Carissa L. Young

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

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility‐enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility‐enhancing and affinity tags. Furthermore, we provide summaries of well‐characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.

High-resolution three-dimensional reconstruction of a whole yeast cell using focused-ion beam scanning electron microscopy.

We developed an approach for focused gallium-ion beam scanning electron microscopy with energy filtered detection of backscattered electrons to create near isometric voxels for high-resolution whole cell visualization. Specifically, this method allowed us to create three-dimensional volumes of high-pressure frozen, freeze-substituted Saccharomyces cerevisiae yeast cells with pixel resolutions down to 3 nm/pixel in x, y, and z, supported by both empirical data and Monte Carlo simulations. As a result, we were able to segment and quantify data sets of numerous targeted subcellular structures/organelles at high-resolution, including the volume, volume percentage, and surface area of the endoplasmic reticulum, cell wall, vacuoles, and mitochondria from an entire cell. Sites of mitochondrial and endoplasmic reticulum interconnectivity were readily identified in rendered data sets. The ability to visualize, segment, and quantify entire eukaryotic cells at high-resolution (potentially sub-5 nanometers isotropic voxels) will provide new perspectives and insights of the inner workings of cells.

Progress toward heterologous expression of active G‐protein‐coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

High‐level expression of mammalian G‐protein‐coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high‐resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A2a (hA2aR) receptor is active for ligand‐binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER‐resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA2aR, as expected, and partially cleaved for hA2bR, hCCR5R, and hD2LR. Ligand‐binding assays conducted on the adenosine family (hA1R, hA2aR, hA2bR, and hA3R) of receptors show that hA2aR and hA2bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae.

A Microphysiological System Model of Therapy for Liver Micrometastases

Metastasis accounts for almost 90% of cancer-associated mortality. The effectiveness of cancer therapeutics is limited by the protective microenvironment of the metastatic niche and consequently these disseminated tumors remain incurable. Metastatic disease progression continues to be poorly understood due to the lack of appropriate model systems. To address this gap in understanding, we propose an all-human microphysiological system that facilitates the investigation of cancer behavior in the liver metastatic niche. This existing LiverChip is a 3D-system modeling the hepatic niche; it incorporates a full complement of human parenchymal and non-parenchymal cells and effectively recapitulates micrometastases. Moreover, this system allows real-time monitoring of micrometastasis and assessment of human-specific signaling. It is being utilized to further our understanding of the efficacy of chemotherapeutics by examining the activity of established and novel agents on micrometastases under conditions replicating diurnal variations in hormones, nutrients and mild inflammatory states using programmable microdispensers. These inputs affect the cues that govern tumor cell responses. Three critical signaling groups are targeted: the glucose/insulin responses, the stress hormone cortisol and the gut microbiome in relation to inflammatory cues. Currently, the system sustains functioning hepatocytes for a minimum of 15 days; confirmed by monitoring hepatic function (urea, α-1-antitrypsin, fibrinogen, and cytochrome P450) and injury (AST and ALT). Breast cancer cell lines effectively integrate into the hepatic niche without detectable disruption to tissue, and preliminary evidence suggests growth attenuation amongst a subpopulation of breast cancer cells. xMAP technology combined with systems biology modeling are also employed to evaluate cellular crosstalk and illustrate communication networks in the early microenvironment of micrometastases. This model is anticipated to identify new therapeutic strategies for metastasis by elucidating the paracrine effects between the hepatic and metastatic cells, while concurrently evaluating agent efficacy for metastasis, metabolism and tolerability.

All-human microphysical model of metastasis therapy

The vast majority of cancer mortalities result from distant metastases. The metastatic microenvironment provides unique protection to ectopic tumors as the primary tumors often respond to specific agents. Although significant interventional progress has been made on primary tumors, the lack of relevant accessible model in vitro systems in which to study metastases has plagued metastatic therapeutic development - particularly among micrometastases. A real-time, all-human model of metastatic seeding and cancer cells that recapitulate metastatic growth and can be probed in real time by a variety of measures and challenges would provide a critical window into the pathophysiology of metastasis and pharmacology of metastatic tumor resistance. To achieve this we are advancing our microscale bioreactor that incorporates human hepatocytes, human nonparenchymal liver cells, and human breast cancer cells to mimic the hepatic niche in three dimensions with functional tissue. This bioreactor is instrumented with oxygen sensors, micropumps capable of generating diurnally varying profiles of nutrients and hormones, while enabling real-time sampling. Since the liver is a major metastatic site for a wide variety of carcinomas and other tumors, this bioreactor uniquely allows us to more accurately recreate the human metastatic microenvironment and probe the paracrine effects between the liver parenchyma and metastatic cells. Further, as the liver is the principal site of xenobiotic metabolism, this reactor will help us investigate the chemotherapeutic response within a metabolically challenged liver microenvironment. This model is anticipated to yield markers of metastatic behavior and pharmacologic metabolism that will enable better clinical monitoring, and will guide the design of clinical studies to understand drug efficacy and safety in cancer therapeutics. This highly instrumented bioreactor format, hosting a growing tumor within a microenvironment and monitoring its responses, is readily transferable to other organs, giving this work impact beyond the liver.

Cassette Series Designed for Live-Cell Imaging of Proteins and High Resolution Techniques in Yeast

During the past decade, it has become clear that protein function and regulation are highly dependent upon intracellular localization. Although fluorescent protein variants are ubiquitously used to monitor protein dynamics, localization and abundance; fluorescent light microscopy techniques often lack the resolution to explore protein heterogeneity and cellular ultrastructure. Several approaches have been developed to identify, characterize and monitor the spatial localization of proteins and complexes at the suborganelle level, yet many of these techniques have not been applied to yeast. Thus, we have constructed a series of cassettes containing codon‐optimized epitope tags, fluorescent protein variants that cover the full spectrum of visible light, a TetCys motif used for fluorescein arsenical hairpin (FlAsH)‐based localization, and the first evaluation in yeast of a photoswitchable variant, mEos2, to monitor discrete subpopulations of proteins via confocal microscopy. This series of modules, complete with six different selection markers, provides the optimal flexibility during live‐cell imaging and multicolour labelling in vivo. Furthermore, high‐resolution imaging techniques include the yeast‐enhanced TetCys motif, which is compatible with diaminobenzidine photo‐oxidation used for protein localization by electron microscopy, and mEos2, which is ideal for super‐resolution microscopy. We have examined the utility of our cassettes by analysing all probes fused to the C‐terminus of Sec61, a polytopic membrane protein of the endoplasmic reticulum of moderate protein concentration, in order to directly compare fluorescent probes, their utility and technical applications. Our series of cassettes expand the repertoire of molecular tools available to advance targeted spatiotemporal investigations using multiple live‐cell, super‐resolution or electron microscopy imaging techniques. Copyright

Protein folding and secretion: mechanistic insights advancing recombinant protein production in S. cerevisiae

The emergence of genomic approaches coupled to recombinant DNA technologies have identified the quality control systems that regulate proteostasis - biological pathways that modulate protein biogenesis, maturation, trafficking, and degradation. The elucidation of these pathways has become of growing importance in therapeutics as loss of proteostasis has been suggested to lead to a number of human diseases including Alzheimers, Parkinsons Disease and Type II Diabetes. We anticipate that the most successful strategies for protein expression and therapeutics development may involve integration of protein engineering strategies with host manipulation, to exploit the cells native stress response pathways and trafficking mechanisms. This review will highlight recent findings and mechanistic detail correlated to quality control in the early secretory pathway of Saccharomyces cerevisiae.

Decreased Secretion and Unfolded Protein Response Upregulation

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 ERs 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.

Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System

In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro–in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography–tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell–mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1β, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-α, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1β, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans.

Analysis of ER Resident Proteins in Saccharomyces cerevisiae: Implementation of H/KDEL Retrieval Sequences

An elaborate quality control system regulates endoplasmic reticulum (ER) homeostasis by ensuring the fidelity of protein synthesis and maturation. In budding yeast, genomic analyses and high‐throughput proteomic studies have identified ER resident proteins that restore homeostasis following local perturbations. Yet, how these folding factors modulate stress has been largely unexplored. In this study, we designed a series of polymerase chain reaction (PCR)‐based modules including codon‐optimized epitopes and fluorescent protein (FP) variants complete with C‐terminal H/KDEL retrieval motifs. These conserved sequences are inherent to most soluble ER resident proteins. To monitor multiple proteins simultaneously, H/KDEL cassettes are available with six different selection markers, providing optimal flexibility for live‐cell imaging and multicolor labeling in vivo. A single pair of PCR primers can be used for the amplification of these 26 modules, enabling numerous combinations of tags and selection markers. The versatility of pCY H/KDEL cassettes was demonstrated by labeling BiP/Kar2p, Pdi1p and Scj1p with all novel tags, thus providing a direct comparison among FP variants. Furthermore, to advance in vitro studies of yeast ER proteins, Strep‐tag II was engineered with a C‐terminal retrieval sequence. Here, an efficient purification strategy was established for BiP under physiological conditions.