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Introduction

Research in the Robinson group centers on three areas in biochemical engineering: understanding and controlling protein aggregation; cellular mechanisms controlling protein quality and human disease; and overcoming obstacles to expression and characterization of G-protein coupled receptors. In each of these areas, we use the tools of molecular and cellular biology, biochemistry, and biophysics, combined with systems biology, mathematical modeling, and engineering analysis, to develop an improved understanding of biological systems. Using this knowledge, we carry out molecular and cellular engineering to develop improved methods, products, and tools for biotechnology, medical, and research applications.

 

 

Understanding and Controlling Protein Aggregation

Aggregation is a long-standing in vitro and in vivo obstacle for studying proteins; it serves as an irreversible, off-pathway process during protein folding, and it is a ubiquitous problem throughout commercial manufacture of protein-based biotechnology products. This is a potentially debilitating setback from a structural biology and protein design perspective, as it can limit the ability of scientists to produce sufficiently high quantities of purified material that are required for subsequent sample preparation and for biophysical characterization. It also greatly limits biotechnology product discovery and development.  From a discovery perspective, only those candidate molecules that can be readily expressed and (re)folded to active forms can be included in screens for improved or novel structure and function.  Even once viable product candidates are identified, elimination of aggregates and prevention of aggregation is one of the most common technical hurdles for product manufacture, formulation, and final product viability.

In collaborative research with the Roberts group, we seek to
1) Understand the time-dependent behavior and structural contributions to protein aggregates,
2) Develop approaches to limit or dissociate aggregation,
3) Predict and test models for protein-protein interactions that will reduce aggregate formation.

Recent and Representative Publications:

  1. Spatara ML, Roberts CJ, Robinson AS* (2009) “Kinetic folding studies of the P22 tailspike beta-helix domain reveal multiple unfolded states.” Biophys Chem. 141(2-3):214-21. PMID: 19258192
  2. Webber T, Gurung S, Saul J, Baker T, Spatara M, Freyer M, Robinson AS, Gage MJ* (2009) “The C-terminus of the P22 tailspike protein acts as an independent oligomerization domain for monomeric proteins.”, Biochem J. Feb 5. [Epub ahead of print] PMID: 19196242

Collaborators:

Chris Roberts
Department of Chemical Engineering
University of Delaware
http://www.che.udel.edu/directory/facultyprofile.html?id=12564

Erik Fernandez
Department of Chemical Engineering
University of Virginia
http://www.che.virginia.edu/people/faculty/fernandez.php

Funding:

MedImmune (w/ CJR)
NSF 0853639 (PI: Roberts) “Collaborative Research: Towards a General Design Approach to Arrest Non-Native Aggregation of Multi-Domain Proteins”

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Cellular Mechanisms Controlling Protein Quality and Human Disease

Schematic of chaperone interactions with tau in refolding and degradation. Adapted from Goryunov and Liem J. Clin. Invest. 117(3): 590-592 (2007). doi:10.1172/JCI31505.Cells are inherently robust to stochastic perturbations, and have evolved to recover readily from short-term exposure to heat, pH changes, and nutrient deprivation during times of stress. This process, termed the stress response, is important in a wide range of basic research and commercial applications since cell growth rates, production of metabolites, and protein expression are all affected by the stress response. Moreover, accumulation of toxic metabolic products or unfolded proteins can in turn induce the stress response, implicated in a number of human diseases. Our two areas of research are:

  1. Understanding the effects of these cellular control mechanisms of protein expression levels and protein quality (activity, post-translational modifications).

  2. Determining how loss of cellular control may lead to a disease state

During protein expression, the stress response to unfolded protein accumulation, termed the unfolded protein response (UPR), resulting in low protein yields during heterologous protein expression. A systematic approach to improving protein production involves understanding on a molecular level how cell regulation mechanisms respond to heterologous protein expression. Key issues include how to maintain reasonable cell health, yet obtain high protein yields and what interactions promote or stabilize formation of active protein with correct post-translation modifications.

Hallmarks of the disease state in the Alzheimer brain, and of several other neurodegenerative diseases including corticobasal degeneration, frontotemporal dementia, and parkinsonism linked to chromosome 17, are the hyperphosphorylation of tau and subsequent formation of insoluble tau aggregates (neurofibrillary tangles or paired helical filaments). In healthy cells, the tau protein binds to and stabilizes microtubules, and is abundant. It is not yet clear whether the problem in these diseases is a loss of tau function (e.g. loss of microtubule stability), or an inherent toxicity of the tau tangles. Major research questions include determining the biochemical and cellular pathways that drive tau homeostasis, including degradation and refolding pathways, and which steps in the tau pathways are the best targets for therapeutic intervention.

Collaborators:

Frank Doyle
Department of Chemical Engineering
University of California at Santa Barbara
http://www.chemengr.ucsb.edu/people/faculty_d.php?id=18

Tunde Ogunnaike
Department of Chemical Engineering
University of Delaware
http://www.che.udel.edu/directory/facultyprofile.html?id=325

Funding:

NIH R01-GM 075297
NSF 1034213

 

Recent and Representative Papers

  1. Spatara, ML and Robinson, AS* (2009) “Transgenic mouse and cell culture models demonstrate a lack of mechanistic connection between endoplasmic reticulum stress and tau dysfunction” Journal of Neuroscience Research, in press.
  2. Xu, P. and Robinson, AS* (2009) “Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast” Biotech & Bioeng, 104(1):20-9.
  3. Hildebrandt, S., D. Raden, L. Petzold, Robinson AS, and F.J. Doyle III* (2008) “A top-down approach to mechanistic biological modeling: application to the single-chain antibody folding pathway”, Biophysical Journal, 95(8):3535-58. Epub 2008 Jul 18.
  4. Xu, P., Raden, D., Doyle, F.J. III, and Robinson AS* (2005) “Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding”, Metabolic Engineering, 7 (4) 269-279.

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Identifying and Overcoming Obstacles to GPCR Expression and Characterization

Membrane proteins are critical for cell-cell recognition, inter and intracellular signaling, and cell homeostasis, and are of great interest in understanding human disease and as drug targets. However, the majority of membrane protein structures have been obtained only from proteins available in natural abundance, due to the major difficulties with heterologous expression to date. Of all unique membrane crystal structures to date, about 10% have been expressed in heterologous systems, and fewer than 5% are of eukaryotic proteins (15 of which are transmembrane proteins: 4 GPCRs, 5 ion channels, 6 aquaporins and other transporters).

Confocal images show changes to trafficking in HEK293 cells expressing cysteine variants of A2aR-CFP. Scale bar = 10 µmG-protein coupled receptors (GPCRs) are membrane proteins expressed in virtually all human tissues, and they transmit a wide variety of signals in response to diverse stimuli (including light, hormones, injury, and inflammation). Although cloning of a mammalian GPCR (β2 adrenergic receptor) was first achieved in the mid-1980s, the first non-rhodopsin GPCR structure only became available almost 20 years later (2007) . There are approximately 360 non-sensory human GPCRs (of ~800-900 total predicted GPCRs), with ligands identified or predicted for only ~2/3 of the non-sensory proteins. Many acute and chronic disease states are linked to GPCR function or malfunction, and 30-60% of commercially available drugs interact with a GPCR, making them targets for nearly 40% of drug discovery efforts worldwide, yet these drugs target less than 10% of all GPCRs. However, efforts to better understand GPCR ligand specificity, structure, stability, and assembly are hampered by the difficulties associated with producing these integral membrane proteins.

Over the last eight years, our laboratory has expressed several GPCRs and other complex proteins in order to determine their relative expressability and to investigate cellular limitations to folding and trafficking to give active protein both from Saccharomyces cerevisiae and E. coli. Our overall goals are to overcome the limitations to expression in S. cerevisiae, identify the mechanisms for membrane insertion and proper trafficking, and biophysical and stability characterization of purified receptors.

Recent and Representative Publications:

  1. O'Malley MA, Mancini JD†, Young CL, McCusker EC, Raden D, Robinson AS*. (2009) “Progress toward heterologous expression of active G-protein-coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking.” Protein Sci. 18(11):2356-70.
  2. McCusker, E., and Robinson, AS.*, (2008) Refolding of G protein α subunits from inclusion bodies expressed in Escherichia coli, Protein Exp. Purif., Apr;58(2): 342-55. Epub 2007 Dec 8.
  3. McCusker, E., Bane, S.E., O’Malley, M., and A.S. Robinson * (2007), “Heterologous GPCR expression: A bottleneck to obtaining crystal structures”, Biotech Progress, May-Jun;23(3):540-7.
  4. O’Malley, M., Lazarova, T., Britton, Z.T., and Robinson, AS * (2007) “High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor”, J. Struct Biol., 159(2): 166-178.
  5. Bane, S.E., Velasquez, J.E. , and Robinson AS* (2007) “Expression and purification of milligram levels of inactive G-protein coupled receptors in E. coli”, Protein Expression and Purification, 52(2):348:355.

Collaborators:

Bramie Lenhoff
Department of Chemical Engineering
University of Delaware
http://www.che.udel.edu/directory/facultyprofile.html?id=252

Funding:

NIH COBRE: P20 RR15588 “Membrane Protein Production and Characterization” Sub-project: "Determinants of GPCR Expression in E. coli and Yeast"

 

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University of Delaware