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
(a)                                       (b)
……………………………………………………………………………………………………………..
(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              -
……………………………………………………………………………………………………………..