Spider Silk-production and Biomedical Applications - Natural Polymers, Biopolymers, Biomaterials, and Their Composites, Blends, and IPNs - page 148

repeat followed by an alanine block appear consecutively about 100 times (Ayoub et
al., 2007).
The MaSps are produced by a single layered epithelium in paired major ampullate
glands that are connected via secretory ducts to the spinnerets on the ventral surface
of the abdomen (Kovoor, 1987). The spidroins can be stored in the glandular lumen
until they are converted into a solid fiber in the end of the secretory duct (Chen et al.,
2002; Work, 1977). Even though the dragline silk proteins are prone to assemble, the
spider manages to keep them in soluble form at very high concentrations (30-50%,
w/v)(Chen et al., 2002; Hijirida et al., 1996). The fiber forming process is well con-
trolled, enabling an almost instantaneous formation of the fiber in a defined segment of
the duct, thus avoiding a fatal spread of the assembly process to the dope in the gland
(Work, 1977). This process probably involves shear forces, dehydration, a drop in pH
and changes in the ion composition along the duct (Chen et al., 2002; Dicko et al.,
2004; Knight and Vollrath, 2001).
Production of recombinant spidroins in different hosts
Two strategies have been most commonly used for recombinant production of spidro-
ins; to express parts of the native sequences, example (Arcidiacono et al., 1998; Stark
et al., 2007), or iterated consensus repeats, example (Bini et al., 2006; Huemmerich et
al., 2004a). Both these strategies have lead to problems such as truncations, translation
pauses and rearrangements (Arcidiacono et al., 1998; Fahnestock and Irwin, 1997).
Moreover, impoverishment of certain tRNA, proteolysis and low solubility of the pro-
tein has been observed (Fahnestock and Irwin, 1997; Prince et al., 1995; Winkler et al.,
1999). There are no known essential post translational modifications of spidroins, al-
though phosphorylation of tyrosine and serine residues in spidroins have been reported
(Michal et al., 1996). Therefore, several expression hosts, including prokaryotes, have
been attempted (reviewed in (Rising et al.)). From the Gram-negative enterobacterium
Escherichia coli ( E. coli ), several successful fermentation processes have been report-
ed (Arcidiacono et al., 1998, 2002; Bini et al., 2006; Brooks et al., 2008; Fukushima,
1998; Huang et al., 2007; Huemmerich et al., 2006; Slotta, 2006; Slotta et al., 2008;
Stephens et al., 2005; Winkler et al., 2000; Zhou et al., 2001). However, low yields
(Arcidiacono et al., 1998; Prince et al., 1995), accumulation of inclusion bodies
(Liebmann, 2008) and low protein solubility (Bini et al., 2006; Fukushima, 1998; Mello
et al., 2004; Szela et al., 2000; Winkler et al., 2000; Wong Po Foo et al., 2006) often
occur. Moreover, the outer membrane of Gram-negative bacteria such as E. coli con-
tains lipopolysaccharides (LPS) that are endotoxic and thus often have to be removed
before the product is used in biomedical applications. Gram-positive bacteria, exam-
ple Bacillus subtilis and Staphylococcus aureus , lack an outer membrane and therefore
LPS (Sandkvist and Bagdasarian, 1996). This trait also simplifies secretion of recom-
binant proteins, although genetic manipulations are not as easy as with E. coli (Wong,
1995). Since expression in yeast gives rise to less translation stops, this host system
might be more suitable than bacteria for long and repetitive sequences, although the
production rate is slower. The methylotrophic yeast Pichia pastoris has been used in
order to produce spidroins as well as amphiphilic silk-like protein (Fahnestock and
Bedzyk, 1997; Werten et al., 2008). Recombinant expression of spidroins in various