283685 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  were designed to test the effects of codon-optimized fluorescent variants, small epitope tags (reviewed in ), polylinker length for N- and C-terminal tags, and the inclusion of essential retrieval sequences for ER luminal chaperones and foldases . In fact, we have assessed many ERQC proteins in yeast that significantly effect cell physiology and disease pathology . S. cerevisiae strains were further engineered to express fluorescent proteins targeted to various organelles . 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 . 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 .
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)
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division - See also TI: Comprehensive Quality by Design in Pharmaceutical Development and Manufacture