(256a) The Exploitation of S. Cerevisiae - Improved Understanding and Optimal Yields of Single-Chain Antibody Fragment (scFv) 4-4-20 | AIChE

(256a) The Exploitation of S. Cerevisiae - Improved Understanding and Optimal Yields of Single-Chain Antibody Fragment (scFv) 4-4-20


Britton, Z. T. - Presenter, University of Delaware
Maurer, R. W. III - Presenter, University of Delaware
Robinson, A. S. - Presenter, University of Delaware

antibody fragments ? for example, the classic monovalent single chain antibody
(scFv) ? are emerging as credible alternatives to
monoclonal antibody (mAb) products. scFv fragments maintain a diverse
range of potential applications in biotechnology and can be implemented as
powerful therapeutic and diagnostic agents. As such, a variety of hosts have
been used to produce antibody fragments resulting in varying degrees of
success. Yeast, Saccharomyces cerevisiae,
is an attractive host for complex proteins like antibodies due to similarities
in the secretory pathway of eukaryotic organisms including analogous mechanisms
for protein synthesis, translocation, maturation, and secretory trafficking. However, the expression of a recombinant
protein in yeast is not trivial; nor are the quality control pathways
simplistic that the cell activates to respond to overwhelming stress, such as
an increased protein load. The endoplasmic reticulum (ER) is a dynamic
organelle, capable of sensing and adjusting its folding capacity in response to
increased demand. When protein abundance or terminally misfolded
proteins overwhelm the ER's capacity, the unfolded protein response (UPR) is
activated. Elucidating the role of ER stress, both physiological and
pathological, will enable the design of new therapeutic modalities aimed at
stress reduction.

have established methodologies for investigating the role of cellular quality
control and its modulation during heterologous protein expression of scFv 4-4-20 tagged variants, focusing specifically on the
UPR, autophagy, and ER associated degradation (ERAD) pathways. Appropriate and
versatile constructs for yeast recombinant protein expression have been
designed to regulate trafficking effects by incorporating selective motifs and
eliminating retrograde transport, resulting in improved secretion of a model
antibody fragment, scFv 4-4-20. Furthermore, we have optimized
methods to monitor intracellular protein expression and trafficking by
developing novel fluorescent proteins and fluorophores
fused to endogenous yeast targets including ER quality control (ERQC) and
organelle markers, and evaluated the UPR by quantitative analysis with
essential controls and purified standards.

Time course analysis, quantitative
PCR, co-immunoprecipitation of select proteins, and yeast deletion strains in
combination with high-resolution imaging techniques have enabled us to evaluate
different expression conditions, minimize UPR, and determine co-localization
with organelles and sub-compartments. Combined with microarray studies, our
data has enabled a better understanding of the role of quality control pathways,
specifically the UPR, autophagy, and ERAD, by analyzing expression profiles of scFv.