Biomedical Engineering Reference
In-Depth Information
plants (tobacco, potato,
Arabidopsis
) has also been attempted (Scheller et al., 2001;
Scheller et al., 2004) (Menassa et al., 2004; Patel et al., 2007; Yang et al., 2005). How-
ever, rather low expression levels are often reported (Scheller et al., 2001; Scheller et
al., 2004; Patel et al., 2007; Yang et al., 2005) and large scale production of spidroins
has so far only resulted in fairly low yields (Menassa et al., 2004). For biomedical ap-
plications, plant antigens, mycotoxins, pesticides, and herbicides can be problematic
(Doran, 2000). Various mammalian cell systems have been used for expression of
spidroins, the most promising being a 60 kDa ADF-3 protein secreted from cell lines
grown in a hollow fiber reactor (Arcidiacono et al., 2002). Since most mammalian
cells require complex media with biological supplement, this production system may
cause contamination by virus, prions, and oncogenic DNA (Ma et al., 2003). Spidroin
production in mammary glands and secretion into milk has been tried in mice (Xu et al.,
2007) and goats (Williams, 2003), although potential contaminations are also a ma-
jor drawback with production in transgenic animals. So far, cytocompatibility studies
have been performed on recombinant spidroins produced in bacteria, yeast, and plants
(see Table 1).
table 1.
Recombinant spider silk proteins in cytocompatibility studies.
Construct
nam
e
Size
(kDa)
Production
host
Type of
silk
Species
Added
motif
Steril-
ization
technique
Tested cell
type
References
SO1
51
Tobacoo
Dragline
silk
(MaSp1)
Nephila clavipes
ELP
filtration
human
primary
chondro-
cytes,
CHO-K1
Scheller et al
2004
4RepCT
21
E.coli
Dragline
silk
(MaSp1)
Euprosthenops
australis
none
filtration,
autoclaving
HEK 293,
human
primary
fibroblasts
Stark et al
2007, Hed-
hammar et al
2008, Widhe
et al 2010
IF9
94
E.coli
Dragline
silk
(MaSp1)
Nephila clavipes
none
96% ethanol 3T3 fibro-
blasts
Agapov et al
2009
15mer
51
E.coli
Dragline
silk
(MaSp1)
Nephila clavipes
RGD,
RGD-R5
(from
silaffin)
ethylene
oxide, 70%
ethanol, UV
hMSC,
MC3T3-E1
osteo-
blastic
precursors,
mesen-
chymal
stem cells
(hMSCs)
Bini et al
2006, Morgan
et al 2008,
Mieszawska
et al 2010
pNSR16
pNSR32
not
specified
E.coli
Dragline
silk
not specified
RGD
not specified
3T3 fibro-
blasts
Wang et al
2009
Control of assembly of recombinant spidroins
As mentioned above, expression of recombinant spidroins has often resulted in wa-
ter insoluble products. Often the purification processes in these cases are obligated
to include solubilisation steps using urea, guanidine hydrochloride, lithium bromide,
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