INTRODUCTION
“Motif” is the versatile functional unit for constructing artificial biomacromolecules. In some cases, motif can endow non-biomolecules with its assigned functions, while in other cases different motifs can be combined to create artificial proteins having novel combinations of functions (Shiba, 2010). Motif sequences are identified both from natural sequences of proteins and from in vitro evolution systems. In the latter cases, a phage display system (Scott & Smith, 1990)) has been employed to create an artificial peptide that binds to biomolecules and non-biological materials such as inorganic materials. The hexapeptidic RKLPDA sequence is one such inorganic material binding peptides. It was originally isolated as a 12-mer peptide binder to a titanium (Ti) surface by using a peptide display system (Sano & Shiba, 2003). Subsequent analyses have revealed that the N-terminal RKLPDA (termed minTBP-1) is both necessary and sufficient for the binding (Sano, Sasaki, & Shiba, 2005). This minTBP-1 motif has been widely employed as an appendix to provide an affinity to Ti in ferritin (Sano, Ajima, et al., 2005), artificial matrix proteins (Kokubun, Kashiwagi, Yoshinari, Inoue, & Shiba, 2008), BMP-2 (Kashiwagi, Tsuji, & Shiba, 2009), antimicrobial peptide (Geng et al., 2018; Yoshinari, Kato, Matsuzaka, Hayakawa, & Shiba, 2010), calcification protein (Tsuji, Oaki, Yoshinari, Kato, & Shiba, 2010), DNA-polymerase (Nishida et al., 2015), carbon nanomaterial-binding peptide (Kokubun, Matsumura, Yudasaka, Iijima, & Shiba, 2018), and self-assembled chemical compounds (Ikemi et al., 2010). Interestingly, it has been revealed that the interaction between minTBP-1 and the surfaces of Ti is governed by reversible interactions (Brandt & Lyubartsev, 2015; Hayashi et al., 2009; Sano, Ajima, et al., 2005; Schneider & Ciacchi, 2012; Skelton, Liang, & Walsh, 2009). In biological systems, weak, specific, reversible binding plays a pivotal role in various biological activities. Indeed, previous studies have shown certain biological activities has been enhanced by adding minTBP-1 to parental biomolecules(Kashiwagi et al., 2009; Kokubun et al., 2008).
Silk fibroin (SF) from Bombyx mori possesses many superior and inherent properties as a biomaterial, including advantageous mechanical properties, environmental stability, biocompatibility, low immunogenicity, and biodegradability (Aigner, DeSimone, & Scheibel, 2018; Asakura & Kaplan, 1994; Asakura, Tanaka, & Tanaka, 2019; Koeppel & Holland, 2017; Koh et al., 2015; Velema & Kaplan, 2006; Vepari & Kaplan, 2007). SF fiber has been used in sutures in the surgical field for more than 2,000 years (Thurber, Omenetto, & Kaplan, 2015). Recently, the representative repeating unit of the crystalline fraction (56% of whole SF) (Strydom, Haylett, & Stead, 1977; Zhou et al., 2001; Zhou et al., 2000), AGSGAG sequence, has been used as a motif to create artificial SFs (Asakura et al., 2005; Asakura & Yao, 2002; Yu Suzuki, Aoki, Nakazawa, Knight, & Asakura, 2010; Yao, Ohgo, Sugino, Kishore, & Asakura, 2004). Motif-based artificial protein constructions provide us with opportunities to create artificial biomolecules having novel biological activities or novel combinations of exiting activities (M. Yang & Asakura, 2005; Mingying Yang, Kawamura, Zhu, Yamauchi, & Asakura, 2009; M. Yang, Muto, Knight, Collins, & Asakura, 2008; M. Yang, Tanaka, et al., 2008; M. Yang, Yamauchi, Kurokawa, & Asakura, 2007). However, the production of these artificial proteins has generally been dependent on microorganisms or cell culture systems, which are obstacles to bulk production.
Recently, a germ line transformation method for the silkworm has been developed using the transposon piggyback (Kojima et al., 2007; Tamura et al., 2000). This enabled the production of functionalized transgenic (TG) SF in a large quantity such as cell-adhesive silks (Asakura et al., 2014; Yanagisawa et al., 2007), colored fluorescent silk (Iizuka et al., 2013), calcium binding silk (Nagano et al., 2011), and silk better suited to artificial blood vessels (Saotome et al., 2015). Such an improvement of the properties of SF for biomaterials has been attained by incorporating suitable DNA sequences. SF consists of a heavy chain (H-chain) and light chain (L-chain) connected by a disulfide bond, as well as a glycoprotein named P25. It is secreted into the posterior silk gland and is thought to be assembled into a high molecular mass elementary unit with a ratio of 6:6:1 (Couble, Chevillard, Moine, Ravel-Chapuis, & Prudhomme, 1985; Takei, Kikuchi, Kikuchi, Mizuno, & Shimura, 1987; Tanaka et al., 1999; Tanaka, Mori, & Mizuno, 1993). The new gene that follows the SF gene is expressed as a new functionalized SF in the posterior silk gland. Subsequently, the SF stored in the middle silk gland is spun out through the anterior silk gland and converted into SF fibers, being coated by silk sericin that emerges in the middle silk gland. After removal of silk sericin by a degumming process, we obtained pure SF. There are many advantages in preparing functionalized SF suitable for biomaterials by the TG technique: (1) the TG silkworms are easy to handle and the larvae are well adapted for artificial rearing; (2) the adult moths cannot fly away and therefore cannot live in the wild; (3) the expression of the recombinant silk proteins can be confirmed visually by fusion of green fluorescent proteins as a marker.
We were interested in the possibility that a combination of the AGSGAG and minTBP-1 motifs could further enhance the functionality of SF when used as a coating agent for titanium substrates. While titanium has already been widely used in dental implants, artificial joints, and vessel stents (Brunette, Tengvall, Textor, & Thomsen, 2001), further improvement of its biocompatibility is still required (Pagel et al., 2016; Schuler et al., 2006; Vidal et al., 2013; Zhang, Zhang, Zhu, Kang, & Neoh, 2008). In the present study, we first synthesized artifice polypeptides containing both AGSGAG and minTBP-1 motifs, and confirmed their reversible interacting ability to titanium, which characterizes the minTBP-1 motif. Then, we constructed the transgenic B. morithat spins the cocoon containing the artificial SF. The modified SF prepared from the cocoons enhanced the ossific differentiation of MC3T3-E1 cells grown on the titanium plates coated by the artificial SF. The binding mechanism of silk-TBP on the surface of TiO2nanoparticles was examined briefly using the model peptides made from the AGSGAG and minTBP-1 motifs, and 13C solid-state NMR.