Elsevier

Methods in Enzymology

Volume 491, 2011, Pages 235-260
Methods in Enzymology

Chapter fourteen - Decreased Secretion and Unfolded Protein Response Upregulation

https://doi.org/10.1016/B978-0-12-385928-0.00014-6Get rights and content

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.

Introduction

Efficient production of heterologous proteins by eukaryotic hosts, including the yeast Saccharomyces cerevisiae, is often hampered by the inefficient secretion of the product. Limitation of protein secretion has often been attributed to a decreased folding rate, leading to novel solutions that have implemented the overexpression of endogenous proteins to support protein folding and maturation (Robinson et al., 1994, Shusta et al., 1998), or the constitutive expression of the unfolded protein response (UPR) transcription factor (TF), Hac1 (Valkonen et al., 2003). Extensive studies conducted with single-chain antibody fragments (scFv) established that overexpression of BiP or PDI increases secretion substantially; and when co-overexpressed, these endoplasmic reticulum (ER)-resident folding assistants act synergistically with the tuning of scFv copy number to increase secretion eightfold (i.e., 20 mg/L; Shusta et al., 1998). Furthermore, our studies have shown that overexpression of PDI or co-overexpression of BiP and PDI significantly reduce the UPR during scFv expression as determined by the canonical UPRE sensor (Mori et al., 1998, Travers et al., 2000). A reduction in the UPR led to an ~ 3.5-fold increase of secreted scFv (Xu et al., 2005).

The comprehensive characterization of genes required for endogenous protein folding (Jonikas et al., 2009), translational behavior (Arava et al., 2003), and cell growth in S. cerevisiae (Brauer et al., 2008) have been described, and are useful for categorizing genes of interest. Therefore, assuming that other components of the secretory pathway affect secretion although are not directly involved in protein folding, one aim of our studies was to evaluate the transcriptome of S. cerevisiae during expression of scFv. On a cellular level, we were also interested in understanding the interactions between BiP and PDI with the scFv during expression. We have determined UPR modulation in different cell strains and evaluated the intracellular retention of scFv based on pulse-chase 35S studies to examine trafficking effects.

This chapter focuses on quality control mechanisms of the secretory pathway, specifically impacting UPR upregulation and decreased secretion of scFv. In this regard, we detail experimental methods used to evaluate the UPR in a population, and appropriate means of quantifying the intracellular concentration of a model antibody fragment, scFv 4-4-20, that may be broadly applied to heterologous protein expression and secretion. Rigorous statistical analysis of microarray and quantitative PCR (q-PCR) data is essential when evaluating global data using either a time-course or static experiment. We have carefully outlined methods and caveats in data analysis and interpretation, and utilize our studies of UPR induction by chemical treatment and expression of scFv as case studies.

Section snippets

Heterologous Protein Expression

Collectively, heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking; thus, a more complete understanding of how these processes interrelate will lead to optimized conditions for scFv expression, secretion, and enhanced activity. In the case of scFv production, there are several reports in literature describing approaches to improve expression: overexpression of folding assistants BiP and PDI (Robinson et al., 1994, Xu et

Quality Control Mechanisms of the Secretory Pathway

The secretory pathway of eukaryotic cells is composed primarily of two organelles, the ER and Golgi, responsible for maintaining the fidelity of protein synthesis and maturation. The environment of the ER is specialized to properly fold secretory proteins due to an oxidizing redox potential, appropriate calcium levels, and dedicated enzymes for protein glycosylation and folding (i.e., chaperones and foldases; van Anken and Braakman, 2005). When abnormalities do occur, such as an overwhelming

Endoplasmic Reticulum Export and Trafficking

Secreted heterologous proteins, such as scFv, enter the secretory pathway at the ER and move via vesicular transport to the Golgi. Export from the ER requires specialized ER-resident proteins including COPII machinery, and receptors such as Erv29 help export certain soluble cargo proteins (Otte and Barlowe, 2004). There is increasing evidence that post-ER quality control mechanisms exist and sort defective proteins from the Golgi to the endosomal system for lysosomal/vacuolar degradation

Experimental Systems to Evaluate Expression, UPR, and Secretory Processing

As a simpler eukaryotic organism, yeast is advantageous to study due to facile genetic manipulation in a sequenced, annotated genome; mammalian-like organelle trafficking of secreted protein with similar mechanisms for protein synthesis, maturation, and secretory trafficking; and microbial growth features. In the following sections, we outline pertinent factors to consider with respect to molecular engineering of constructs for heterologous protein expression and selection of the most

S. cerevisiae Strains Used for Optimal Expression

A yeast strain should be selected based on its suitability for the process being studied, efficiency of transformation, and flexibility with respect to selection. Difficulties associated with the expression level of a recombinant protein, effect of growth rates, and proteases are aspects that should be considered. The choice of an appropriate host strain, induction media, and expression plasmid (i.e., 2 μm, low-copy, or multicopy δ integrating plasmids) can overcome most obstacles. Usually it is

Plasmid Design

For the maximal expression of a recombinant protein, the 2 μm is generally considered. Yet, high-level expression will overwhelm the capacity of the secretory pathway; therefore, alternatives include low-copy plasmids (i.e., pRS300 series; Sikorski and Hieter, 1989), direct chromosomal integration, or the flexibility provided by δ integration. Comparison of these molecular engineered constructs are examined in Parekh et al., 1996, Shusta et al., 1998, Kauffman et al., 2002 by evaluating

Strain growth, expression, and isolation of intracellular heterologous protein

The following time-course protocol is specified for the expression of a heterologous protein and evaluation of the UPR (Fig. 14.2) although it can be modified for any experimental system. Synthetic media has been described elsewhere (Sherman, 2002).

  • 1.

    Following the restreak of your desired strain onto a selective agar plate (e.g., SD-URA-LEU), resuspend a single colony in 1 mL of appropriate dextrose media and measure the optical density at 600 nm (OD600). Estimate, a priori, the volume of the

Statistical Analysis of Microarray Results

The advent of high-throughput microarray technology to simultaneously measure the expression of thousands of genes has revolutionized the study of biological systems. A single microarray experiment provides an abundance of information from which diverse analyses are possible, from large-scale characterization of the effects of the experimental condition on known cellular processes to network inference efforts in which the regulatory pathways responsible for the system's response to the imposed

Acknowledgment

The authors were supported in part for the experimental studies by NIH R01 GM 075297.

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