title: 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.
authors.affiliation: Department of Chemical Engineering, University of Delaware, Newark, Delaware, USA.
authors.forename: Carissa L
authors.initials: CL
authors.lastname: Young
authors.forename: Theresa
authors.initials: T
authors.lastname: Yuraszeck
authors.forename: Anne S
authors.initials: AS
authors.lastname: Robinson
mesh_terms: D000818:Animals; D015870:Gene Expression; D006801:Humans; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism; D056811:Unfolded Protein Response; D015854:Up-Regulation
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies