Biomaterials 30 (2009) 1166 1175
Contents lists available at ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Directional BMP-2 for functionalization of titanium surfaces
a,b a,b a,b,*
Kenji Kashiwagi , Toru Tsuji , Kiyotaka Shiba
a
Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan
b
CREST, Japanese Science and Technology Corporation Agency, Japan
a r t i c l e i n f o a b s t r a c t
Article history:
Efficient immobilization of biomacromolecules on material surfaces is a key to development in areas of
Received 7 August 2008
regenerative medicine and tissue engineering. However, strong and irreversible immobilization of
Accepted 17 October 2008
cytokines on surfaces often diminishes their biological functionality. A destructive hydrophobic inter-
Available online 20 November 2008
action between the material surface and the biomolecule may underlie this inactivation. Alternatively,
dissociation of the cytokine from the material may be necessary for signal transduction. Here we propose
Keywords:
a new method for immobilizing cytokines on material surfaces: a material-binding artificial peptide is
Artificial protein
used to mediate reversible interaction between the cytokine and the material surface. We created
BMP (bone morphogenetic protein)
artificial proteins that contained three copies of a Ti-binding motif, and fused them to the N-terminal of
Interface
BMP-2. The engineered BMP-2 showed reversible binding to Ti surfaces and induced BMP signaling
Motif-programming
activity. When a hydrophobic protein devoid of the Ti-binding motif was fused to BMP-2, the protein
Osseointegration
Titanium tightly bound to Ti surfaces but showed little BMP activity, confirming the importance of the mode of
immobilization.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction materials and biomolecules continue to make this a challenging
problem. It is known, for example, that hydrophobic interactions
Titanium (Ti) is presently being widely used in various areas of between a protein and the surface of an inorganic material some-
medicine, including artificial joints, dental implants and cardiac times result in deactivation of the protein due to the collapse of its
stents [1]. The reasons Ti is preferred over other materials include tertiary structure [5]. Furthermore, some cytokines are inactivated
its biocompatibility, resistance to corrosion and low allergenicity, when irreversibly immobilized on a material surface. Therefore, to
among others. Despite these superior properties, there is still ample maximize the function of biomolecules, an optimized method for
room to improve Ti devices. For instance, when Ti is used in a dental their immobilization that would avoid denaturation of the proteins
implant, patients are generally obliged to endure restricted masti- and, when necessary, allow dissociation from the surface would be
cation for several weeks until the tight adhesion between the Ti highly desirable.
implant and gnathic bone (termed osseointegration) is established In recent years, peptide aptamers (źbinders) that specifically
[2]. Thus shortening the time required for osseointegration through bind to the surfaces of various inorganic materials have been
appropriate modification of the implant could substantially attracting attention in the field of bionanotechnology [6]. These
improve patients quality of life. Toward that end, a variety methods artificial peptides are created using evolutionary engineering
for surface modification of Ti implants have been proposed, methods such as peptide phage systems, in which phage clones that
including plasma flame spraying, sandblasting, acid-etching and attach a desired target material at neutral pH and detach it at acidic
coating with ceramic [3]. In addition to these physical and chemical pH are selected from a pool of phages displaying random peptide
methods, modification using natural proteins such as collagen, cell sequences [7]. TBP-1 is one such material-binding peptide that was
adhesive molecules or growth factors has also been tried [4]. isolated as an aptamer against Ti [8]. Although originally isolated as
Although these biological modification methods can make the most a 12-mer peptide, further mutational analyses revealed that a hex-
use of our accumulated knowledge of molecular biology, the often- apeptide sequence, RKLPDA (named minTBP-1), is sufficient for the
unpredictable effects of the interaction between inorganic binding [8]. These analyses also showed that the first arginine (R1),
fourth proline (P4) and fifth aspartate (D5) all play important roles
in the recognition, which suggests an electrostatic interaction is the
dominate molecular mechanism via which the peptide recognizes
* Corresponding author. Department of Protein Engineering, Cancer Institute,
Ti [8,9].
Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550,
In the present study, we first used the minTBP-1 peptide motif to
Japan. Tel.:þ81 3 3570 0489; fax:þ81 3 3570 0461.
create a modified BMP-2 endowed with directionality toward Ti
E-mail address: [e-mail] (K. Shiba).
0142-9612/$  see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2008.10.040
K. Kashiwagi et al. / Biomaterials 30 (2009) 1166 1175 1167
37 C. The soluble fractions enriched with wild-type or modified BMP-2 were then
surfaces. We then investigated the effects of the attached protein
recovered after removing the residual insoluble debris by centrifugation.
on BMP signaling and cell differentiation in vitro.
2.2.4. Refolding of wild-type and modified BMP-2
2. Materials and methods
The soluble fractions prepared as above were quickly diluted in refolding buffer
(1 ML-arginine, 0.1 M Tris HCl (pH 8.0), 5 mM EDTA, 5 mM oxidized glutathione and
2.1. Materials
2mM reduced glutathione [12]) and stirred for 7 days at 4 C. The refolded proteins
were then concentrated using Centriprep-10 (Millipore, Billerica, CA) until the total
2.1.1. Cell line and its maintenance
solution volumes were less than 1 mL. The correctly folded proteins were separated
C2C12 cells (ATCC CRL-1772) were cultured in low-glucose Dulbecco s modified
from the misfolded ones using a heparin agarose (H 0402, Sigma) as described
Eagle s medium (DMEM; Gibco, Grand Island, NY) supplemented with 15% heat
previously [12]. After equilibrating the column with buffer A (0.1 M Tris-acetate (pH
inactivated fetal bovine serum (FBS; Premium Fetal Bovine Serum, Cambrex Bio
5.5), 5 mM EDTA, 6 M urea), the protein solution diluted 1:10 with buffer A was
Science Walkersville, Walkersville, MD), penicillin (Banyu pharmaceutical, Tokyo,
applied. The column was then washed with 10 bed volumes of buffer A, and the
Japan) and streptomycin (Meiji Seika Kaisha, Tokyo, Japan) in a humidified incubator
bound proteins were eluted with a stepwise NaCl gradient (0.1 1.0 M NaCl in 0.1-M
at 37 C under a 5% CO2/95% air atmosphere. The cultures were passaged (1:10)
steps). Fractions containing BMP-2 dimer were confirmed by SDS-PAGE under
every 2 days and were used for experimentation up to passage 20.
nonreducing conditions. These fractions were then dialyzed against 10 mM HCl and
lyophilized. The proteins were dissolved in 10 mM HCl when used.
2.1.2. Ti plates
Wrought Ti plates that had been polished using a buff and ultrasonically cleaned
2.2.5. Expression and purification of the artificial proteins
in ethanol and distilled water were obtained from Shinkinzoku Co., Ltd. (JIS, Japan
E. coli strain XL1-Blue (Invitrogen, Carlsbad, CA) harboring pTT047, pTT048 or
Industrial Specification H4600, 99.9 mass % Ti, 10 10 1 mm, Osaka). The plates
pTT061 was inoculated into 25 mL of LB medium containing 50 mg/mL carbenicillin
were then cleaned further by sonication in acetone followed by ethanol and Milli-Q
and 0.2% glucose (Nacalai Tesque, Kyoto) and incubated overnight at 37 C. The
water before use.
overnight culture was inoculated into 500 mL of prewarmed LB containing carbe-
nicillin and glucose and incubated at 37 C until the OD660 reached 0.35 (approxi-
2.2. Genetically modified BMP-2s
mately 3 4 h), at which time IPTG was added to a concentration of 1 mM to induce
protein expression. After an additional 1 h, the cells were harvested by centrifuga-
2.2.1. Construction of genes encoding minTBP-1-appended BMP-2s
tion and stored at 80 C until use. The artificial proteins were purified under
The DNA encoding human mature BMP-2 (which excludes its pro-sequence) was
denaturing conditions using TALON resin (Clontech, Palo Alto, CA) as described
amplified from the cDNA (IMAGE cDNA collections, ID: 6303163) using PCR with
previously [13]. Once purified, the proteins were dialyzed against 10 mM HCl and
primers KY-1435 (50-AGA GAA CAT ATG CAA GCC AAA CAC AAA CAG CGG-30; the NdeI
lyophilized for storage. The proteins were dissolved in 10 mM HCl for use.
site is underlined) and KY-1436 (50-TTT GGA TCC TCA GCG ACA CCC ACA ACC CTC-30;
the BamHI site is underlined). The amplified fragment was digested using NdeI and
2.3. QCM measurements
BamHI, electrophoresed through 1% agarose gel, and purified using a Gene Clean II
kit (Qbiogene, Carlsbad, CA). The isolated DNA was then ligated into the NdeI BamHI
Quartz Crystal Microbalance (QCM) measurements were carried out using Q-
sites of pET-20b plasmid (Novagen, Madison, WI) to obtain pKK141, after which the
sense D300 equipment and a Ti sensor (Q-Sense, Västra Frölunda), which was
coding frame of the BMP-2 was confirmed by sequencing. Starting from pKK141, we
cleaned using a UV-ozone ProCleaner system (BioForce Nanosciences, Ames, IA)
first constructed BMP-2 derivatives in which a single or multiple copies of the
prior to use. Before making measurements, the Ti-sensor and the chamber were
minTBP-1 hexapeptide motif was appended at their N- or C-terminal. For N-terminal
fusion, an oligonucleotide duplex (KY-1339: 50P-TA TGC GCA AAC TTC CGG ATG C-30 equilibrated in TBS containing 0.5% Tween 20 at 25 C. The artificial protein samples
were prepared in a 1-mL volume at a concentration of 0.1 mg/mL in TBS containing
and KY-1340: 50P-TA GCA TCC GGA AGT TTG CGC A-30) was inserted at the NdeI site
0.5% Tween 20.
of pKK141. Clones with single, double or triple copies of the insertion were termed
pKK144, pKK161 and pKK162, respectively. For the C-terminal fusion, BMP-2 gene
was amplified with oligonucleotide KY-1437 (50-GA GCC ATG GCG CAA GCC AAA CAC 2.4. Characterization of the Ti-directionality of the modified BMP-2s
AAA CAG CGG-30; the NcoI site is underlined) and KY-1438 (50-TTT GGA TCC TCA CAT
ATG GCG ACA CCC ACA ACC CTC-30; the NdeI site is underlined) and cloned into 2.4.1. Coating Ti plates with the modified BMP-2s
NcoI NdeI site of pET-19b vector to obtain pKK142. Then an oligonucleotide duplex For protein coating, the plates were first incubated for 30 min in 0.5 ml of 0.5%
of KY-1339 and KY-1340 was inserted at the NdeI site of pKK142 to obtain pKK145. bovine serum albumin (BSA, Chon fraction V, Iwai chemicals, Tokyo) in TBS and then
washed three times in binding buffer (5 M urea, 0.2 M Tris HCl (pH 9.5), 0.1% Tween
2.2.2. Construction of the genes for fusion of artificial proteins with BMP-2 20). The wild-type and modified BMP-2s were dissolved to the indicated concen-
The details of the construction of artificial proteins will be published elsewhere. trations in binding buffer, after which 10 mL of the protein solution was placed onto
Briefly, a microgene encoding the minTBP-1 motif in one of its reading frames was each Ti plate and left for 1 h at ambient temperature. The plates were then rinsed
designed and polymerized using the MPR method [10] to construct a combinatorial twice with binding buffer and three times with TBS.
library encoded by the microgene in the three coding frames. In this study, we used
clones pTT047, pTT048 and pTT061 to produce the artificial proteins #55, #56 and 2.4.2. Assaying modified BMP-2s
#61, respectively. Modified BMP-2s were quantified using a Human BMP-2 ELISA development kit
The genes for the fusion of artificial protein with BMP-2 were constructed as (PeproTech, Rocky Hill, NJ) after preparation of standard curves for each (data not
follows. The NdeI BamHI fragment of pKK141, which encodes BMP-2, was filled-in at shown). To estimate the amounts of BMP-2 released from Ti plates, the protein-
both ends using with Klenow fragment (New England Biolabs) and inserted in to the coated plates were incubated in DMEM supplemented with 15% FBS for 2 days at
SmaI site (located at the 30end of artificial gene) of pTT047 and pTT048, or the blunt- 37 C, and the proteins recovered from the medium were quantified after 1:10
ended BamHI site of pTT061 to give pKK171, pKK170 and pKK169, respectively. dilution in phosphate buffered saline (PBS [11]) containing 0.1% BSA. At the same
time, the amount of protein retained on the plates was estimated after dissolving the
2.2.3. Expression and purification of wild-type and modified BMP-2 adsorbed protein in 5 M urea, 0.2 M HCl and 0.1% Tween 20 at room temperature for
The wild-type BMP-2 expression plasmid (pKK141) or a modified plasmid 30 min. This eluate was diluted 1:20 in PBS containing 0.1% BSA before carrying out
(pKK144, pKK161, pKK162) was introduced into Escherichia coli strain BL21(DE3)- ELISAs.
pLysS (Novagen), after which the cells were inoculated into 5 mL of LB medium [11]
containing 50 mg/mL carbenicillin (Carbþ, Sigma chemicals, St. Louis, MO) and 34 mg/ 2.4.3. Evaluation of the binding of a BMP receptor to immobilized BMP-2s
mL chloramphenicol (Chlþ, Sigma) and incubated overnight at 37 C. The overnight The binding of a BMP receptor 1A (BMPR-1A)/Fc chimeric protein (R&D Systems,
culture was inoculated into 500 mL of LB CarbþChlþmedium and incubated at 37 C Minneapolis, MN) to BMPs immobilized on a Ti surface was evaluated as follows. Ti
with shaking until the OD660 reached 0.35, at which time protein expression was plates coated with the modified BMPs were blocked for 30 min with 0.5% BSA in TBS
induced by addition isopropyl-b-D-thio-galactopyranoside (IPTG, TaKaRa-Bio, Shiga, and then washed three times with TBS containing 0.1% Tween 20 (TBS-T). The coated
Japan) to a concentration of 1 mM. After an additional 2 h, the cells were harvested plates were incubated for 30 min with 0.5 mL of 0.1 mg/mL BMPR-1A/Fc chimera in
by centrifugation and stored at 80 C. TBS-T containing 0.5% BSA (TBS-TB), and then washed three times with TBS-T. The
Under the conditions summarized above, the proteins formed inclusion bodies bound BMPR-1A/Fc was reacted with an anti-human Fc antibody HRP conjugate
within the cells. Purification of these proteins was therefore started by collecting the (1:1000 dilution; Sigma) in TBS-BT and washed three times with TBS-T. The Ti plates
precipitated proteins. For this purpose, the frozen cell pellets were thawed in Tris were then transferred to a new 24 well plate, and ABTS solution [0.22 mg/mL ABTS
buffered saline (TBS; 50 mM Tris HCl (pH 7.5) and 150 mM NaCl) containing 1% (2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), Sigma), 50 mM sodium
Triton-X100 and sonicated for 30 s using a Sonifier. From the disrupted cells, the citrate (pH 4.0) and 0.05% hydrogen peroxide] was added to the cells to estimate HRP
insoluble fractions were collected by centrifugation (6000 g for 15 min) and washed activity. In separate preparations, amounts of modified BMP-2 on Ti plates were
with TBS containing 1% Triton-X100, after which this cycle was repeated three times. estimated using an anti-His 6 tag antibody (1:5000 dilution. Clontech) and anti-
The insoluble fractions were then incubated in 2 mL of lysis buffer (6 M guanidium mouse IgG HRP conjugate (1:2000 dilution; Bio-Rad laboratories, Hercules, CA) as
hydrochloride, 20 mM Tris HCl (pH 8.0) and 14 mM 2-mercaptoethanol) for 1 h at the primary and secondary antibodies, respectively. In this case, we found that
1168 K. Kashiwagi et al. / Biomaterials 30 (2009) 1166 1175
blocking solution containing 0.5% skim milk (BD Difco, Franklin Lakes, NJ) gave 2.4.6. Enzymatic staining for ALP and immunofluorescent staining of myotubes
better results than 0.5% BSA (data not shown). C2C12 cells were cultured on Ti plates pretreated with various BMPs for 9 days
under the same conditions described in Section 2.4.3. During that period the
2.4.4. Monitoring BMP signaling using a tester cell line medium was refreshed every 2 days. The cells were then fixed for 15 min with 4%
We used a tester cell line, BRE-Luc C2C12 (BRE, BMP responsive element; Luc, paraformaldehyde in PBS. For enzymatic staining of ALP, cells were incubated for
luciferase, [14]) developed by T. Murakami (Cancer Institute) to monitor the acti- 30 min with BCIP/NBT solution (prepared from Fast BCIP/NBT buffered substrate
vation in BMP signaling. Details of the development of the cell line will be reported tablet, Sigma). For immunostaining of myotubes, the fixed cells were permeabilized
elsewhere. with 0.25% Triton-X100 in PBS and incubated with monoclonal anti-myosin heavy
To measure the activity of the soluble form of the modified BMP-2s, aliquots of chain in culture supernatant (MF20, Developmental Studies Hybridoma Bank,
suspended BRE-Luc C2C12 cells (100 mL at a concentration of 1.0 105 cells/mL) University of Iowa) [15] diluted 1:100 in PBS containing 1% horse serum (GIBCO),
were seeded onto a 96-well plate. After incubation for 24 h, the indicated amounts followed by incubation with 4 mg/mL biotinylated goat anti-mouse IgG (Santa Cruz
of each BMP were added and incubated for an additional 24 h. The cells were then Biotechnology, Santa Cruz, CA) and 10 mg/mL Streptavidin Alexa Fluor 594 conju-
washed once with PBS, disrupted with lysis reagent (CCLR, Promega, Madison, WI), gate (Molecular Probes, Eugene, OR) in PBS. For nuclear staining, 0.5 mM SYTO Green
and the luciferase activity in the lysate was measured using a Luciferase assay 11 (Cambrex, East Rutherford, NJ) was also included. The stained cells were photo-
reagent kit (Promega) with a Mithras LB940 luminescence plate reader (Berthold graphed under a DMLP reflection microscope (Leica Microsystems, Bensheim, Ger-
Technologies, Bad WildBad, Germany). The observed activity was normalized to the many) equipped with a DC480 or DFC350 FX digital camera.
protein content in the cell lysate, which was determined using a BCA protein assay
kit (Pierce, Rockfold, IL).
3. Results
To measure the activity of the modified BMP-2s immobilized on Ti, the Ti plates
were incubated with 0.5 mL of tester cell suspension (1.0 105 cells/mL) in the wells
3.1. BMP-2 modified with minTBP-1 peptides
of a 24-well plastic plate. After incubation for 48 h, the cells were detached from the
Ti plates by washing with 0.5 mL of PBS and transferred to new 24-well plates. The
luciferase activity in the cells was then measured as described above.
To endow BMP-2 with the ability to bind Ti, we first
appended the minTBP-1 hexapeptide at either end of BMP-2
2.4.5. Alkaline phosphatase assay
protein. Native BMP-2 is known to be synthesized in a pro form
C2C12 cells were seeded onto a 96-well plate to a density of 1.0 104 cells/well
and cultured for 24 h in DMEM supplemented with 15% FBS. The medium was then that has a 259-amino acid N-terminal propeptide [16]. The
changed to DMEM supplemented with 2% FBS, 50 mg/mL ascorbic acid (Sigma) and
propeptide is cleaved by a proprotein convertase to release
10 mM b-glycerophosphate (Sigma), which is known to induce ossification. At the
mature BMP-2 from the cell membrane into the extracellular
same time, modified BMP-2 were added, after which the cells were cultured for an
fluid [12,17]. Despite the complex synthetic pathway of the
additional 72 h, washed once with TBS and lysed in TBS containing 0.2% Triton X-
native protein, recombinant BMP-2 devoid of the propeptide is
100. The alkaline phosphatase (ALP) activity in the lysates was measured using the
fluorescent substrate 4-methylumbelliferyl phosphate (4-MUP, Sigma). Aliquots
biologically active if it is properly refolded after heterologous
(100 mL) of cell lysate and 50 mM 4-MUP were added to the reaction buffer (1.5 M
expression in bacteria [18]. With that in mind, we inserted
Tris HCl (pH 8.5), 1 mM MgCl2) in a 96-well plate, mixed, and incubated at 37 C. The
a DNA cassette encoding minTBP-1 peptide at the N- or C-
fluorescent signal from the substrate was then measured using a Biolumin 960
terminal of the coding sequence of mature BMP-2 to construct
fluorescence microplate reader (Molecular Dynamics, Sunnyvale, CA) equipped with
a 360 10 nm excitation filter and a 485 10 nm emission filter. TBP-BMP-2 or BMP-2-TBP (Fig. 1B). When we evaluated the
BMP-2
A C
TBP(x2)-BMP-2
TBP
His x 6
MRKLPDAMRKLPDA
tag
-(BMP-2)
#56-BMP-2
#69-BMP-2
Artificial Protein
MRGSHHHHHHGSVDWRSPV--- MRGSHHHHHHTDPSTWADPP
----------------GAGFSS
QASRRSESQESDRSLSRSRILL
QASRRSESQESDRSLSRSRILT
ASFPTLRIAGIGQESQSEQDS
RKLPDAPNRRNRTGVSVGAGFS QASRRSESQESDRSLSR----T
BMP-2 BMP-2
B
RKLPDAPNRRNRTGVSVGAGFS ----TLRIAGIGQESQSEQDSQ
BMP-2-TBP BMP-2
RKLPDAPNRRNRTGVSVGAGFS
QASRRSESQESDRSLSRSRILSS
TBP
ASFPTLRIAGIGQESQSEQDSH QASRRSESQESDRSLSRG
TBP-BMP-2 BMP-2
ASFPTLRIAGIGQESQSEGYVP -(BMP-2)
TBP
-(BMP-2)
TBP(x2)-BMP-2 BMP-2
TBP TBP
TBP(x3)-BMP-2 BMP-2
#55-BMP-2
TBPTBPTBP
MRGSHHHHHHGIRRQWQIPLI-
Artificial protein
ASFPTLRIAGIGQESQSEQDSL
Charged residues
#56-BMP-2 BMP-2
ASFPTLRIAGIGQESQSEQDSL
Hydrophobic residues
His x 6 TBP TBP TBP
ASFPTLRIAGIGQESQSEQDSL
Tag
minTBP-1 motif
BMP-2
#55-BMP-2 ASFPTLRIAGIG----------
BMP-2
-KLPDAPNRRNRTGVSVGAGFS
SQASRRSESQESDRSLSRSGYP
#69-BMP-2
BMP-2
-(BMP-2)
Fig. 1. Schematic diagram of structures of the modified BMP-2s constructed in this study. (A) Conceptual diagram of Ti-directed BMP-2. Dimeric BMP-2 (yellow) was fused with an
artificial protein containing multiple copies of a Ti-binding motif (minTBP-1, green). The protein reversibly associated with a Ti surface, most probably via minTBP-1 sequences. (B)
Schematic diagram of the structures of the modified BMP-2s. The mature portions of native BMP-2 and the artificial proteins are indicated by orange and pink boxes, respectively.
The locations of the minTBP-1 motif within the artificial proteins are indicated by green bars. (C) Amino acid sequences of #56-BMP-2, #55-BMP-2 and #69-BMP-2. Charged and
hydrophobic amino acids are colored in red and green, respectively.
K. Kashiwagi et al. / Biomaterials 30 (2009) 1166 1175 1169
activity of the modified BMP-2s after purification and refolding, 3.3. Association and dissociation behavior of artificial proteins
we found that BMP-2-TBP, whose C-terminal was appended with observed by QCM
minTBP-1, had lost its ability to activate BMP signaling (data not
shown), which is consistent with the notion that the C-terminal We initially used a QCM equipped with a Ti sensor to assess the
end of BMP-2 is sensitive to chemical modification. In subse- association and dissociation of the artificial proteins from a Ti
quent experiments, therefore, we focused on the Ti-binding surface (Fig. 2). Injection of 0.1 mg/mL #56 or #55 into the
capacity of TBP-BMP-2, though initially we were unable to measurement cell elicited an immediate reduction in the resonance
obtain results confirming that protein would bind to a Ti plate frequency of the sensor, indicating that both proteins rapidly
(data not shown). associated with the Ti surface. However, when buffer solution was
During its selection process, TBP-1 showed the capacity to then injected into the measurement cell, #69 dissociated faster
moor a huge phage body (w16 MDa) at a Ti surface and with- than #56 did. The rapid association and relatively slow dissociation
stand substantial thermal disturbance. We would therefore shown by #56 was also observed with other artificial proteins
expect that TBP-1 would strongly bind to Ti. Nevertheless, we programmed with minTBP-1 motif (Kokubun et al., in press).
noticed that the synthetic TBP-1 peptide only weakly bound to Because minTBP-1 is thought to interact with Ti surfaces via an
Ti  i.e., the Kd for the peptide binding to Ti was only electrostatic interaction, and minTBP-1 ornamented nanoparticles
13.2 4.0 mM [19]. Diminished binding affinity of a peptide rapidly associated with and slowly dissociated from Ti, we believe
aptamer accompanied by detachment of the phage body from that #56 inherited the Ti-binding property of minTBP-1. In contrast
a surface often observed in peptide phage systems. One possible to #56 and #69, #55 more slowly associated with Ti, but once
explanation for strong binding by a peptide aptamer is the fact associated, the protein did not dissociate, which is typical of
that it is displayed multivalently on the phage [20]. Indeed, hydrophobic interactions between proteins and the surfaces of
when minTBP-1 was multivalently displayed on the nanocage materials [31].
protein ferritin, strong binding to Ti (Kdź3.82 nM) was restored Because the energy dissipation shifts elicited by the injections
[21]. Wild-type BMP-2 dimerizes by forming an intermolecular were less than 1 10 6 for all proteins, Sauerbrey s equation could
disulfide bond between two subunits, and it was the dimeric be applied [32], and the calculated maximum mass gains were 610,
form of the modified BMP-2 that we purified as described in 458 and 539 ng/cm2 for #56, #55 and #69, respectively. If we
Section 2. Consequently, the modified TBP-BMP-2 should display presume that similar amounts of water were bound to the three
minTBP-1 at a valency of 2, which was apparently insufficient to proteins, these data indicate that much more #56 was adsorbed
endow BMP-2 with Ti-directionality. onto the Ti than the other proteins.
To increase the multivalency of minTBP-1 in the modified BMP-
2, we inserted two or three copies of the DNA sequence encoding
3.4. Biological activities of BMP-2 fused to an artificial protein
the peptide into the BMP-2 gene to produce TBP( 2)-BMP-2 and
TBP( 3)-BMP-2, respectively. Unfortunately, TBP( 3)-BMP-2 was
Because the N-terminal end of mature BMP-2 can tolerate fusion
not expressed in bacterial cells, and TBP( 2)-BMP-2 showed little
with a foreign polypeptide, we constructed the #55-BMP-2, #56-
ability to bind Ti, as is described below.
BMP-2 and #69-BMP-2 fusion proteins as described in Section 2.
The fusion proteins were well expressed in E. coli and were able to
3.2. The minTBP-1-programmed artificial protein #56
be purified as mature BMP-2. Because at the ends of the modified
BMP-2s there was a large extra domain that could not be tightly
The well known difficulty of expressing tandem repeats of
folded, we were not sure whether the modified BMP-2s would
a peptide [22,23] prevented us from appending more than two
dimerize after the in vitro refolding reaction. However, SDS-PAGE
copies of minTBP-1 to BMP-2 in tandem form. To solve this
carried out under reducing conditions revealed that large fractions
expression problem and to bring out the latent competence of
of the proteins were recovered as dimers (Supplementary figure 1).
inconspicuous motifs, we employ the MolCraft system, which is
a method for constructing artificial proteins by rationally
programming functional motif(s) such that they occur in a combi-
800
natorial manner in a repetitive artificial protein [10,24]. The overall
repetitiousness of proteins created in this way is thought to
contribute to the formation of structures [13,25], and we can even
600
rationally embed secondary structure in the proteins [10,26]. Using
the MolCraft system, we have created a variety of functional arti-
ficial proteins by programming one or more functional motifs
400
[23,26 30]. In one experiment, we created artificial proteins that
endowed Ti with the ability to bind cells by programming the
minTBP-1 motif together with the RGD motif, a sequence that
200
mediates cell attachment (Kokubun et al., in press). In another
experiment, we focused on motifs related to biomineralization and
created mineralization enhancing proteins by programming two #55
0
#56
short motifs derived from dentin matrix protein [9]. In that exper-
#69
iment, we embedded the minTBP-1 motif such that the proteins
could be used to coat a Ti surface with crystals of calcium phos- -200
0 10203040
phate. Some of the proteins created in that experiment contained
multiple copies of minTBP-1 in their sequences. Among them, we
time(min)
focused on #56, which contains three copies of minTBP-1, and used
Fig. 2. QCM analyses of the interaction between Ti and the artificial proteins. Injection
it as a Ti-binding protein in the present experiment. As controls, we
of a protein (0.1 mg/mL; black, #55; red, #56; green, #69) into the QCM measuring
also used #55 and #69, neither of which contain a minTBP-1 motif,
chamber (closed arrowhead) immediately reduced the resonance frequency of the
but the amino acid composition of #55 is more hydrophobic than
sensor. Dissociation was observed upon injection of TBS-Tween 20 into the chamber
that of #69 (Fig. 1B and C). (open arrowheads).
2
adsorbed amount (ng/cm )
1170 K. Kashiwagi et al. / Biomaterials 30 (2009) 1166 1175
Apparently the large appendix at the N-terminal did not prevent proteins directionality toward Ti under physiological conditions. To
dimerization of BMP-2 in vitro. that end, we dissolved the BMP derivatives in 5 M urea, which
We next used two methods to assess the biological activities of enabled solubilization of the proteins at high concentrations. The
the purified dimeric fusion proteins. We first used a tester cell urea was not expected to disturb the electrostatic interaction
containing the luciferase gene under the control of BMP responsible between minTBP-1 and Ti, and at a concentration of 5 M it does not
element (BRE) [14]. Incubating the cells with increasing amounts of completely abolish the proteins structures [12]. The pH of the
the modified BMP-2s led to dose-dependent induction of luciferase experiment was set at 9.5, which we thought would not disrupt the
activity, indicating the proteins stimulated the BMP signaling amphoteric nature of minTBP-1 needed to interact with Ti. Under
pathway (Fig. 3A). We then used C2C12 cells to test the proteins these conditions, the modified BMPs were incubated with Ti plates
ability to induce ALP activity, which is an indicator of early osteo- for 1 h, after which unassociated proteins were washed out. The
blast differentiation, and found that the modified proteins dose- adsorbed proteins were then evaluated using a sandwich ELISA for
dependently induced ALP activity. Thus the extra artificial domain BMP-2 after eluting them with a solution of 0.2 M HCl, 5 M urea and
does not abolish the biological activity of BMP-2. 0.1% Tween 20 (Fig. 4A). The results showed that wild-type BMP-2,
TBP( 2)-BMP-2 and #69-BMP-2 were all adsorbed onto the Ti at
a density of only about 100 ng/cm2. By contrast, the densities of two
3.5. Ti directionality of the fused BMP-2
of the artificial protein-fused BMPs, #56-BMP-2 and #55-BMP-2,
were 459 56 ng/cm2 and 544 56 ng/cm2, respectively, indi-
At high concentration, recombinant BMP-2 is known to aggre-
cating the Ti-binding ability of these two proteins.
gate at neutral pH [12]. To avoid this aggregation, the protein was
The QCM experiments shown in Fig. 2 indicate that #55 disso-
usually dissolved in acidic solution (e.g., dilute HCl). This charac-
ciated very slowly from Ti, which might have caused the apparent
teristic of BMP-2 made it difficult to assess the ability of the
Ti-binding of #55-BMP-2 in the above assay. To confirm that the
modified BMP-2s to bind Ti because, like other protein aptamers,
relatively rapid and very slow dissociation from Ti observed with
TBP-1 does not bind to Ti at low pH. For that reason, we sought
#56 and #55, respectively, was recapitulated in their fusion
conditions that would enable us to overcome the limitation of BMP-
proteins, we measured the amounts of the proteins released from
2 s poor solubility at neutral pH and to evaluate the modified
a Ti surface into the medium. For this purpose, we incubated Ti
plates that had each been precoated with one of the fusion proteins
in DMEM containing 15% FBS for 2 days at 37 C, after which the
14000
A
BMP2
TBP(x2)-BMP2
12000
#69-BMP2
700
A
#56-BMP2
10000
600
#55-BMP2
8000
500
400
6000
300
4000
200
2000
100
0 0
10 100 1000 10000
protein conc [ng/mL]
450000
B
BMP2
160
400000 B
TBP(x2)-BMP2
140
350000
#69-BMP2
120
#56-BMP2
300000
100
#55-BMP2
250000
80
200000
60
150000
40
100000
20
0
50000
0
10 100 1000 10000
protein conc. [ng/mL]
Fig. 3. Biological activities of the modified BMP-2s. (A) Induction of luciferase activity Fig. 4. Quantification of proteins adsorbed onto Ti and subsequently released. (A) The
in BRE-luciferase tester cells was measured as a function of protein concentration. (B) amounts of modified BMP-2 on Ti surfaces were estimated as described in Section 2.
Induction of ALP activity in C2C12 cells was assayed as a function of protein (B) The amounts of modified BMP-2 released from Ti into culture medium were also
concentration. measured. The error bars represent the standard deviation for five experiments.
2
ng/ cm
RLU/ lysate proein [µg]
2
ng/ cm
RFU/ lysate protein [µg]
BMP-2
TBP(x2)-
BMP-2
#69-BMP-2
#56-BMP-2
#55-BMP-2
BMP-2
TBP(x2)-
BMP-2
#56-BMP-2
#55-BMP-2
#69-BMP-2
K. Kashiwagi et al. / Biomaterials 30 (2009) 1166 1175 1171
amounts of protein released were estimated using an ELISA 3.7. Biological activity of BMP-2s adsorbed onto Ti surfaces
(Fig. 4B). As predicted, approximately 30% of the #56-BMP-2
immobilized on the Ti was released into the medium, while less Given that the mode of binding appears to influence the
than 5% of #55-BMP-2 was released, which is consistent with the accessibility of BMPR to the immobilized BMP, we next investigated
tight binding of #55-BMP-2 to Ti. We then confirmed that the extent to which the difference in binding mode affected the
substantial amounts of protein remained on the Ti after incubation biological activity of the immobilized BMP-2. After coating Ti plates
for 2 days by detaching it with a solution of 0.2 M HCl, 5 M urea and with #56-BMP-2 or #55-BMP-2, tester cells (BRE-luciferase, see
0.1% Tween 20 (Supplementary figure 2). above) were seeded directly onto the plates and incubated for 2
days. The attached cells were then lysed and their luciferase
activities were measured (Fig. 6). We found that cells grown on
#56-BMP-2-coated Ti plates showed much greater luciferase
3.6. Accessibility of the BMP receptor to the immobilized BMP-2
activity than those on #55-BMP-2-coated plates, indicating that
#56-BMP-2 was more active than #55-BMP-2 when immobilized
The experiments summarized in Fig. 4 show that both #56-
on a Ti surface.
BMP-2 and #55-BMP-2 bind to Ti surfaces, though the modes of
their binding differ: whereas #56-BMP-2 reversibly associates with
Ti, #55-BMP-2 is irreversibly bound. The latter is most likely 3.8. Staining ALP and myotubes in differentiated cells
mediated by a strong hydrophobic interaction between #55 and the
Ti surface. To determine the effect of the differences in the binding We also assessed the ability of cells to differentiate on Ti plates
mode on the biological activity of BMP-2, we next evaluated the coated with BMP-2. Premyoblastic C2C12 cells were cultured on
accessibility of the immobilized BMP-2 to a recombinant extracel- BMP-2-coated Ti plates for 9 days in medium that allowed the cells
lular domain of BMPR-1A. After coating Ti plates with each of the to differentiate into an osteoblastic state [34]. Subsequent BCIP/NBT
modified BMP-2s, the plates were incubated with a BMPR-1A/Fc staining revealed high levels of ALP activity in cells cultured on
chimera known to bind to active BMP-2 [33]. The bound chimeric #56-BMP-2-coated Ti plates, indicating that BMP-2-induced
protein was then assayed using an ELISA, which showed that only differentiation had occurred. Substantial ALP activity was also
#56-BMP-2 was bound to BMPR-1A (Fig. 5). observed in cells grown on TBP( 2)-BMP-2- and #55-BMP-2-
A
BMPR-1A/Fc
BMP-2
Artificial Protein
B
1.2
1.2
1
0.8
1
0.6
0.4
0.8
0.2
0
0.6
0.4
0.2
0
(-) #69-BMP-2 #56-BMP-2 #55-BMP-2
Fig. 5. Recognition of immobilized BMP-2 by BMPR-1A. (A) Schematic drawing of the recognition between #56-BMP-2 and BMPR-1A/Fc chimeric protein. (B) The amounts of BMPR-
1A/Fc adsorbed onto the indicated surface-coated Ti plates. The inset shows the amounts of protein coating the Ti plates. The methods of quantification are described in Section 2.
The error bars represent the standard deviation for five experiments.
405
OD
405
OD
(-)
#55-BMP2
#69-BMP2
#56-BMP2
1172 K. Kashiwagi et al. / Biomaterials 30 (2009) 1166 1175
the development of new biomaterials. To realize such reversible
*** ***
4000
immobilization, we have been focusing on peptide aptamers that
3500
interact with the surface of inorganic materials [7, 40]. Peptide
aptamers are created using in vitro evolution systems. Among
3000
the most widely used is a peptide phage system in which pools
2500
of phages displaying various peptides on their surfaces are
incubated with target materials, after which the bound phages
2000
are recovered [6]. Acid treatment is generally used during the
1500
recovery step, which means that a selected peptide is able to
bind to a target molecule under the conditions used for selec-
1000
tion, but detaches under acidic conditions. Thus peptide
500
aptamers inherently bind to targets in a reversible manner.
TBP-1 is a 12-mer peptide aptamer that rapidly associates with
0
Ti surfaces and then dissociates relatively slowly. These character-
#69-BMP2 #56-BMP2 #55-BMP2
istics can be transferred to foreign molecules by appending the
Fig. 6. Induction of luciferase activity in BRE-Luc C2C12 tester cells grown on protein-
hexapeptide minTBP-1 to them [21]. In the present study, we used
coated Ti plates. The error bars indicate the standard deviation for five experiments.
minTBP-1 to endow BMP-2 with the capacity to reversibly bind Ti.
***p<0.001.
Although the strength of the reversible binding by peptide
aptamers is much weaker than covalent linkage, several proto-
cols have been proposed to enhance aptamer binding to a prac-
coated Ti plates, but the apparent ALP activity was most prominent
tical level. Among those increasing is the valency of the
with #56-BMP-2 (Fig. 7A). By contrast, no ALP activity was
displayed peptide. For instance, Sano et al. were able to increase
observed in cells grown on untreated Ti plates or plates treated
the affinity of a Ti-binding peptide (minTBP-1) approximately
with BMP-2 or #69-BMP-2.
1000 by using a ferritin multipeptide display system [21].
C2C12 cells are known to form myotubes (long, fused multi-
Similarly, artificial proteins containing multiple copies of
cellular structures) when cultured for several days at a confluent
minTBP-1 are able to bind to and functionalize Ti surfaces
density, and this myotube formation can be blocked by activation of
(Kokubun et al., in press). Our present results further support
the BMP pathway, which redirects the cells to differentiate from the
the utility of multivalency for increased binding. By fusing an
myoblastic to the osteoblastic lineage [34]. We therefore tested
artificial protein (#56) containing three copies of the minTBP-1
whether this blockade of myoblastic differentiation occurred in
sequence to the N-terminal of BMP-2, we were able to obtain
cells grown on BMP-coated Ti plates. Immunostaining for myosin
a modified BMP-2 that reversibly interacted with Ti. Although
heavy chain revealed that myotubes formed from cells grown on
we did not systematically compare Ti binding by BMP-2 modi-
untreated Ti plates (Fig. 7B), and similar tubular structures were
fied with an artificial protein or the minTBP-1 peptide, we did
also observed when cells were grown on #69-BMP-2-treated
find that addition of two copies of minTBP-1 in tandem was not
plates. By contrast, cells grown on Ti plates pretreated with BMP-2,
sufficient to endow BMP-2 with practical affinity for Ti.
TBP( 2)-BMP-2, #56-BMP-2 or #55-BMP-2 did not form myotubes,
We used our MolCraft system to construct the artificial
indicating that the BMP pathway was activated in those cells.
proteins. In MolCraft, we first design a microgene in which we
pre-embed necessary motif(s) for a particular experiment [24].
4. Discussion In this case, we embedded the minTBP-1 motif. Starting with the
designer microgene, we used microgene polymerization reaction
The efficient clinical utilization of the osteoinductive cytokine (MPR) to prepare tandem polymers of the microgene [10].
BMP has been a long-awaited goal in the area of regenerative Because MPR conditions allow random insertions and/or dele-
medicine. In the area of regenerative ossification, a key focus has tions of nucleotides at the junctions of microgene blocks, the
been the combination of BMP with inorganic materials such as resultant polymers comprise a combinatorial library of three
Ti or various polymers and ceramics [35]. To date, several peptides encoded by a single microgene, and the clones within
cytokines have been successfully immobilized on inorganic the library contain various numbers of the minTBP-1 motif [10].
materials via chemical linkage [36]. To our knowledge, however, Here, we focused on three artificial proteins, #55, #56 and #69,
there has been only a single report in which a BMP was which were chosen based on their good yields and solubility.
chemically linked to an inorganic material [37]. In that report, Because the artificial proteins are encoded by combinations of
the authors claimed BMP-4 could be chemically linked to reading frames different from the one coding minTBP-1, they
a plasma-treated Ti alloy; however, no direct evidence showing provided the opportunity to collect interesting control proteins
the chemical anchorage of BMP-4 was presented. What makes for comparison  e.g., #55 irreversibly bound to Ti, probably
BMP so difficult to handle is its hydrophobic nature. Tight because of its hydrophobic nature, whereas #69 did not asso-
linkage between a protein and a hydrophobic surface increases ciate with Ti. Comparison of #56-BMP-2 and #55-BMP-2 was
the likelihood the protein will become denatured [38]. In addi- especially interesting because it highlighted the importance of
tion, it may be necessary for BMP to dissociate from the surface reversible immobilization on a material surface. In our experi-
once it is recognized by its receptor on a cell surface [39], which ment using BMPR-1A, the immobilized #55-BMP-2 was inac-
is impossible when the protein is covalently linked. cessible to the receptor (Fig. 5B), suggesting the structure of
Biological activities such as cell signaling and cell movement BMP-2 is somehow disrupted by the strong binding between
are accomplished through complex interactions among a multi- #55 and Ti. On the other hand, we doubt that #55, itself, had
tude of biomolecules, and these interactions are mostly revers- a disruptive effect on BMP-2 because #55-BMP-2 showed BMP
ible  i.e., they reflect a combination of electrostatic interactions, activity in its soluble form. It may be that appropriate interac-
hydrogen bonding, van der Waals s force and hydrophobic tion between BMP-2 and its receptor requires transient detach-
interactions, which make short-range, weak and reversible ment of BMP-2 from the Ti.
associations. In that context, we suggest that immobilization We compared the biological activities of #56-BMP-2 and #55-
based on reversible binding should be a focus of research into BMP-2 in two different assays, and both indicated that #56-BMP-2
RLU / lysate protein [µg]
K. Kashiwagi et al. / Biomaterials 30 (2009) 1166 1175 1173
A
BMP-2 TBP(x2)-BMP-2
(-)
#56-BMP-2 #55-BMP-2
#69-BMP-2
B
BMP-2 TBP(x2)-BMP-2
(-)
#56-BMP-2 #55-BMP-2
#69-BMP-2
Fig. 7. Enzymatic and immunochemical staining of cells grown on protein-coated Ti plates. (A) ALP activity was assessed by staining with BCIP/NBT. (B) Myotubes formed in the cells
were stained with an anti-myosin heavy chain antibody (Red). Nuclei were stained with SYTO Green 11 (Green).
was more active than #55-BMP-2. It has been proposed that internalization of the BMP-2 BMPR complex, even if #55-BMP-2
internalization of BMP upon binding to BMPR is necessary for full was recognized by the receptor on the cell surface.
activation of the BMP pathway [39]. Our data are consistent with Several methods for immobilizing BMP on Ti have been repor-
that idea. Irreversible binding of #55-BMP-2 to Ti would prevent ted, including incorporation into calcium phosphate deposit [41],
MF20
SYTO Green 11
MF20
SYTO Green 11
1174 K. Kashiwagi et al. / Biomaterials 30 (2009) 1166 1175
[10] Shiba K, Takahashi Y, Noda T. Creation of libraries with long ORFs by
linkage to a monolayer of a phosphonic acid derivative of Ti [42],
polymerization of a microgene. Proc Natl Acad Sci U S A 1997;94(8):
and adsorption onto chromosulfuric acid-treated Ti [43]. In addi-
3805 10.
tion, attempts have been made to attach BMP to various other
[11] Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. New
York: Cold Spring Harbor Laboratory Press; 2001.
materials, including collagen sponge [44], block copolymers [45],
[12] Hillger F, Herr G, Rudolph R, Schwarz E. Biophysical comparison of BMP-2,
gelatin hydrogel [46] and silk protein [47]. Our method provides
ProBMP-2, and the free pro-peptide reveals stabilization of the pro-peptide by
a new approach to the immobilization of biomolecules on the
the mature growth factor. J Biol Chem 2005;280(15):14974 80.
surfaces of inorganic materials. The strength of our method is the [13] Shiba K, Takahashi Y, Noda T. On the role of periodism in the origin of proteins.
J Mol Biol 2002;320(4):833 40.
programmability of the functional motifs, which enables modula-
[14] Korchynskyi O, ten Dijke P. Identification and functional characterization of
tion of binding mode, binding affinity and/or binding target using
distinct critically important bone morphogenetic protein-specific response
appropriate designs and selection of artificial proteins. This degree elements in the Id1 promoter. J Biol Chem 2002;277(7):4883 91.
[15] Bader D, Masaki T, Fischman DA. Immunochemical analysis of myosin heavy
of flexibility would be difficult to achieve with other existing
chain during avian myogenesis in vivo and in vitro. J Cell Biol 1982;95(3):
methods.
763 70.
[16] Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel
regulators of bone formation: molecular clones and activities. Science
5. Conclusions
1988;242(4885):1528 34.
[17] Cui Y, Jean F, Thomas G, Christian JL. BMP-4 is proteolytically activated by furin
Here we have described the construction of engineered BMP-
and/or PC6 during vertebrate embryonic development. Embo J
1998;17(16):4735 43.
2 that has directionality toward Ti surfaces. This modified BMP-2
[18] Ruppert R, Hoffmann E, Sebald W. Human bone morphogenetic protein 2
was endowed with the ability to reversibly interact with Ti
contains a heparin-binding site which modifies its biological activity. Eur J
surfaces by appending an N-terminal artificial protein domain
Biochem 1996;237(1):295 302.
[19] Sano K, Sasaki H, Shiba K. Specificity and biomineralization activities of Ti-
(#56) created by programming the Ti-binding motif minTBP-1.
binding peptide-1 (TBP-1). Langmuir 2005;21(7):3090 5.
For comparison, we examined the effects #55, which has no
[20] Terskikh AV, Le Doussal JM, Crameri R, Fisch I, Mach JP, Kajava AV.   Pepta-
minTBP-1 motif and binds to Ti surfaces irreversibly, probably
body  : a new type of high avidity binding protein. Proc Natl Acad Sci U S A
because of its hydrophobic nature. Both #56-BMP-2 and #55- 1997;94(5):1663 8.
[21] Sano K, Ajima K, Iwahori K, Yudasaka M, Iijima S, Yamashita I, et al. Endowing
BMP-2 bound to Ti, but whereas a large fraction of the bound
a ferritin-like cage protein with high affinity and selectivity for certain inor-
#56-BMP-2 was released into medium after incubation for 2
ganic materials. Small 2005;1(8 9):826 32.
days, most #55-BMP-2 was retained on the Ti surface, indicating [22] Shen SH. Multiple joined genes prevent product degradation in Escherichia
coli. Proc Natl Acad Sci U S A 1984;81(15):4627 31.
that the respective binding modes of #56 and #55 were
[23] Shiba K, Minamisawa T. A synthesis approach to understanding repeated
inherited by the fusion proteins. In vitro experiments revealed
peptides conserved in mineralization proteins. Biomacromolecules 2007;8(9):
more prominent activation of BMP signaling and induction of
2659 64.
[24] Shiba K. MolCraft: a hierarchical approach to the synthesis of artificial
differentiation in cells grown on #56-BMP-2-coated Ti than in
proteins. J Mol Catal B 2004;28(4 6):145 53.
those grown on #55-BMP-coated Ti. Collectively, these data
[25] Shiba K, Shirai T, Honma T, Noda T. Translated products of tandem microgene
indicate the importance of reversible immobilization when using
repeats exhibit diverse properties also seen in natural proteins. Protein Eng
2003;16(1):57 63.
biomolecules to modify the surfaces of inorganic materials.
[26] Kashiwagi K, Isogai Y, Nishiguchi K, Shiba K. Frame shuffling: a novel method
for in vitro protein evolution. Protein Eng Des Sel 2006;19(3):135 40.
Acknowledgement
[27] Saito H, Honma T, Minamisawa T, Yamazaki K, Noda T, Yamori T, et al.
Synthesis of functional proteins by mixing peptide motifs. Chem Biol
2004;11(6):765 73.
We thank Dr. T. Murakami for kindly providing BRE-Luc C2C12
[28] Saito H, Kashida S, Inoue T, Shiba K. The role of peptide motifs in the evolution
cells, and Drs. T. Imamura, S. Maeda, Y. Takaoka and K. Sano for their
of a protein network. Nucleic Acids Res 2007;35(19):6357 66.
helpful discussions. This work was partly supported by Grant-in- [29] Saito H, Minamisawa T, Shiba K. Motif programming: a microgene-based
method for creating synthetic proteins containing multiple functional motifs.
Aid for Scientific Research on Priority Areas (20034057 to K. K.) and
Nucleic Acids Res 2007;35(6):e38.
Grant-in-Aid for Young Scientists (B) (18710164 to T. T.) from
[30] Saito H, Minamisawa T, Yamori T, Shiba K. Motif-programmed artificial protein
Ministry of Education, Science, Sports and Culture, Japan. induces apoptosis in several cancer cells by disrupting mitochondria. Cancer
Sci 2008;99(2):398 406.
[31] Karlsson M, Ekeroth J, Elwing H, Carlsson U. Reduction of irreversible protein
Appendix. Supplementary figures
adsorption on solid surfaces by protein engineering for increased stability. J
Biol Chem 2005;280(27):25558 64.
[32] Hook F, Kasemo B, Nylander T, Fant C, Sott K, Elwing H. Variations in coupled
Supplementary figures associated with this article can be found,
water, viscoelastic properties, and film thickness of a Mefp-1 protein film
in the online version, at doi:10.1016/j.biomaterials.2008.10.040.
during adsorption and cross-linking: a quartz crystal microbalance with
dissipation monitoring, ellipsometry, and surface plasmon resonance study.
Anal Chem 2001;73(24):5796 804.
References
[33] Wendler J, Vallejo LF, Rinas U, Bilitewski U. Application of an SPR-based
receptor assay for the determination of biologically active recombinant bone
[1] Brunette D, Tengvall P, Textor M, Thomsen P. Titanium in medicine. Berlin &
morphogenetic protein-2. Anal Bioanal Chem 2005;381(5):1056 64.
Heidelberg: Springer-Verlag; 2001.
[34] Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda T, et al.
[2] Brunski JB. In vivo bone response to biomechanical loading at the bone/
Bone morphogenetic protein-2 converts the differentiation pathway of
dental-implant interface. Adv Dent Res 1999;13:99 119.
C2C12 myoblasts into the osteoblast lineage. J Cell Biol 1994;127(6 Pt 1):
[3] Le Guehennec L, Soueidan A, Layrolle P, Amouriq Y. Surface treatments of
1755 66.
titanium dental implants for rapid osseointegration. Dent Mater
[35] Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and
2007;23(7):844 54.
regeneration. Nat Biotechnol 1998;16(3):247 52.
[4] Schuler M, Trentin D, Textor M, Tosatti SG. Biomedical interfaces: titanium
[36] Metzger W, Grenner N, Motsch SE, Strehlow R, Pohlemann T, Oberringer M.
surface technology for implants and cell carriers. Nanomedicine
Induction of myofibroblastic differentiation in vitro by covalently immobilized
2006;1(4):449 63.
transforming growth factor-b1. Tissue Eng 2007;13(11):2751 60.
[5] Roach P, Farrar D, Perry CC. Surface tailoring for controlled protein adsorption:
[37] Puleo DA, Kissling RA, Sheu MS. A technique to immobilize bioactive proteins,
effect of topography at the nanometer scale and chemistry. J Am Chem Soc
including bone morphogenetic protein-4 (BMP-4), on titanium alloy. Bioma-
2006;128(12):3939 45.
terials 2002;23(9):2079 87.
[6] Baneyx F, Schwartz DT. Selection and analysis of solid-binding peptides. Curr
[38] Nakanishi K, Sakiyama T, Imamura K. On the adsorption of proteins on solid
Opin Biotechnol 2007;18(4):312 7.
surfaces, a common but very complicated phenomenon. J Biosci Bioeng
[7] Smith GP, Petrenko VA. Phage display. Chem Rev 1997;97(2):391 410.
2001;91(3):233 44.
[8] Sano K, Shiba K. A hexapeptide motif that electrostatically binds to the surface
[39] Hartung A, Bitton-Worms K, Rechtman MM, Wenzel V, Boergermann JH,
of titanium. J Am Chem Soc 2003;125(47):14234 5.
Hassel S, et al. Different routes of bone morphogenic protein (BMP)
[9] Hayashi T, Sano K, Shiba K, Kumashiro Y, Iwahori K, Yamashita I, et al.
receptor endocytosis influence BMP signaling. Mol Cell Biol 2006;26(20):
Mechanism underlying specificity of proteins targeting inorganic materials.
7791 805.
Nano Lett 2006;6(3):515 9.
K. Kashiwagi et al. / Biomaterials 30 (2009) 1166 1175 1175
[40] Kase D, Kulp III JL, Yudasaka M, Evans JS, Iijima S, Shiba K. Affinity selection of [44] Morin R, Kaplan D, Perez-Ramirez B. Bone morphogenetic protein-2 binds as
peptide phage libraries against single-wall carbon nanohorns identifies a multilayers to a collagen delivery matrix: an equilibrium thermodynamic
peptide aptamer with conformational variability. Langmuir 2004;20(20): analysis. Biomacromolecules 2006;7(1):131 8.
8939 41. [45] Saito N, Okada T, Horiuchi H, Murakami N, Takahashi J, Nawata M, et al. A
[41] Liu Y, de Groot K, Hunziker EB. BMP-2 liberated from biomimetic implant biodegradable polymer as a cytokine delivery system for inducing bone
coatings induces and sustains direct ossification in an ectopic rat model. Bone formation. Nat Biotechnol 2001;19(4):332 5.
2005;36(5):745 57. [46] Takahashi Y, Yamamoto M, Yamada K, Kawakami O, Tabata Y. Skull bone
[42] Adden N, Gamble LJ, Castner DG, Hoffmann A, Gross G, Menzel H. Phosphonic regeneration in nonhuman primates by controlled release of bone morphoge-
acid monolayers for binding of bioactive molecules to titanium surfaces. netic protein-2 from a biodegradable hydrogel. Tissue Eng 2007;13(2):293 300.
Langmuir 2006;22(19):8197 204. [47] Karageorgiou V, Meinel L, Hofmann S, Malhotra A, Volloch V, Kaplan D. Bone
[43] Jennissen HP, Zumbrink T, Chatzinikolaidou M, Steppuhn J. Biocoating of morphogenetic protein-2 decorated silk fibroin films induce osteogenic
implants with mediator molecules: surface enhancement of metals by treat- differentiation of human bone marrow stromal cells. J Biomed Mater Res A
ment with chromosulfuric acid. Materwiss Werksttech 1999;30(12):838 45. 2004;71(3):528 37.