Decreased Secretion and Unfolded Protein Response Upregulation

Abstract

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 due to quality control mechanisms of the secretory pathway that ensure secreted proteins are properly folded. However, the expression of a recombinant protein in yeast is not trivial; neither are the quality control mechanisms the cell initiates to respond to overwhelming stress, such as an increased protein load, 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. In the guidelines presented here, we discuss varying aspects of quality control, its modulation, and ways to design appropriate constructs for yeast recombinant protein expression. Furthermore, we have provided protocols and methods to monitor intracellular protein expression and trafficking as well as evaluation of the UPR, with essential controls. The latter part of this chapter will review considerations for the experimental design of microarray and quantitative polymerase chain reaction (q-PCR) techniques while suggesting appropriate means of data analysis.

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…Recently, the intrinsic fluorescence of GFP and its variants have been used to rapidly evaluate positive clones by in-gel fluorescence techniques [167,168], in addition to the use of GFP fluorescent standards and established protocols [169] permitting the estimation of recombinant protein concentration via western blot analysis.…”

Cassette series designed for live‐cell imaging of proteins and high‐resolution techniques in yeast

“…A detailed protocol has been described elsewhere (Young, et al, 2011). Briefly, overnight cultures of each sample were grown to mid-log phase (0.8 ≤ OD 600 ≤ 1.0) in 5 mLs of YPD media (30°C, 275 rpm).…”

Recombinant G Protein-Coupled Receptor Expression in Saccharomyces cerevisiae for Protein Characterization

“…Addition of CHS in the solubilization of A 2 aR in DDM may be beneficial to function due to the effect on micelle shape, specifically by causing an increase in ellipsoidal character, with a thickness approaching that of mammalian membranes (O'Malley et al, 2011a). The trial and error approach necessary to discover detergent systems capable of solubilizing membrane proteins, while retaining the protein stability and function has made the use of more native-like environments such as liposomes, bicelles, and nanodiscs more attractive (Allen et al, 2009;Orwick-Rydmark et al, 2012;Robinson, 2011). Typically, reconstitution of membrane proteins in bicelles and liposomes takes place following solubilization in detergent micelles (Allen et al, 2009).…”

“…Despite the low stress on the secretory pathway, levels of A 2 bR still remain lower than A 2 aR, indicating that an attribute in the protein sequence or tertiary structure impacts expression. Minimizing cell stress is one approach that should yield the highest protein levels (Young et al, 2011); however, low stress alone is not enough to ensure high yields of A 2 bR.…”

“…The DNA cassettes were transformed into BY4742 + UPRE-yEGFP strain using the lithium acetate transformation method (Gietz & Woods, 2002). This strain of yeast incorporates four unfolded protein response elements (UPRE) in series coupled to yEGFP allowing yEGFP expression upon activation of the unfolded protein response (UPR) (Young, Yuraszeck, & Robinson, 2011). In Fig.…”