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MONDAY, AUGUST 20

Protein purification has reached a plateau where favored steps, affinity tags, kits and protocols are widely accepted and used. Yet challenges remain, especially for novel and difficult proteins, such as membrane proteins. While cell culture processes have been refined to reach higher densities, greater demands are placed on purification to keep up with the booming biologics industry. High-throughput purification provides solutions leading to greater efficiencies while also posing additional challenges.

Emerging technologies present unrealized potential for monitoring processes, analyzing conditions, and identifying optimal strategies. Automation and robotics are revolutionizing protein industrialization. This meeting will explore purification challenges and how to overcome those challenges drawing on new technologies and innovative methods. Please join the discussion of next-generation strategies and technologies in the quest for purified proteins.

11:30 - 5:30 pm Main Conference Registration

CONQUERING CHALLENGES

1:00 Chairperson’s Opening Remarks

» 1:10 Opening Keynote Presentation:

Addressing Challenges of Membrane Protein Expression, Purification and Stabilization

Lawrence J. De Lucas, Ph.D., Director, Center for Macromolecular Crystallography (CMC), University of Alabama, Birmingham

This presentation will compare strategies using a variety of protein/peptide tags to provide an early quality assessment for protein expression, potential yield, purity and homogeneity of integral membrane proteins (IMPs). In addition, several examples will be presented for high-throughput self-interaction chromatography used as a method to improve membrane protein solubility and stability.

1:45 Expression and Purification of Diacylglycerol Acyltransferases

Heping Cao, Ph.D., Principal Research Scientist, Southern Regional Research Center, U.S. Department of Agriculture

Diacylglycerol acyltransferases (DGATs) are integral membrane proteins that catalyze the last and rate-limiting step of triacylglycerol biosynthesis in eukaryotic organisms. DGAT knockout mice are resistant to diet-induced obesity and lack milk secretion. DGAT genes have been identified from at least 83 organisms, but none of the enzymes from any organism had been expressed and purified from bacterial expression systems. We developed the first procedure for producing full-length recombinant DGAT proteins from any species using an E. coli expression system.

2:15 QbD and PAT Approaches to Purify Protein in E.coli

Ji Zheng, Ph.D., Senior Scientist, Biologics Development, Allergan, Inc.

2:45 Sponsored Presentation (Opportunity Available)

3:00 Refreshment Break

AFFINITY PURIFICATION

3:15 The Exploitation of S. cerevisiae: A Comprehensive Design of Affinity Tags and Overview of Microbial Applications

Carissa Young, Ph.D., Research Scientist, Delaware Biotechnology Institute, Department of Chemical and Biomolecular Engineering, University of Delaware

To improve the functional production of secreted or membrane proteins (e.g., ligand-binding yields indicative of active receptors), we have generated versatile yeast expression cassettes that incorporate numerous tags for identification and purification, and to assess protein interactions, protein structure/function, and intracellular localization. To reference selective organelles in S. cerevisiae, endogenous proteins were designed with codon-optimized fluorescent protein variant resulting in a valid assessment of heterologous protein subcellular localization. Furthermore, we have established methodologies to investigate the role of cellular quality control and its modulation during heterologous protein expression, focusing specifically on the unfolded protein response (UPR), autophagy, and ER associated degradation (ERAD) pathways.

3:45 Analyzing Cell Signaling Pathways with Affinity Purification-Mass Spectrometry

Alexey Veraksa, Ph.D., Assistant Professor, Biology, College of Science & Mathematics, University of Massachusetts, Boston

The study of cell communication mechanisms has been facilitated by the advances in protein complex purification and analysis. In my laboratory, we use affinity purification followed by mass spectrometry to map the structure of signaling networks, with follow-up functional studies in model systems. I will present the evolution of preferred tags used in my lab for affinity purification of protein complexes from higher eukaryotic cells and tissues, and will discuss the advantages and potential limitations of this approach.

5:15 Breakout Group Summaries

5:30 Grand Opening Reception with Exhibit and Poster Viewing

7:00 End of Day

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