Figure captions
Figure 1. (a) Construction flow of the recombinant plasmids
from genetic blocks coding for the crystalline region of silk fibroin
((AGSGAG)3AS, blue arrow) and for the minimal titanium
binding motif (RKLPDA , orange arrow). B, N, S, and H representBam HI, Nhe I, Spe I, and Hind III,
respectively. From the resultants plasmids, the His-tagged proteins,
(Bc3) 2, (Bc3) 4, (Bc3)2T, and (Bc3) 4T were produced in
BL21(DE3)pLysS and purified by nickel-chelate chromatography, whose
domain structures are schematically illustrated by the colored ellipses
(indicated in the inner box). (b) The structure of the vector for
constructing the transgenic silkworm. The gene for
[TS[(AGSGAG)3AS]2RKLPDA]8is under the control of Fibroin H promoter. (c) The structure of
proteins translated from pBac[HC-TiBP-3xP3EGFP], which has the
N-terminal domain (NTD), the C-terminal domain (CTD), and C-terminal
His-tag, which were derived from the parental vector,
pBac[3xP3-EGFPafm] (Horn et al., 2002). Arrows indicate right and
left arms of the transposon piggyBac. EGFP was used for screening the
transgenic silkworm.
Figure 2. (a) Schematic drawings representing the changes in
frequency (∆f; left axis) of titanium sensor upon (i) buffer injection,
(ii) sample injection, (iii) buffer wash and (iv) water wash in QCM
analyses. (b) Changes in resonance frequency with 0.5 µm (Bc3)2 (blue) and (Bc3) 2T (green). (c)
Changes in resonance frequency with 0.5 µm (Bc3) 4(blue) and (Bc3) 4T (green).
Figure 3. (a) SDS-PAGE and Coomassie Brilliant Blue R250
staining for the functionalized SF, TiBP-SF, and its parental SF. The
arrow indicates the artificial protein containing
[TS[(AGSGAG)3AS]2RKLPDA]8(Figure 1 c). The open circle, the closed circle, and the triangle
indicate Fibroin H-chain protein, Fibroin L-chain protein, and Fibro
hexamerin protein (Inoue et al., 2000), respectively. (b) Western
blotting using anti-His antibody, which recognizes the C-terminal
His-tag of the artificial SF.
Figure 4. (a) Changes in resonance frequency of titanium sensor
with 1 mg/mL of SF (blue) and TiBP-SF (green). Details of experimental
procedures are described in Figure 1 caption and MATERIALS AND METHODS.
(b) Dispersion of TiO2 particles (21 nm) with TBP
(RKLPDA) peptide, Silk-TBP (AGSGAGGRKLPDAGGAGSGAG) peptide, Silk
((AGSGAG)2 ) peptide and no peptide (control). Details
of experiments are described in MATERIALS AND METHODS.
Figure 5. (a) SEM images (magnification of 2000×) of non-coated
(cont), SF-coated and TiBP-SF-coated polished Ti surfaces after
cultivating MC3T3-E1 cells for 14 or 21 days. The bars indicate 10 µm.
(b) Effects of TiBP-SF coating on the alkaline phosphatase (ALP)
activity and the expression level of osteocalcin (OC) of MC3T3-E1 cells
after 14 and 21 days incubation. Values are expressed as means ± SD (n =
3). Asterisks indicate P < 0.05 in the t-test. (c) Effect of
TiBP-SF coating on the mineralization in MC3T3-E1 cells. Cells were
stained with Alizarin Red (left) and quantified as described in the
MATERIALS AND METHODS after 14 and 21 days incubation. Asterisks
indicate P < 0.05 in the t-test. (d) Expression levels of mRNA
for Runx2, Osterix, ALP) Collagen Type I (Col I), Osteopontin (OPN) and
OC, which were quantified by quantitative RT-PCR, in MC3T3-E1 cells
after 14 and 21 days incubation on non-coated (control), SF-coated, and
TiBP-SF-coated polished Ti surfaces. The relative mRNA expression levels
were calculated as a ratio to β-actin expression of each sample. Values
represent means ± SD from three independent experiments. Asterisks
indicate P < 0.05 in the t-test.
Figure 6. 13C CP/MAS NMR spectra (10–70 ppm)
of (a) 13C Silk-TBP
(AGSG[1-13C]AGGRKLPD[3-13C]AGGAGSGAG)
and (b) 13C Silk-TBP adsorbed on the surface of
TiO2 nanoparticles together with the assignments.
Table 1. 13C chemical shifts and assignments of13C CP/MAS NMR spectra of (a) 13C
Silk-TBP and (b) 13C Silk-TBP adsorbed on the surface
of TiO2 nanoparticles.
Chemical Shift Assignment
……………………………………………………………………………………………………………..
(b) 14.5
ppm Ala 13Cb a-helix
(a) 16.2 ppm Ala 13Cb random
coil
(b) 21
ppm Leu
Cd1,d2 Lys Cg
(b) 25.5
ppm Lys
Cd
(a) 42 ppm (broad) Gly Ca
(b) 42–43 ppm
(broad) Gly Ca
(a) 49 ppm (broad) Ala Ca random coil -
……………………………………………………………………………………………………………..