S. Park et al. / Biochemical Engineering Journal 128 (2017) 1218

Fig. 5. Binding activity test of SpAs to immunoglobulins after SFP-mediated encapsulation for immunoprecipitation (IP).
A: Blocking with 1% BSA to reduce non-specic binding. R5: SpA-R5@SiO2 , EctP1: SpA-EctP1@SiO2 , Agarose: SpA-Agarose bead(commercial product) as a control. B: Silicied
SpA-SFPs and SpA-Agarose were incubated with a 1:200 diluted NIR-labeled Ab. The NIR uorescence signal was measured at 800 nm wavelength after each experimental
step (removal of unbound agents, 3 washing steps, and nal IP assay). The binding activities of SpA-SFP@SiO2 and SpA-Agarose were analyzed by densitometry of scanned
images using ImageJ.

were observed during the 3 washing steps. As shown in Fig. 5,
SpA-SFP@SiO2 particles had a higher efciency for IP than the
commercial SpA-Agarose beads. From our observations, the use
of SpA-SFP@SiO2 instead of SpA-Agarose beads in IP assays may
improve the reliability of the assays.
3.4. IP of a target protein (GFP)
Finally, we performed IP assays using SpA-SFP@SiO2 to precip-
itate an Ag-Ab complex. GFP and an anti-GFP mAb were used as
the model molecules for a target antigen (Ag) and an Ab with afn-
ity to that Ag, respectively. IP assays were performed as follows.
SFP-mediated SpA-silica matrices (SpA-SFP@SiO2 ) were prepared
by incubating SpA-SFP with TMOS for 1 h. GFP and anti-GFP mAb
were incubated for 2 h at 25 C to induce Ag-Ab complex formation.
Ag-Ab complex solution was incubated with SpA-SFP@SiO2 for 2 h.
Precipitates of Ag-Ab complex were obtained by centrifugation at
14,000 g for 10 min at 4 C. Finally, the presence of the Ag (GFP)
was conrmed by SDS-PAGE and if necessary, western blots were
also performed.
We compared the capacities of SpA-SFP@SiO2 and SpA-Agarose
beads for use in IP by testing 1:10 and 1:100 dilutions of cell
lysate containing GFP from a 200-mL culture broth of recom-
binant E. coli (Fig. 6). With the 1:100-diluted cell lysate, both
kinds of SpA-SFP@SiO2 could mediate effective IP results including
the purication of a large amount of target protein (GFP). Under
the same conditions, SpA-Agarose could not well precipitate GFP,
with quite low detection of GFP by western blot analysis. When
SpA-Agarose was used for IP with the 1:10-diluted cell lysate, it
precipitated a detectable amount of GFP, but the amount was still
lower than that obtained using SpA-SFP@SiO2 . Considering the
results from western blots after IP assays, SpA-SFP@SiO2 showed an
approximately 300% higher precipitation of GFP than the commer-
cially obtained SpA-Agarose beads. These ndings demonstrated
that both types of SpA-SFP@SiO2 have a high Ab-binding capac-
ity and a high performance for the recovery of target protein in IP
assays.
Thorough the SFP-mediated biosilicication, high amount of
SpA can be immobilized (over 90%) at biosilica. In addition, sphere-
shaped biosilica can effectively provide large surface area to the
immobilized SpA. Several previous reports showed that biosilica
has a high porosity as well as a large surface area [1421]. Thanks
to high-amount immobilization and high porosity/large surface of
biosilica, SpA-SFP@SiO2 is more likely to facilitate proteinprotein
interactions and have a high afnity for binding to one another.
Moreover, SFP-mediated SpA immobilization is carried out under
mild and ambient conditions, in which no chemical cross-linking
reaction is involved. In contrast, SpA-Agarose bead(the commercial
one) was manufactured by chemical crosslink process. Therefore,
SpA-SFP@SiO2 matrices are more likely to exist as more active
forms and show higher Ab-binding capacity than SpA-Agarose
bead.
4. Conclusions
SpA-immobilized matrices are widely used for the purication
of Ab or the immunoassay of target proteins because of their high
afnity to the Fc region of Abs. In this study, an SFP-mediated
 K.S. Park et al. / Biochemical Engineering Journal 128 (2017) 1218 17

Fig. 6. IP assay test using Anti-GFP-IgG/SpA-SFP@SiO2 complex.

A: Anti-GFP IgG2 from mouse was used as a primary antibody to recover GFP by IP and an NIR uorescence-labeled secondary Ab was used to produce a detectable signal for
western blot analysis. We tested the IP procedure using two SpA-SFP@SiO2 and SpA-Agarose (commercial product as a control) with GFP-expressing E. coli cell lysates diluted
at 1:100 or 1:10. Lane R5: SpA-R5@SiO2 , EctP1: SpA-EctP1@SiO2 , Agarose: SpA-Agarose. The results showed clear bands for GFP at the correct molecular weight (predicted
molecular weight, 27 kDa), demonstrating that IP with SpA-SFP@SiO2 was able to isolate a target protein from cell lysates. B: The isolation efciencies of SpA-R5@SiO2,
SpA-EctP1@SiO2 and SpA-Agarose were analyzed by band densitometry of western blot images using ImageJ.

Fig. 7. Overview of IP assay using SFP-mediated biosilica (SpA-SFP@SiO2 ).

silica-encapsulation method was employed for the design of new says (Fig. 7). The preparation of SpA-SFP@SiO2 is easy and scalable.
SpA-immobilized matrices. SFP peptides (R5 and EctP1) were Biosilica materials with such high Ab-binding ability can be use-
separately introduced genetically at the C-terminus of SpA to gen- fully employed for various applications such as biosensors, drug
erate two fusion proteins, SpA-SFPs (SpA-R5 or SpA-EctP1). When delivery, and cell/tissue scaffolds.
employed on a solid surface, the SpA-SFPs showed a higher Ab-
binding ability than intact SpA. Similar results, higher Ab-binding
Acknowledgments
performances, were observed when they were encapsulated in a sil-
ica matrix (SpA-SFP@SiO2 ). Moreover, in IP assays, SpA-SFP@SiO2
This work was supported by the Basic Core Technology Develop-
matrices showed an approximately 300% higher precipitation of a
ment Program for the Oceans and the Polar Regions of the National
target protein than commercially obtained SpA-Agarose beads, and
Research Foundation (NRF) funded by the Ministry of Science, ICT
the SpA-SFP@SiO2 matrices recovered detectable amounts of tar-
& Future Planning, Korea (NRF-2015M1A5A1037054) and a Marine
get protein even when a small amount of cell lysate sample was
Biomaterials Research Center grant from the Marine Biotechnology
used. These ndings demonstrated that SpA-SFPs could be useful
Program funded by the Ministry of Oceans and Fisheries, Korea.
biomolecules for the development of more efcient immunoas-
This work was also supported by the Energy Efciency & Resources
18 K.S. Park et al. / Biochemical Engineering Journal 128 (2017) 1218
Core Technology Program (20142020200980) of the Korea Institute
of Energy Technology Evaluation and Planning grant funded by the
Ministry of Trade, Industry and Energy, Korea and BK21 plus.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bej.2017.08.017.
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