Construction of transgenic Bombyx mori that spins the cocoons containing the functionalized silk fibroin (TiBP-SF).
The QCM analyses indicated that the artificial proteins composed of SF core motifs and minTBP-1 motif have the abilities to bind to (and detach from) the titanium surface. To evaluate the effect of the appended minTBP-1 motif on the nourishing ability of SF for cells, we decided to construct the large-scale production system for SF-minTBP-1 proteins. For this experiment, we selected the (Bc3)2T = [TS[(AGSGSG)3AS]2RKLPDA] as a core unit. This was because in the QCM experiments, (Bc3)2T bound in larger quantities and detached faster (especially in water) than (Bc3)4T, suggesting the properties of minTBP-1 was more manifested in this formula.
We have already established the transgenic B. mori system, which has enabled us to prepare large amounts of engineered SF (Asakura et al., 2014; Iizuka et al., 2013; Kojima et al., 2007; Nagano et al., 2011; Saotome et al., 2015; Tamura et al., 2000; Yanagisawa et al., 2007). In this study, a DNA fragment coding [TS[(AGSGSG)3AS]2RKLPDA]8((Bc3)2T) was inserted into the plasmid pHC-EGFR(Kojima et al., 2007) to construct pBac[HC-TiBP-3xP3EGFP] (Figure 1 b), which was injected into the eggs of the silkworm, and transgenic silkworms were selected as described previously (Kojima et al., 2007; Tamura et al., 2000; Yanagisawa et al., 2007). The established transgenic silkworms spin cocoons that are apparently indistinguishable from wild-type samples (data not shown). From the cocoons, SF proteins were prepared following standard procedures described in MATERIALS AND METHODS, and the obtained fibroin proteins (named TiBP-SF) were analyzed by SDS-PAGE followed by CBB staining (Figure 3 a). From the TiBP-SF sample, in addition to SF H-chain and SF L-chain proteins, an additional protein band was observed that was not detected from the wild-type SF sample. Western blotting using an anti-His tag antibody confirmed that the protein was derived from a transgenic gene (Figure 3 b).
The apparent molecular weight of the appendix protein (75 K) was larger than that of the estimated molecular weight (52 kD). Similar abnormal mobilities of the modified SFs in SDS-PAGE have also been reported in other experiments (Asakura et al., 2014; Iizuka et al., 2013; Kojima et al., 2007; Saotome et al., 2015; Teule et al., 2012; Yanagisawa et al., 2007), which may be due to either skewed amino acid compositions of the proteins or particular secondary structures. Because the ratio of minTBP-1 motif to SF motif should be extremely small (approximately 0.1 to 0.9 % in a similar system (Yanagisawa et al., 2007)) compared to the model artificial proteins used in the experiments of Figure 2, we were interested in the effect of minTBP-1 on the binding ability of SF to titanium. As shown in Figure 4 a, QCM analyses indicated that TiBP-SF decreased the amounts of protein absorbed on the titanium sensor, which agreed well with the results obtained from (Bc3)2T and (Bc3)4T. This means that with a decreased ratio of minTBP-1/silk motif, the minTBP-1 motif masks the binding ability of sill peptide to the material surface. Thus, doping SF with minTBP-1 dramatically changed the binding properties of SF. This notion was further confirmed by the observation that the Silk model peptide (AGSGAG)2 was not able to disperse Ti particles in aqueous solution, whereas Silk-TBP model peptide AGSGAGGRKLPDAGGAGSGAG can endow the Ti particle with dispersibility similar to the mimTBP-1 peptide (Figure 4 b).