Protein Expression and Purification 71 (2010) 91–95
Contents lists available at ScienceDirect
Protein Expression and Purification
journal homepage: www.elsevier.com/locate/yprep
Single-step affinity purification of recombinant proteins using the silica-binding
Si-tag as a fusion partner
Takeshi Ikeda a,b,*, Ken-ichi Ninomiya a, Ryuichi Hirota a, Akio Kuroda a,b
a
b
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan
Research Institute for Nanodevice and Bio Systems, Hiroshima University, 1-4-2 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8527, Japan
a r t i c l e
i n f o
Article history:
Received 20 October 2009
and in revised form 16 December 2009
Available online 23 December 2009
Keywords:
Affinity purification
Affinity tag
Disordered protein
Silanol group
Silica-binding protein
Si-tag
a b s t r a c t
We previously reported that a silica-binding protein, designated Si-tag, can be used as a fusion tag to
immobilize functional proteins on silica surfaces. In this study, by taking advantage of the strong affinity
of Si-tag for silica, we developed a single-step purification method for Si-tagged fusion proteins. We utilized unmodified bare silica particles as a specific adsorbent and a high concentration of MgCl2 solution as
an elution buffer. A fusion protein of Si-tag and immunoglobulin-binding staphylococcal protein A, designated Si-tagged protein A, was recovered with a purity of 87 ± 3% and yield of 84 ± 4% from a crude
extract of recombinant Escherichia coli. The simplicity of our method enables rapid, cost-effective purification of Si-tagged fusion proteins. We also discuss the mechanism of binding and dissociation of Si-tag
and silica surfaces, and we suggest that the unusual basicity and disordered structure of the Si-tag polypeptide play important roles in the binding to silica.
! 2009 Elsevier Inc. All rights reserved.
Introduction
Affinity tags are highly efficient tools for protein purification.
They allow the purification of nearly any protein without any prior
knowledge of its biochemical properties. A variety of proteins, domains, or peptides have been used as affinity tags to facilitate the
purification of proteins of interest from crude extracts (reviewed in
Refs. [1–4]). For example, a hexahistidine tag (His-tag)1 is widely
used for affinity purification of recombinant proteins by immobilized
metal–ion affinity chromatography. This technique is based on the
affinity of neighboring histidine residues for chelated transition
metal ions (e.g., Ni2+, Co2+, and Cu2+) immobilized on a chelating
resin such as a Ni2+–nitrilotriacetic acid (Ni2+–NTA) resin [5,6].
Glutathione S-transferase and maltose-binding protein are also used
as affinity tags, with their biological binding partners (glutathione
and amylose, respectively) as the ligand [7,8].
Recently, we found that bacterial ribosomal protein L2 binds
strongly to silica surfaces [9]. The binding of L2 fusion proteins to silica surfaces does not require chemical modification, pre-treatment,
or any specific conditions. L2 fusion proteins can be bound to a silica
surface simply by mixing them with or spotting them on a target
* Corresponding author. Address: Department of Molecular Biotechnology,
Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1
Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan. Fax: +81 82 424
7047.
E-mail address: ikedatakeshi@hiroshima-u.ac.jp (T. Ikeda).
1
Abbreviations used: CBB, Coomassie brilliant blue; His-tag, hexahistidine tag; IgG,
immunoglobulin G; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel
electrophoresis.
1046-5928/$ - see front matter ! 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.pep.2009.12.009
even in the presence of high concentrations of salt and detergent
in the protein solution. We have designated the silica-binding protein L2 as ‘‘Si-tag” when used as a fusion tag to immobilize functional
proteins on silica surfaces [9]. Si-tag can be functionally fused to
either terminus of the target protein. We further demonstrated that
Si-tag is a useful fusion tag for oriented immobilization of functional
proteins, which achieves good steric accessibility of their active sites
to target molecules in solution and enhances interaction between
immobilized proteins and target molecules [10]. Si-tag should be a
powerful tool for developing silicon-based biodevices, such as nanowire field effect transistors [11,12] and silicon-based optical sensors
[13], because it can be used to directly functionalize silica surfaces
with functional proteins [14].
The high affinity of Si-tag for silica also prompted us to develop
a new affinity purification method for Si-tagged fusion proteins. In
this study, we developed a single-step purification method, wherein unmodified bare silica particles and MgCl2 are used as a specific
adsorbent and eluent, respectively. By using this method, Si-tagged
fusion proteins can be purified with high yield in an inexpensive
way. We also discuss the mechanism of binding and dissociation
of the Si-tag polypeptide and silica surfaces.
Materials and methods
Materials
Silica particles (a-quartz) with a diameter of approximately
0.8 lm were purchased from Soekawa Chemical Co., Ltd. (Tokyo,
Japan) and were used without any pre-treatment. The packed
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T. Ikeda et al. / Protein Expression and Purification 71 (2010) 91–95
wet volume of the particles was approximately 1.0 ml per gram dry
weight. Because Si-tag also binds to glass surfaces [9], all protein
manipulations were carried out using polypropylene tubes and
tips. All chemicals used in this study were of analytical grade.
Bacteria, plasmids, and growth conditions
The pET-21b-derivative plasmid, pET-SpA-Sitag, was constructed previously [10]. This plasmid carries the gene encoding
the fusion protein of immunoglobulin-binding staphylococcal protein A and Si-tag with a C-terminal His-tag (Si-tagged protein A).
Escherichia coli Rosetta(DE3)pLysS (Novagen, Madison, WI, USA)
was used as a host for the plasmid and was grown in 2 ! YT
medium [15] at 37 "C. When necessary, ampicillin (100 lg/ml)
and chloramphenicol (10 lg/ml) were added to the medium. When
the optical density at 600 nm reached 0.5, 1 mM isopropyl-b-Dthiogalactopyranoside was added to the medium. After another
6 h of cultivation, cells were harvested by centrifugation and then
stored at "80 "C until use.
Dissociation of Si-tag from silica
Recombinant Si-tag (273 aa, corresponding to 30 kDa) was expressed alone (i.e., not fused to another protein) and purified by
cation exchange chromatography as described previously [10]. Silica particles (10 mg) were mixed with 10 lg of the purified Si-tag
in 1 ml of binding buffer (25 mM Tris–HCl buffer [pH 8.0] containing 2 M NaCl and 0.5% [v/v] Tween 20) and incubated with rotary
mixing for 30 min at 4 "C. The silica particles with the bound Sitag were collected by centrifugation at 5000g for 2 min and
washed twice with 1 ml of wash buffer (25 mM Tris–HCl buffer
[pH 8.0] containing 0.5 M NaCl and 0.5% [v/v] Tween 20). To identify the conditions for dissociation of Si-tag from silica, the particles were suspended and incubated in various solutions for
10 min. The particles were collected again and washed with 1 ml
of wash buffer. After the supernatant was carefully removed, the
Si-tag still bound to the particles was released by boiling in Laemmli sample buffer [16] for 5 min and was analyzed by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE;
12.5%).
Affinity purification using silica particles
Recombinant E. coli cells (typically 1–1.5 g wet weight) expressing Si-tagged protein A were suspended in 10 ml of binding buffer.
The cells were disrupted by sonication with a Digital Sonifier 450
(Branson Ultrasonics Corporation, Danbury, CT, USA) and centrifuged at 40000g for 20 min to remove cell debris. The supernatant
was incubated with silica particles (approximately 1.2 g dry weight
of silica per gram wet weight of cells) with rotary mixing for
30 min at room temperature. The particles were collected by centrifugation at 5000g for 2 min and washed twice with 10 ml of
wash buffer. Next, the particles were suspended in a small volume
(approximately 500 ll) of wash buffer and further suspended in
5 ml of 50 mM Tris buffer containing 2 M MgCl2 (pH #8)2. After a
10-min incubation, the suspension was centrifuged at 5000 ! g for
2 min. Next, the supernatant containing the released proteins was
collected and dialyzed against 25 mM Tris–HCl buffer (pH 8.0) containing 0.5% Tween 20 to remove MgCl2, which interferes with the
migration of proteins on SDS–PAGE.
2
The pH of the solution was not adjusted with acid or base because the pH was
approximately 8 when Tris base and MgCl2$6H2O were dissolved at the above
concentrations in distilled water and because further addition of base caused
precipitation.
His-tag purification
Si-tagged protein A (containing a C-terminal His-tag) was also
purified by immobilized metal–ion affinity chromatography on a
HisTrap HP column (7 ! 25 mm) with an ÄKTApurifier system
(GE Healthcare UK Ltd., Buckinghamshire, UK) according to the
manufacturer’s recommendations. Proteins were eluted with a linear gradient from 20 to 500 mM imidazole in 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl.
Protein assays
Protein concentrations were determined using Bio-Rad protein
assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as the standard. The purity of the proteins
was analyzed by SDS–PAGE (12.5%) and Coomassie brilliant blue
(CBB) staining, followed by densitometric analysis with ImageJ
software, version 1.41 [17].
Silica- and immunoglobulin G-binding (IgG-binding) assay of Sitagged protein A
Silica particles (2 mg) were mixed with 0.7 lg of Si-tagged protein A, 2 lg of mouse IgG2a, or both in 1 ml of wash buffer and then
incubated with rotary mixing for 30 min at 4 "C. The silica particles
were collected by centrifugation at 5000g for 2 min and washed
twice with 1 ml of wash buffer. The proteins that bound to the particles were released by boiling in Laemmli sample buffer [16] for
5 min and were analyzed by SDS–PAGE (12.5%).
Results
Primary structure of Si-tagged protein A
In this study, we used a fusion protein of Si-tag and immunoglobulin-binding protein A as a model protein. Protein A is a cell
wall component of the bacterium Staphylococcus aureus that binds
specifically to many mammalian immunoglobulins, most notably
IgG. The fusion protein, Si-tagged protein A, can be used for oriented immobilization of antibodies on a silicon wafer [10]. The
structural genes for immunoglobulin-binding domains of protein
A (spa) and for Si-tag (E. coli ribosomal protein L2; rplB) were
amplified by PCR. The resultant PCR products were inserted together into a C-terminal His-tag plasmid pET21-b via the appropriate restriction sites [10]. The constructed gene encodes a 541-aa
fusion protein composed of four immunoglobulin-binding domains
of protein A (domains E, D, A, and B [18]) and Si-tag, followed by a
C-terminal His-tag (Fig. 1).
Affinity purification of Si-tagged protein A using silica particles as an
adsorbent
Si-tagged protein A was expressed in E. coli Rosetta(DE3)pLysS
under the control of the T7 promoter of the pET vector. The recombinant protein was expressed as a soluble form in the cytosolic
fraction of E. coli (Fig. 2, lane 1). To purify Si-tagged protein A from
Fig. 1. Schematic representation of the primary structure of Si-tagged protein A.
The recombinant protein contains four immunoglobulin-binding domains of
protein A (domains E, D, A, and B), followed by Si-tag and His-tag.
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T. Ikeda et al. / Protein Expression and Purification 71 (2010) 91–95
Table 1
Si-tag purification of Si-tagged protein A from E. colia.
Step
Total
Amount of Si-tagged Purity Purification Yield
protein (mg) protein A (mg)b
(%)b
(-fold)
(%)
Cell extract
61.7
MgCl2-eluted 15.1
a
b
Fig. 2. SDS–PAGE analysis (12.5%) of the affinity purification of Si-tagged protein A
using silica particles as an adsorbent. Lane 1, cell extract; lane 2, silica-unbound
fraction; lane 3, silica-bound fraction (proteins bound to the silica particles were
released by boiling in Laemmli sample buffer [16]); lane 4, MgCl2-eluted fraction;
lane M, molecular mass markers. Proteins were stained with CBB R-250.
the crude extract of recombinant E. coli, we used silica particles as
an adsorbent. Silica particles were mixed with the crude extract
and incubated with rotary mixing for 30 min at room temperature
in the presence of 2 M NaCl and 0.5% (v/v) Tween 20. The salt and
detergent (2 M NaCl and 0.5% [v/v] Tween 20, respectively) were
added to minimize adsorption of host proteins to the particles. Under these conditions, Si-tagged protein A was the major protein
that bound to silica particles (Fig. 2, lanes 2 and 3). The particles
with bound proteins were collected by centrifugation and washed
twice with wash buffer.
Before attempting to recover functional Si-tagged protein A
from silica particles, we investigated elution conditions for Si-tag.
We found that divalent cations Mg2+ and Ca2+ could release Sitag at neutral pH. At the concentration of 2 M, these cations were
the most effective at dissociating Si-tag from silica (Fig. 3). We
used Mg2+ for elution because it has a higher ionic potential than
Ca2+ (see Discussion).
The particles with the bound Si-tagged protein A were suspended in 50 mM Tris buffer containing 2 M MgCl2 (pH #8). After
incubation for 10 min, the supernatant containing the released Sitagged protein A was collected (Fig. 2, lane 4). Table 1 shows typ-
16.0
13.0
26
86
1
3.3
100
81
The starting material was 1.3 g wet weight of cells.
Determined by densitometric analysis of the protein bands on a SDS–PAGE gel.
ical results from the purification. The purity of the protein was
evaluated by densitometric analysis of the CBB-stained protein
bands on a SDS–PAGE gel (Fig. 2). Si-tagged protein A was recovered with a purity of 87 ± 3% and yield of 84 ± 4% (mean ± standard
deviation from three independent experiments) from the crude extract of the recombinant E. coli by this single-step purification. The
purified fraction showed no visible band on an ethidium bromidestained agarose gel (data not shown), indicating little, if any, contamination with DNA and RNA. Although not only the heterologously expressed Si-tagged protein A but also intrinsic ribosomal
protein L2 of the host E. coli should exist in the crude extract, the
latter protein, with a molecular mass of 30 kDa, was not visible
in the purified fraction on SDS–PAGE (Fig. 2, lane 4). This is probably because the amount of the intrinsic L2 was much less than that
of the overexpressed recombinant protein, and, moreover, the
intrinsic L2 tightly binds to rRNA [19] so that it could not bind to
the silica surfaces.
Although the maximum binding capacity of the silica particles
for a purified 60-kDa Si-tagged protein is reported to be #30 mg
per gram dry weight [9], 1 g dry weight of the particles could bind
only #9 mg of Si-tagged protein A even in the presence of an excess
of this protein in the crude extract (data not shown). This difference could be attributed to the presence of E. coli cellular substances and to the more stringent binding conditions of our
purification method (i.e., NaCl concentration was increased from
0.5 M [9] to 2 M).
Comparison with His-tag purification
The dual-tagged structure of Si-tagged protein A (Fig. 1) enabled
direct comparison between our Si-tag purification and the widely
used His-tag purification. For comparison, a crude extract of
E. coli containing the expressed recombinant protein was divided
in half: one half was purified using Si-tag and silica as described
above (Fig. 4, lanes 1–3), and the other half was subjected to
immobilized metal–ion affinity chromatography using His-tag
and a HisTrap HP column (Fig. 4, lanes 1, 4, and 5, and Table 2).
These results indicate that Si-tag purification results in similar
purity and higher yield than His-tag purification (Fig. 4, and
compare Tables 1 and 2).
Silica- and IgG-binding assay of the purified Si-tagged protein A
Fig. 3. Dissociation conditions of Si-tag from silica. Silica particles with the bound
Si-tag were suspended and incubated for 10 min in the following solutions at room
temperature: 50 mM Tris buffer (pH 8.0) containing 5 M NaCl (lane 2), 2 M MgCl2
(lane 3), or 2 M CaCl2 (lane 4); 1 N HCl (lane 5); 1 N NaOH (lane 6). Silica particles
were collected by centrifugation, and then the Si-tag still bound to the particles was
analyzed by SDS–PAGE (12.5%). Lane 1 is a control without the above elution
procedure. Lane M, molecular mass markers. Proteins were stained with CBB R-250.
We examined whether exposure of the protein to high concentrations of MgCl2 affect the silica- and IgG-binding activity of the
purified Si-tagged protein A. The purified fraction containing Sitagged protein A was dialyzed to eliminate MgCl2 and then mixed
with silica particles and/or IgG. Almost all of the purified Si-tagged
protein A bound to silica particles in the presence of 0.5 M NaCl
and 0.5% (v/v) Tween 20 (Fig. 5, compare lanes 1 and 2), indicating
that elution of Si-tag by MgCl2 is reversible. Under these conditions, IgG bound to silica particles only in the presence of Si-tagged
protein A (Fig. 5, compare lanes 3 and 8), indicating that IgG was
immobilized to the silica surface via Si-tagged protein A. The protein purified using Si-tag had comparable silica- and IgG-binding
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T. Ikeda et al. / Protein Expression and Purification 71 (2010) 91–95
Fig. 4. Comparative analysis of Si-tag purification and His-tag purification by SDS–
PAGE (12.5%). Si-tagged protein A was purified from the recombinant E. coli by Sitag purification (lanes 2 and 3) or His-tag purification using a HisTrap HP column
(lanes 4 and 5). Lane 1, cell extract; lane 2, silica-unbound fraction; lane 3, MgCl2eluted fraction; lane 4, flow-through fraction of the HisTrap HP column; lane 5,
imidazole-eluted fraction; lane M, molecular mass markers. Proteins were stained
with CBB R-250.
Fig. 5. Silica- and IgG-binding assay of the purified Si-tagged protein A. Si-tagged
protein A purified by Si-tag purification (lanes 1–3) or His-tag purification (lanes 4–
6) was used as a sample. Lanes 1 and 4, purified Si-tagged protein A (0.7 lg); lanes 2
and 5, silica particles were mixed with Si-tagged protein A (0.7 lg); lanes 3 and 6,
silica particles were mixed with Si-tagged protein A and mouse IgG (0.7 and 2 lg,
respectively); lane 7, mouse IgG (2 lg); lane 8, silica particles were mixed with
mouse IgG (2 lg). For lanes 2, 3, 5, 6, and 8, the proteins that bound to the particles
were released by boiling in Laemmli sample buffer [16] and were analyzed by SDS–
PAGE (12.5%). Lane M, molecular mass markers. Proteins were stained with CBB R250.
Table 2
His-tag purification of Si-tagged protein A from E. colia.
Step
Total protein Amount of Si-tagged Purity Purification Yield
(mg)
protein A (mg)b
(%)b
(-fold)
(%)
Cell extract 61.7
HisTrap HP
8.7
a
b
16.0
8.1
26
94
1
3.6
100
51
The starting material was 1.3 g wet weight of cells.
Determined by densitometric analysis of the protein bands on a SDS–PAGE gel.
activities as the protein purified using His-tag (Fig. 5, compare
lanes 1–3 and 4–6). These results indicate that Si-tagged protein
A retains both silica- and IgG-binding activities after the purification procedures.
Discussion
In this study, we developed a new purification method for silica-binding Si-tagged fusion proteins that utilizes inexpensive
commercial silica particles as a specific adsorbent. Although sil-
ica-based materials are widely used as chromatographic supports
because of their superior stability, rigidity, and inertness [20,21],
we utilize unmodified bare silica itself as a specific adsorbent, taking advantage of the high affinity of Si-tag for silica.
Some glycoproteins that are involved in cell adhesion, such as
vitronectin and fibronectin, have affinity for glass and can be partially purified using glass beads as an adsorbent [22–25]. However,
this method shows poor purification efficiency, and, moreover,
these glycoproteins are unsuitable as affinity tags because they require post-translational modification. In contrast, Si-tag does not
require such modification, and is therefore suitable for heterologous expression as a fusion with a protein of interest.
In aqueous solutions, the outermost layer of silica (SiO2) is hydrated to form silanol groups ("SiOH). The silanol group has a
pKa value of #7 [26], so that under neutral and basic conditions,
it dissociates to "SiO- and H+, meaning that the silica surface is
negatively charged and can act as a cation exchanger [20]. Conversely, Si-tag is a highly basic protein that contains many positively charged residues (29 Arg, 25 Lys, and 9 His residues out of
273 aa) with a theoretical isoelectric point of 10.9, suggesting that
positively charged Si-tag binds electrostatically to negatively
charged ionized silanol groups on the silica surface.
Other researchers suggest the importance of steric geometry of
amino acid residues in addition to their electric charge in adhesion
to inorganic surfaces [27–29]. We previously reported that the Nterminal 60-aa region and C-terminal 70-aa region of Si-tag, where
many positively charged residues are clustered, play important
roles in silica binding [9]. The central region excluding these Nand C-terminal regions has been crystallized, whereas attempts
to crystallize the intact whole protein have been unsuccessful
[30,31], indicating that the N- and C-terminal regions of Si-tag do
not form an organized structure [32,33]. In contrast to globular
proteins with well-defined three-dimensional structures, these
disordered (unstructured) regions are thought to exist as nonglobular extensions adopting highly flexible structures in aqueous solution [33–35]. Although globular proteins interact with solid
surfaces via only a limited number of surface residues displayed
on a certain side of the protein molecule, Si-tag should be able to
interact with a larger area of a silica surface via many more residues in the extended, disordered regions [35,36]. We believe that
the strong binding of Si-tag to silica surfaces is due to the presence
of the clustered positively charged residues in the disordered regions forming dense, multiple ionic bonds with ionized surface silanol groups (8.0 ± 1.0 lmol m"2, corresponding to 4.8 ± 0.6 silanol
groups nm"2 [20]).
Although strong acid and alkaline solutions (1 N HCl and 1 N
NaOH, respectively) were able to release Si-tag from the particles
(Fig. 3, lanes 5 and 6), these harsh conditions are destructive for
proteins. We found that high concentrations of MgCl2 and CaCl2
also released Si-tag, even at neutral pH (Fig. 3, lanes 3 and 4). In
contrast, little Si-tag was released at as high as 5 M NaCl (Fig. 3,
lane 2), suggesting that divalent cations play an important role in
dissociating Si-tag from silica. These divalent cations have much
higher ionic potential (charge density at the surface of the ion, z/
r) than Na+ ion (+2.78 for Mg2+ and +2.00 for Ca2+ vs. +0.98 for
Na+ [37]). Therefore, divalent cations are electrostatically attracted
to negatively charged silica surfaces more strongly than monovalent Na+. Accordingly, we suspect that Si-tagged proteins electrostatically adsorbed on silica surfaces are dissociated by an ion
exchange effect, wherein Mg2+ and Ca2+ act as competing ions for
silanol groups. Moreover, after the dissociation of Si-tag, the divalent cations are expected to bind to the surface silanol group, with
each alkaline earth metal ion linked to approximately one silanol
group as follows [26,38]:
"SiOH þ M 2þ ! "SiOM þ Hþ
þ
T. Ikeda et al. / Protein Expression and Purification 71 (2010) 91–95
This positively charged surface site should neutralize the total
surface charge, repel the Si-tag polypeptide with a net positive
charge, and therefore prevent re-adsorption of the released Sitag, resulting in the efficient elution of Si-tag. After elution of Sitagged proteins, the silica particles can be regenerated by washing
with 1 N HCl followed by binding buffer (data not shown).
The method developed in this study accomplishes high-purity
and high-yield purification for Si-tagged fusion proteins. The purity
and yield of our method are as high as reported by Lichty et al. for
conventional elutable affinity tags, including His-tag [39]. Moreover, our method is much less expensive than conventional affinity
purification methods, which require a ligand to be immobilized on
a resin [21], because the silica itself serves as both a resin and ligand for Si-tag. On the basis of costs reported by Lichty et al., our
method is the least expensive of several commonly used methods
for isolating tagged proteins; the cost was approximately $3 per
10 mg of 30-kDa polypeptide using silica particles (calculated from
the retail price of $1.2 per gram dry weight of the particles used),
whereas the conventional methods cost $12 to $5000 to purify
the same amount of tagged polypeptide [39].
The purified Si-tagged proteins can be used for our original purpose of developing Si-tag, i.e., the direct immobilization of functional proteins on silica materials, such as glass slides and silicon
wafers with a silica surface layer. However, for other applications,
because of its large size (273 aa, corresponding to 30 kDa), the
fused Si-tag is likely to affect the intrinsic properties of the target
protein. To apply our Si-tag purification for more general uses,
we are therefore currently investigating techniques for site-specific cleavage to separate intact target proteins from the purified
Si-tagged fusion proteins, a method commonly used for other fusion proteins [4,40].
Acknowledgments
The authors thank Dr. Yoshio Takahashi (Department of Earth
and Planetary Systems Science, Hiroshima University) for helpful
discussions. This study was supported in part by the Special Coordination Funds for Promoting Science and Technology from the
Ministry of Education, Culture, Sports, Science, and Technology of
Japan; a Grant-in-Aid for Young Scientists (B) from Japan Society
for the Promotion of Science (No. 21780096 to T.I.); and Industrial
Technology Research Grant Program in 2009 from New Energy and
Industrial Technology Development Organization (NEDO) of Japan
(No. 09C46130a to T.I.).
References
[1] J. Nilsson, S. Ståhl, J. Lundeberg, M. Uhlén, P.-Å. Nygren, Affinity fusion
strategies for detection, purification, and immobilization of recombinant
proteins, Protein Expr. Purif. 11 (1997) 1–16.
[2] M.T.W. Hearn, D. Acosta, Applications of novel affinity cassette methods: use of
peptide fusion handles for the purification of recombinant proteins, J. Mol.
Recognit. 14 (2001) 323–369.
[3] K. Terpe, Overview of tag protein fusions: from molecular and biochemical
fundamentals to commercial systems, Appl. Microbiol. Biotechnol. 60 (2003)
523–533.
[4] J. Arnau, C. Lauritzen, G.E. Petersen, J. Pedersen, Current strategies for the use of
affinity tags and tag removal for the purification of recombinant proteins,
Protein Expr. Purif. 48 (2006) 1–13.
[5] E. Hochuli, H. Döbeli, A. Schacher, New metal chelate adsorbent selective for
proteins and peptides containing neighbouring histidine residues, J.
Chromatogr. 411 (1987) 177–184.
[6] E. Hochuli, W. Bannwarth, H. Döbeli, R. Gentz, D. Stüber, Genetic approach to
facilitate purification of recombinant proteins with a novel metal chelate
adsorbent, Bio/Technology 6 (1988) 1321–1325.
[7] D.B. Smith, K.S. Johnson, Single-step purification of polypeptides expressed in
Escherichia coli as fusions with glutathione S-transferase, Gene 67 (1988) 31–40.
[8] C. di Guan, P. Li, P.D. Riggs, H. Inouye, Vectors that facilitate the expression and
purification of foreign peptides in Escherichia coli by fusion to maltose-binding
protein, Gene 67 (1988) 21–30.
95
[9] K. Taniguchi, K. Nomura, Y. Hata, T. Nishimura, Y. Asami, A. Kuroda, The Si-tag
for immobilizing proteins on a silica surface, Biotechnol. Bioeng. 96 (2007)
1023–1029.
[10] T. Ikeda, Y. Hata, K. Ninomiya, Y. Ikura, K. Takeguchi, S. Aoyagi, R. Hirota, A.
Kuroda, Oriented immobilization of antibodies on a silicon wafer using Sitagged protein A, Anal. Biochem. 385 (2009) 132–137.
[11] F. Patolsky, G. Zheng, C.M. Lieber, Fabrication of silicon nanowire devices for
ultrasensitive, label-free, real-time detection of biological and chemical
species, Nat. Protoc. 1 (2006) 1711–1724.
[12] E. Stern, J.F. Klemic, D.A. Routenberg, P.N. Wyrembak, D.B. Turner-Evans, A.D.
Hamilton, D.A. LaVan, T.M. Fahmy, M.A. Reed, Label-free immunodetection
with CMOS-compatible semiconducting nanowires, Nature 445 (2007) 519–
522.
[13] S.M. Weiss, G. Rong, J.L. Lawrie, Current status and outlook for silicon-based
optical biosensors, Physica E 41 (2009) 1071–1075.
[14] S. Yamatogi, Y. Amemiya, T. Ikeda, A. Kuroda, S. Yokoyama, Si ring optical
resonators for integrated on-chip biosensing, Jpn. J. Appl. Phys. 48 (2009)
04C188.
[15] J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual, third ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001.
[16] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head
of bacteriophage T4, Nature 227 (1970) 680–685.
[17] M.D. Abramoff, P.J. Magelhaes, S.J. Ram, Image processing with ImageJ,
Biophotonics Int. 11 (2004) 36–42.
[18] S. Löfdahl, B. Guss, M. Uhlén, L. Philipson, M. Lindberg, Gene for staphylococcal
protein A, Proc. Natl. Acad. Sci. USA 80 (1983) 697–701.
[19] K. Watanabe, M. Kimura, Location of the binding region for 23S ribosomal RNA
on ribosomal protein L2 from Bacillus stearothermophilus, Eur. J. Biochem. 153
(1985) 299–304.
[20] J. Nawrocki, The silanol group and its role in liquid chromatography, J.
Chromatogr. A 779 (1997) 29–71.
[21] J.E. Schiel, R. Mallik, S. Soman, K.S. Joseph, D.S. Hage, Applications of silica
supports in affinity chromatography, J. Sep. Sci. 29 (2006) 719–737.
[22] R. Holmes, Preparation from human serum of an alpha-one protein which
induces the immediate growth of unadapted cells in vitro, J. Cell Biol. 32
(1967) 297–308.
[23] D. Barnes, R. Wolfe, G. Serrero, D. McClure, G. Sato, Effects of a serum spreading
factor on growth and morphology of cells in serum-free medium, J. Supramol.
Struct. 14 (1980) 47–63.
[24] D.W. Barnes, J. Silnutzer, Isolation of human serum spreading factor, J. Biol.
Chem. 258 (1983) 12548–12552.
[25] D.W. Barnes, L. Mousetis, B. Amos, J. Silnutzer, Glass-bead affinity
chromatography of cell attachment and spreading-promoting factors of
human serum, Anal. Biochem. 137 (1984) 196–204.
[26] R.K. Iler, The Chemistry of Silica, John Wiley & Sons, Inc., New York, 1979.
[27] K.-I. Sano, K. Shiba, A hexapeptide motif that electrostatically binds to the
surface of titanium, J. Am. Chem. Soc. 125 (2003) 14234–14235.
[28] R.L. Willett, K.W. Baldwin, K.W. West, L.N. Pfeiffer, Differential adhesion of
amino acids to inorganic surfaces, Proc. Natl. Acad. Sci. USA 102 (2005) 7817–
7822.
[29] T. Hayashi, K.-I. Sano, K. Shiba, K. Iwahori, I. Yamashita, M. Hara, Critical amino
acid residues for the specific binding of the Ti-recognizing recombinant ferritin
with oxide surfaces of titanium and silicon, Langmuir 25 (2009) 10901–10906.
[30] T. Nakashima, M. Kimura, A. Nakagawa, I. Tanaka, Crystallization and
preliminary X-ray crystallographic study of a 23S rRNA binding domain of
the ribosomal protein L2 from Bacillus stearothermophilus, J. Struct. Biol. 124
(1998) 99–101.
[31] A. Nakagawa, T. Nakashima, M. Taniguchi, H. Hosaka, M. Kimura, I. Tanaka, The
three-dimensional structure of the RNA-binding domain of ribosomal protein
L2; a protein at the peptidyl transferase center of the ribosome, EMBO J. 18
(1999) 1459–1467.
[32] N. Ban, P. Nissen, J. Hansen, P.B. Moore, T.A. Steitz, The complete atomic
structure of the large ribosomal subunit at 2.4 Å resolution, Science 289 (2000)
905–920.
[33] V.N. Uversky, Natively unfolded proteins: a point where biology waits for
physics, Protein Sci. 11 (2002) 739–756.
[34] P.E. Wright, H.J. Dyson, Intrinsically unstructured proteins: re-assessing the
protein structure–function paradigm, J. Mol. Biol. 293 (1999) 321–331.
[35] A.K. Dunker, C.J. Brown, J.D. Lawson, L.M. Iakoucheva, Z. Obradović, Intrinsic
disorder and protein function, Biochemistry 41 (2002) 6573–6582.
[36] K. Gunasekaran, C.-J. Tsai, S. Kumar, D. Zanuy, R. Nussinov, Extended
disordered proteins: targeting function with less scaffold, Trends Biochem.
Sci. 28 (2003) 81–85.
[37] D. Langmuir, Aqueous Environmental Geochemistry, Prentice-Hall, Inc., Upper
Saddle River, 1997.
[38] H. Cheng, K. Zhang, J.A. Libera, M. Olvera de la Cruz, M.J. Bedzyk,
Polynucleotide adsorption to negatively charged surfaces in divalent salt
solutions, Biophys. J. 90 (2006) 1164–1174.
[39] J.J. Lichty, J.L. Malecki, H.D. Agnew, D.J. Michelson-Horowitz, S. Tan,
Comparison of affinity tags for protein purification, Protein Expr. Purif. 41
(2005) 98–105.
[40] R.J. Jenny, K.G. Mann, R.L. Lundblad, A critical review of the methods for
cleavage of fusion proteins with thrombin and factor Xa, Protein Expr. Purif. 31
(2003) 1–11.