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) – 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.

We 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.