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.