Biomedical Engineering Reference
In-Depth Information
Our strategy for the spontaneous dimerization of EGF was to incorporate an
a
-helical oligopeptide with the ability to participate in forming a coiled-coil structure
[
99
]. We implemented a heterodimerization system in which (KELASVK)
5
(K5) and (EKLASVE)
5
(E5) peptides could form stable coiled-coil heterodimers.
We then synthesized two chimeric proteins that contained EGF attached to either
the K5 (EGF-K5-His) or the E5 (EGF-E5-His). Both had the hexahistidine
sequence added to the C-terminus for anchoring through coordination with Ni
2+
ions fixed to a substrate.
Analyses by native polyacrylamide gel electrophoresis and circular dichroism
(CD) spectroscopy revealed that spontaneous coiled-coil associations between
EGF-E5-His and EGF-K5-His promoted heterodimer (dEGF-His) formation. The
CD spectroscopic analysis suggested that the E5 peptide in monomeric EGF-E5-
His had a disordered structure. However, the
a
-helical structure was induced in the
E5 peptide when it associated with EGF-K5-His. These findings are shown
schematically in Fig.
6
.
We tested adhesion and proliferation of rat NSCs on surfaces with immobilized
dEGF-His, EGF-K5-His, EGF-E5-His, or EGF-His. The surface with immobilized
dEGF-His provided the highest efficiency for selective expansion of NSCs. On this
substrate, cells expanded 60-fold over 96 h of culture. Of note, over 98% of the
expanded cells expressed nestin, but not
b
III.
The monomeric EGF-His appeared to be limited in its capacity for promoting
EGFR dimerization, most likely due to the reduced mobility of surface-anchored
EGF. In contrast, when dEGF-His was anchored to the surface, EGFR dimerization
was most likely enhanced because the EGF domains were paired in close proximity.
Presumably, this would promote efficient proliferation of NSCs, as observed on the
surface with immobilized dEGF-His. We estimated that the distance between two
EGF domains in the dEGF-His dimer was approximately comparable to the dis-
tance between the two binding sites in dimerized EGFR [
100
]. We further observed
that both EGF-E5-His and EGF-K5-His, anchored as single components, provided
more efficient substrates than EGF-His alone. This might be explained by the E5
and K5 peptides inserted between the EGF domain and the His sequence; this extra
component may have increased the mobility of the EGF domain and thus enhanced
its accessibility. Consistent with that notion, the effects of these peptides on cell
proliferation were not detectable when the chimeric proteins were used as unat-
tached, diffusible factors.
3.6 Modules for Culturing NSCs in a Closed System
The method for immobilizing EGF-His on SAMs gave rise to the need for
fabricated cultureware that could allow large-scale expansion of pure NSCs. We
attempted to construct culture modules with surface areas much larger than the
laboratory-scale substrates described above. For uniformly anchoring EGF-His
over a large area, we utilized a glass plate with amine functionalities on the surface.
