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SHORT COURSE 6: STRATEGIES FOR PURIFYING PROTEINS: OPTIMIZING BUFFERS AND OVERCOMING AGGREGATION 

 

Sunday, January 12 | 5:00-8:00 pm


This course will provide strategies for purifying proteins, such as identifying and overcoming aggregation and stabilizing proteins through buffer optimization, including analyzing quality, pH, salt, stabilizing elements, buffering systems and identifying solubility-promoting buffers.

5:00 pm A Rapid Filtration-Based Assay to Identify Conditions Promoting Protein Solubility Before, During, or After Purification

Sarah E. Bondos, Ph.D., Assistant Professor, Molecular and Cellular Medicine, Texas A&M Health Science Center

  • Good sign of solubility: proper oligomeric conformation
  • Checking and Optimizing Solubility at the Expression Level
  • Checking: SDS-PAGE crude vs clarified cell lysate
  • Optimizing 1: alter expression rate
  • Optimizing 2: peptide/protein fusion
  • Aggregation during processing
  • Define aggregation vs precipitation
  • Potential sources of problems
  • Types of solubility problems
  • Co-solvents that can help (or harm) solubility
  • Methods for Rapidly Screening Solubility Conditions:
  • Visual or microscope screen in 24 or 96 wells, including monitor over time or at different temperatures
  • Ultrafiltration assays
  • Commercial
  • Our filtration-based solubility assay
  • Final concern: soluble does not guarantee native structure

5:45 Protein Solution Nonideality and High Concentration Solutions

Thomas Laue, Ph.D., Professor, Biochemistry and Molecular Biology; Director, Biomolecular Interaction Technologies Center (BITC), University of New Hampshire

The thermodynamic nonideality of protein solutions determines their colloidal stability. This talk will introduce the fundamentals of thermodynamic nonideality and measurements of nonideality.  There will be a description of what protein features contribute to solution nonideality, and how they may be measured. Finally, how the solvent affects nonideality will be addressed.

6:30 Dinner Break

7:00 Construct Design and Manipulation of Protein Expression and Purification Conditions to Maximize Recombinant Protein Yields

Mark Arbing, Ph.D., Director, Protein Expression Technology Center, UCLA-DOE Institute for Genomics & Proteomics

The heterologous expression of target proteins in soluble form is a major bottleneck for research projects requiring large amounts of purified protein. The solubility of target proteins expressed in Escherichia coli, the most common prokaryotic expression host, is affected by a wide variety of factors at the translational and posttranslational level. This talk focuses on construct engineering, including the use of fusion proteins, and strategies for bacterial expression and purification that maximize soluble protein yields.  

7:45 Extended Q & A

8:00 Close of "Strategies for Purifying Proteins" Short Course

Speaker Biographies  

Sarah E. BondosSarah E. Bondos, Ph.D., Assistant Professor, Molecular and Cellular Medicine, Texas A&M Health Science Center 

Dr. Sarah Bondos first became interested in protein aggregation while working with challenging mutants of HIV Reverse Transcriptase as an undergraduate at the University of North Carolina.  She pursued protein folding and aggregation studies by examining the impact of Hofmeister ions and hydrostatic pressure on insoluble protein folding intermediates as a graduate student at the University of Illinois, where she completed a Ph.D. in Biochemistry in 1998 working with Dr. Stephen Sligar and Dr. Jiri Jonas.  As a postdoc in Dr. Kathleen Matthews’ lab at Rice University, Dr. Bondos developed a filter-based protein solubility assay which allowed her to purify soluble, full length Hox transcription factors for subsequent biophysical characterization.  She is currently an Assistant Professor of Molecular and Cellular Medicine at the Texas A&M Health Science Center, where her lab manipulates the solubility of the Hox transcription factor Ultrabithorax to create ordered, functionalized biomaterials for tissue engineering scaffolds.

Thomas LaueThomas Laue, Ph.D., Professor, Biochemistry and Molecular Biology; Director, Biomolecular Interaction Technologies Center (BITC), University of New Hampshire

Tom Laue is the Carpenter Professor of Molecular, Cellular and Biomedical Sciences, and professor of Material Sciences at the University of New Hampshire. He is the Director of both the Center to Advance Molecular Interaction Science and the Biomolecular Interaction Technologies Center. He received his bachelor’s degree in Natural Sciences from the Johns Hopkins University in 1971 and his Ph.D. in Biophysics and Biochemistry from the University of Connecticut in 1981. His post-doctoral studies were conducted at the University of Oklahoma. Between 1969 and 1975, he worked as a technician in the deep space program of NASA. He joined the University of New Hampshire in 1984 as an Assistant Professor, and teaches both undergraduate and graduate courses in biochemistry and biophysics. His research focuses on the development of instrumentation and methods that use the fundamental properties of mass and charge for examining macromolecular interactions. These instruments provide unique insights into these interactions, and have resulted in extensive collaborations with both academic and industrial labs. Tom has over 120 publications, serves on several editorial boards, and gives over one hundred lectures, seminars and workshops a year.

Mark ArbingMark Arbing, Ph.D., Director, Protein Expression Technology Center, UCLA-DOE Institute for Genomics & Proteomics

Dr. Arbing received his Ph.D. in 2002 for his studies on bacterial outer membrane proteins in the lab of James Coulton at McGill University. From 2002-2007 he was a postdoctoral research scientist with John F. Hunt at Columbia University where he used X-ray crystallography and biochemical and biophysical methods to characterize eukaryotic ion channels and bacterial proteins involved in stress response. Since 2007 he has been the director of the Protein Expression Technology Center (PETC) of the UCLA-DOE Institute for Genomics and Proteomics. The PETC supports research projects in bioenergy, structural biology, and proteomics which are related to the overall mission of the Institute for Genomics & Proteomics. One major research project at the PETC is the support of Dr. David Eisenberg’s participation in the Tuberculosis Structural Genomics Consortium which focuses on determining the structures of mycobacterial protein complexes. Dr. Arbing leads the cloning and protein expression/purification efforts for this project and is using X-ray crystallography to determine the structures of secreted mycobacterial protein complexes.

  

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