Anne S. Robinson

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.

Thermolabile folding intermediates: inclusion body precursors and chaperonin substrates.

An unexpected aspect of the expression of cloned genes is the frequent failure of newly synthesized polypeptide chains to reach their native state, accumulating instead as insoluble inclusion bodies. Amyloid deposits represent a related state associated with a variety of human diseases. The critical folding intermediates at the juncture of productive folding and the off‐pathway aggregation reaction have been identified for the phage P22 tailspike and coat proteins. Though the parallel β coil tailspike is thermostable, an early intracellular folding intermediate is thermolabile. As the temperature of intracellular folding is increased, this species partitions to inclusion bodies, a kinetic trap within the cell. The earliest intermediates along the in vitro aggregation pathway, sequential multimers of the thermolabile folding intermediates, have been directly identified by native gel electrophoresis. Tem‐ perature‐sensitive folding (tsf) mutations identify sites in the β coil domain, which direct the junctional intermediate down the productive pathway. Global suppressors of tsf mutants inhibit the pathway to inclusion bodies, rescuing the mutant chains. These mutants identify sites important for avoiding aggregation. Coat folding intermediates also partition to inclusion bodies as temperature is increased. Coat tsf mutants are suppressed by overexpression of the GroE chaperonin, indicating that the thermolabile intermediate is a physiological substrate for GroE. We suggest that many proteins are likely to have thermolabile intermediates in their intracellular folding pathways, which will be precursors to inclusion body formation at elevated temperatures and therefore substrates for heat shock chaperonins.—King, J., Haase‐Pettingell, C., Robinson, A. S., Speed, M., Mitraki, A. Thermolabile folding intermediates: inclusion body precursors and chaperonin substrates. FASEB J. 10, 57‐66 (1996)

Heterologous GPCR Expression: A Bottleneck to Obtaining Crystal Structures

G protein‐coupled receptors (GPCRs) are an important, medically relevant class of integral membrane proteins. Laboratories throughout all disciplines of science devote time and energy into developing practical methods for the discovery, isolation, and characterization of these proteins. Since the crystal structure of rhodopsin was solved 6 years ago, the race to determine high‐resolution structures of more GPCRs has gained momentum. Since certain GPCRs are currently produced at sufficient levels for X‐ray crystallography trials, it is speculated that heterologous expression of GPCRs may no longer be a bottleneck in obtaining crystal structures. This Review focuses on the current approaches in heterologous expression of GPCRs and explores the problems associated with obtaining crystal structures from GPCRs expressed in different systems. Although milligram amounts of certain GPCRs are attainable, the majority of GPCRs are still either produced at very low levels or not at all. Developing reliable expression techniques for GPCRs is still a major priority for the structural characterization of GPCRs.

Hydrostatic pressure rescues native protein from aggregates

Misfolding and misassembly of proteins are major problems in the biotechnology industry, in biochemical research, and in human disease. Here we describe a novel approach for reversing aggregation and increasing refolding by application of hydrostatic pressure. Using P22 tailspike protein as a model system, intermediates along the aggregation pathway were identified and quantitated by size-exclusion high-performance liquid chromatography (HPLC). Tailspike aggregates were subjected to hydrostatic pressures of 2.4 kbar (35,000 psi). This treatment dissociated the tailspike aggregates and resulted in increased formation of native trimers once pressure was released. Tailspike trimers refolded at these pressures were fully active for formation of infectious viral particles. This technique can facilitate conversion of aggregates to native proteins without addition of chaotropic agents, changes in buffer, or large-scale dilution of reagents required for traditional refolding methods. Our results also indicate that one or more intermediates at the junction between the folding and aggregation pathways is pressure sensitive. This finding supports the hypothesis that specific determinants of recognition exist for protein aggregation, and that these determinants are similar to those involved in folding to the native state. An increased understanding of this specificity should lead to improved refolding methods.

Decreased protein expression and intermittent recoveries in BiP levels result from cellular stress during heterologous protein expression in Saccharomyces cerevisiae.

Cells are inherently robust to environmental perturbations and have evolved to recover readily from short‐term exposure to heat, pH changes, and nutrient deprivation during times of stress. The stress of unfolded protein accumulation has been implicated previously in low protein yields during heterologous protein expression. Here we describe the dynamics of the response to this stress, termed the unfolded protein response (UPR), during the expression of the single chain antibody 4–4–20 (scFv) in Saccharomyces cerevisiae. Expression of scFv decreased the growth rate of yeast cells whether the scFv was expressed from single‐copy plasmids or integrated into the chromosome. However, the growth rates recovered at longer expression times, and surprisingly, the recovery occurred more quickly in the high‐copy integration strains. The presence of a functional UPR pathway was necessary for a recovery of normal growth rates. During the growth inhibition, the UPR pathway appeared to be activated, resulting in decreased intracellular scFv levels and intermittent recovery of the chaperone BiP within the endoplasmic reticulum. Intracellular scFv was observed primarily in the endoplasmic reticulum, consistent with activation of the UPR pathway. Although the intracellular scFv levels dropped over the course of the expression, this was not a result of scFv secretion. A functional UPR pathway was necessary for the drop in intracellular scFv, suggesting that the decrease was a direct response of UPR activation. Taken together, these results suggest that control of heterologous gene expression to avoid UPR activation will result in higher production levels.

Conformational Features of Tau Fibrils from Alzheimer’s Disease Brain Are Faithfully Propagated by Unmodified Recombinant Protein

Fibrils composed of tau protein are a pathological hallmark of several neurodegenerative disorders including Alzheimers disease (AD). Here we show that when recombinant tau protein is seeded with paired helical filaments (PHFs) isolated from AD brain, the amyloid formed shares many of the structural features of AD PHFs. In contrast, tau amyloids formed with heparin as an inducing agent-a common biochemical model of tau misfolding-are structurally distinct from brain-derived PHFs. Using ultrastructural analysis by electron microscopy, circular dichroism, and chemical denaturation, we found that AD seeded recombinant tau fibrils were not significantly different than tau fibrils isolated from AD brain tissue. Tau fibrils produced by incubating recombinant tau with heparin had significantly narrower fibrils with a longer periodicity, higher chemical stability, and distinct secondary structure compared to AD PHFs. The addition of heparin to the reaction of recombinant tau and AD PHFs also corrupted the templating process, resulting in a mixture of fibril conformations. Our results suggest that AD-isolated PHFs act as a conformational template for the formation of recombinant tau fibrils. Therefore, the use of AD PHFs as seeds to stimulate recombinant tau amyloid formation produces synthetic tau fibers that closely resemble those associated with AD pathology and provides a biochemical model of tau misfolding that may be of improved utility for structural studies and drug screening. These results also demonstrate that post-translational modifications such as phosphorylation are not a prerequisite for the propagation of the tau fibril conformation found in AD.

Competing aggregation pathways for monoclonal antibodies.

Aggregation is mediated by local unfolding to allow aggregation “hot spot(s)” to become solvent exposed and available to associate with a hot spot on another partially unfolded protein. Historically, the unfolding of either the crystallizable fragment (Fc) or the antigen binding fragment (Fab) regions of a given monoclonal antibody (MAb) has been implicated in aggregation, with differing results across different proteins. The present work focuses on separately quantifying the aggregation kinetics of isolated Fc, isolated Fab, and intact MAb as a function of pH under accelerated (high temperature) conditions. The results show that both Fab and Fc are aggregation prone and compete within the same MAb.

Identification of manipulated variables for a glycosylation control strategy

N‐linked glycan distribution affects important end‐use characteristics such as the bioactivity and efficacy of many therapeutic proteins, (including monoclonal antibodies), in vivo. Yet, obtaining desired glycan distributions consistently during batch‐to‐batch production can be challenging for biopharmaceutical manufacturers. While an appropriately implemented on‐line glycosylation control strategy during production can help to ensure a consistent glycan distribution, to date no such strategies have been reported. Our goal is to develop and validate a comprehensive strategy for effective on‐line control of glycosylation, the successful achievement of which requires first identifying appropriate manipulated variables that can be used to direct the glycan distribution to a desired state. While various culture conditions such as bioreactor process variables, media type, and media supplements have been shown to affect the glycan distribution, in this study we focus on the latter. Specifically, we implemented a statistically designed series of experiments to determine the significant main effects (as well as interaction effects) of media supplementation with manganese, galactose, ammonia and found that each had significant effects on certain glycans. We also include data indicating the glycosylation enzyme gene transcript levels as well as the intracellular nucleotide sugar concentrations in the presence of the media supplements to provide insight into the intracellular conditions that may be contributing to the changes in glycan distribution. The acquired experimental data sets were then used to identify which glycans can be controlled by the media supplements and to what degree. We determined that MnCl2 can be used as a manipulated variable to increase the relative abundance of M51 and decrease FA2 simultaneously, and galactose can be used as a manipulated variable to increase the relative abundance of FA2G1 and decrease FA2 and A2 simultaneously. Biotechnol. Bioeng. 2014;111: 1957–1970.

Computational Design and Biophysical Characterization of Aggregation-Resistant Point Mutations for γD Crystallin Illustrate a Balance of Conformational Stability and Intrinsic Aggregation Propensity

γD crystallin is a natively monomeric eye-lens protein that is associated with hereditary juvenile cataract formation. It is an attractive model system as a multidomain Greek-key protein that aggregates through partially folded intermediates. Point mutations M69Q and S130P were used to test (1) whether the protein-design algorithm RosettaDesign would successfully predict mutants that are resistant to aggregation when combined with informatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (ΔΔG(un)) and intrinsic aggregation propensity (IAP). M69Q was predicted to have ΔΔG(un) ≫ 0, without significantly affecting IAP. S130P was predicted to have ΔΔG(un) ∼ 0 but with reduced IAP. The stability, conformation, and aggregation kinetics in acidic solution were experimentally characterized and compared for the variants and wild-type (WT) protein using circular dichroism and intrinsic fluorescence spectroscopy, calorimetric and chemical unfolding, thioflavin-T binding, chromatography, static laser light scattering, and kinetic modeling. Monomer secondary and tertiary structures of both variants were indistinguishable from WT, while ΔΔG(un) > 0 for M69Q and ΔΔG(un) < 0 for S130P. Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its IAP compared to WT and M69Q.

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.