16 / 27
Page 72
allied
academies
J Pharmacol Ther Res 2017 Volume 1 Issue 2
November 02-03, 2017 Chicago, USA
4
th
International Congress on
International Conference and Exhibition on
Drug Discovery, Designing and Development
Biochemistry, Molecular Biology: R&D
&
T
he endoplasmic reticulum (ER) plays a critical role in
protein folding, protein secretion, calcium homeostasis,
and lipid biosynthesis. Mammalian cells are often used for
the production of recombinant biotherapeutic proteins
where the secretory pathway machinery, including the
ER, is essential to the correct folding, assembly and post-
translation modifications required of the target protein.
However, expression of recombinant proteins in high
amounts in mammalian cells can result in ER stress, which
can result in cellular responses and multiple stimuli from the
ER that activate the unfolded protein response (UPR), slow
protein synthesis and can negatively impact upon protein
yields and quality. The maintenance of the ER and secretory
pathway system requires a carefully coordination of lipid
biosynthesis. Here we investigate approaches and strategies
to design new hosts and cellular circuits to reprogramme
the CHO cell ER with a view to either expanding its capacity
and/or subsequent secretory vesicle system to improve cell
growth, yields and quality of recombinant secreted proteins.
Our hypothesis is that controlled manipulation of lipid
biosynthesis will result in an enhancement of the efficiency
of the CHO platform as a recombinant protein expression
system. Here we report on the manipulation of the CHO
lipid biosynthesis machinery by altering key components.
We have transiently and stably over-expressed two proteins
in particular reported to led to expansion of the ER in CHO
cells. Stable cell pools have subsequently been cloned via
limited dilution cloning to obtain clonal cell lines. Over-
expression of the lipid biosynthesis proteins did not impact
upon cell growth behaviour, however transient expression of
two model recombinant proteins (EPO and Etanercept – a
TNFR-Fc fusion protein) that are difficult to express in CHO
cells was enhanced in CHO cells engineered to over-express
the lipid biosynthesis proteins. Here we present implications
for this and potential applications.
e:
faa8@kent.ac.ukReprogramming lipid synthesis in Chinese Hamster Ovary (CHO) cells for enhanced recombinant protein
production
Folasade Ajayi
University of Kent, UK