(518e) The Exploitation of S. Cerevisiae - Improved Understanding and Optimal Yields of Single-Chain Antibody Fragment (scFv) 4-4-20

Recombinant antibody fragments – for
example, the classic monovalent single chain antibody (scFv) – have
emerged as credible alternatives to monoclonal antibody (mAb) products. scFv fragments maintain a diverse range of potential
applications in biotechnology, specifically as therapeutic and diagnostic
agents. As such, a variety of hosts have produced antibody fragments with
varying degrees of success. Yeast, Saccharomyces
cerevisiae, is an attractive microbe 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 recombinant proteins in yeast is not trivial;
neither are cellular quality control pathways simplistic. 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.

We
have established methodologies for investigating the role of cellular quality
control and its modulation during heterologous protein expression of scFv
4-4-20, focusing specifically on the UPR, autophagy, and ER-associated
degradation (ERAD). By
implementing DNA recombination strategies combined with high-resolution imaging
techniques, we have identified the intracellular localization of scFv and determined
the extent of protein interactions with ER folding factors. In pursuit of a
thorough analysis of protein distribution at the subcellular level, yeast
expression cassettes [1] were designed to test the effects of codon-optimized
fluorescent variants, small epitope tags (reviewed in [2]), polylinker length
for N- and C-terminal tags, and the inclusion of essential retrieval sequences
for ER luminal chaperones and foldases [3]. In fact, we have assessed many ERQC
proteins in yeast that significantly effect cell physiology and disease
pathology [4]. S. cerevisiae
strains were further engineered to express fluorescent proteins targeted to
various organelles [5]. To investigate discrete subpopulations of tagged
proteins using live-cell imaging methods and super-resolution techniques (e.g.
Fluorescence-Photoactivation Localization Microscopy, F-PALM), a
photoconvertible GFP variant (i.e. mEos2) and six-residue tetracysteine motif
required for FlAsH (fluorescein arsenical helix binder)-based technology were
implemented. For yeast recombinant protein expression, versatile constructs were
designed to regulate trafficking effects. Consequently, the implementation of
motifs and elimination of retrograde transport have resulted in improved
secretion of the model antibody fragment, scFv 4-4-20 [6]. Furthermore, we have
assessed the UPR at the cDNA and protein levels, as well as quantified total scFv
production versus secretion utilizing novel 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 systems have
facilitated an improved understanding of the pivotal role of cellular quality
control, specifically the UPR, autophagy, and ERAD [6].

Publications

1.    
C. L. Young, D. Raden, J. Caplan, K. Czymmek, A. S.
Robinson Optimized Cassettes for
Live-Cell Imaging of Proteins and High Resolution Techniques in Yeast,
Yeast, 2012 doi:10.1002/yea.2895. [Epub 2012 Apr 4]

2.    
C. L. Young, Z. T. Britton, A. S.
Robinson Recombinant
Protein Expression and Purification: A Comprehensive Review of Affinity Tags
and Microbial Applications, Biotechnology
Journal, 7(4), Jan 10 2012 doi:10.1002/biot.201100155.
[Epub ahead of print]

3.    
C. L. Young, D. L. Raden, A. S. Robinson, Analysis of Endoplasmic Reticulum Resident
Proteins in S. cerevisiae: Implementation of H/KDEL Retrieval Sequences,
2012 (submitted).

4.    
C. L. Young, D. L. Raden, J. Caplan, B. Chung, K.
Czymmek, A. S. Robinson Dynamics of
Endoplasmic Reticulum Resident Proteins and Organelle Morphology in S.
cerevisiae, 2012 (in preparation).

5.     C.
Young, Z.
Britton, J. Caplan, K. Czymmek, A. Robinson Exploiting
S. cerevisiae: Cellular Systems & Techniques Aimed at Identifying the
Localization of Targeted Proteins, 2012 (in preparation).

6.     C. Young, R. Maurer, P. Xu, A. Robinson scFv: the model single-chain antibody
fragment, 2012 (in preparation)