Molecular Gels for Tissue Engineering - Soft Fibrillar Materials: Fabrication and Applications - page 152

gel (P-gel). Protein expression was achieved by incubating P-gel with wheat germ
lysates, consisting of RNA polymerase and ribosomes, for 24 h. The amount and
functionality of the proteins produced by the P-gels were analyzed, quantified, and
compared with those proteins synthesized from commercially available cell-free
solution-based protein expression systems. It was revealed that P-gel was able to
produce up to 5mg mL -1 of functional proteins, a number that is significantly
higher than the levels achievable by conventional solution processes, at a faster rate
and for a longer duration.
The high yield and efficiency of protein expression were hypothesized to be due
to the following reasons. Firstly, the stability of the protein genomic information is
enhanced due to the protective effect conferred by covalently cross-linked X-DNA
linkers that make the gene less susceptible to enzymatic and hydrolytic degradation.
Secondly, there is an increase in overall gene concentration in a gel phase compared
with that in a solution phase, which possesses additional solvent volume. Thirdly,
an elevation in the turnover rate of the biosynthetic machinery is apparent as a
result of closer gene proximity within a gel structure. Lastly, high abundance of
bivalent cations inside the hydrogel matrix, due to the presence of polyanionic DNA,
enhances the activity of transcription machineries by mimicking the condensed
phase of chromatin in a normal cell nucleus with high ionic concentration [88].
In addition to renilla luciferase , 16 other functional proteins, including membrane
proteins, receptor proteins, and even toxic proteins, have been synthesized by
altering the gene sequences incorporated into the P-gels, with similar high yield
and efficiency. This highlighted the highly flexible and adaptable nature of P-
gel to produce a wide variety of complex proteins and even toxic proteins that
cannot be otherwise synthesized using a cell-based system, suggesting its potential
application in the field of onsite protein production and high-throughput protein
engineering.

4.5
Summary

Here we focus on the recent strategies in designing biomaterials into molecular
gels and their present and potential applications in tissue engineering. Compared
to polymer-based hydrogels, which do not usually degrade under physiological

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 4.11 (A) Oligonucleotide sequences of
the DNA building blocks for the DNA hydro-
gels. (B) External morphologies and internal
structures of different DNA hydrogels; FE-SEM
images of dried X-DNA (a), T-DNA (b), and
T-DNA (c) hydrogels; Confocal microscopic im-
ages of the swollen X-DNA (d), Y-DNA (e), and
T-DNA (f ) hydrogels; The scale bars are 200 μ m

for (a,d,f), 50 μ m for (b), 15 μ m for (c), and
10 μ m for (e). (C) Fluorescent images of live
CHO cells encapsulated into a X-DNA hydrogel.
CHO cells were stained red by CellTrackerTM
Red CMTPX Probes while the DNA hydrogel
was stained green with SYBR I dye. Scale bar =
100 μ m. (Figure adapted from Ref. [86] (Copy-
right 2006) Nature Publishing Group.)