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Affinity Protein Purification - Day 2



Affinity Protein Purification 


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Tuesday, August 23

7:30 am Breakfast Presentation (Sponsorship Opportunity Available) or Morning Coffee

 

OPTIMIZING PURIFICATION 

8:25 Chairperson's Remarks

8:30 Protein Production in S. cerevisiae for Systems Biology Studies

Naglis Malys, Experimental Officer, Faculty of Life Sciences, University of Manchester

Proteins, collectively with metabolites, nucleic acids, lipids and other intracellular molecules, form biological systems that involve networks of functional and physical interactions. To understand these interactions and the many other characteristics of proteins in the context of biochemical networks and systems biology, research aimed at studying medium and large sets of proteins is required. This either involves an investigation focused on individual protein activities in the mixture (e.g. cell extracts) or a protein characterisation in the isolated form. Here I provide an overview on the currently available resources and strategies for isolation of proteins from Saccharomyces cerevisie. The use of standardised gene expression systems is discussed and protein production protocols applied to the data generation pipeline for systems biology are described in detail.

9:00 Engineering Proteases with Switchable Activity

Philip N. Bryan, Ph.D., Professor, Department of Bioengineering, University of Maryland; Owner, Potomac Affinity Proteins, LLC

We will describe the engineering of highly-specific proteases which are tightly regulated by small molecules. The ability to control protease specificity and activity creates a vast potential for building enzyme-based nanodevices. The substrate protein contains the cognate sequence of the protease. The trigger of the protease is a specific small molecule. In this scheme the protease occupies a role analogous to a transistor in an electronic circuit and is able to mediate such functions as detection, purification, activation, or inactivation (destruction) of other proteins. The substrate protein and the triggering molecule will vary from application to application. For example, we have developed a protein purification system (the Profinity eXact Purification System,  Bio-Rad). This method uses a simple protease machine for protein purification and provides the conceptual foundation for other devices based on triggered proteases. 
 

9:30 Identification of an FHL1 Protein Complex Containing ACTN1, ACTN4, and PDLIM1 Using Affinity Purifications and Mass Spectrometry Based Protein–Protein Interaction Analysis

Anthony Gramolini, Ph.D., Professor, Department of Physiology, University of Toronto

Affinity tagged constructs of cardiac and skeletal muscle-relevant proteins were expressed, and metal affinity chromatography was used to purify the protein together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS) we identified putative interacting complexes. Follow-up biochemical and imaging experiments were used to validate and extend these findings.

10:00 Networking Coffee Break with Exhibit and Poster Viewing

10:45 Substrate Selectivity and Kinetics for Fe(II)-Dependent gamma-Hydroxybutyrate Dehydrogenase from Ralstonia Eutropha

Stanley M. Parsons, Ph.D., Professor, Department of Chemistry and Biochemistry, Neuroscience Research Institute, University of California, Santa Barbara

The gene for iron(II)-dependent gamma-hydroxybutyrate dehydrogenase (GHB-DH; EC 1.1.1.61) from the bacterium Ralstonia eutropha was fused to the gene for glutathione S -transferase in an expression vector, and 161 ± 32 mg of fusion protein was isolated from 1 L of induced E. coli. The purified fusion protein was used to test 42 natural and synthetic compounds structurally related to the human drug of abuse GHB for substrate activity. When testing otherwise normal human urine or blood using a prescribed protocol, GHB-DH will detect only ingested GHB.

11:15 Application of Thermal Shift Assay in the Stability Optimization of Aurora B Kinase Domain

Payal Sheth, Ph.D., Investigator, Protein Science Department, Merck Research Laboratory

 

AFFINITY CHROMATOGRAPHY 

11:45 pm Classical and Affinity Chromatography Employed in Obtaining BACE and JAK Proteins

Thomas L. Emmons, Ph.D., Senior Scientist, Pfizer, Inc.

The catalytic domains of tagged aspartyl protease BACE-1 and BACE-2 and tyrosine kinases JAK-2/-3 and TYK-2 have been purified to homogeneity by using a combination of classical and tag affinity chromatography, as well as (in the instance of BACE-1 and BACE-2) a ligand specific affinity chromatography. Tag removal by BACE-1 and BACE-2 autocleavage or by JAK-2/-3 and TYK-2 cleavage with specific proteases was performed as needed. High quality enzymes were obtained for kinetic and inhibition studies and to obtain crystals that yielded high resolution 3D structures for inhibitor optimization. The purification of a double tagged full length kinase will be discussed as well. Finally, yields, optimization, throughput, reproducibility and scalability of the purification processes will be presented along with in depth characterizations of the purified product to establish its authenticity and activity.

12:15 Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own 

1:55 Chairperson's Remarks

2:00 A Recovery Platform for the Initial Purification of His-Tagged Proteins Derived from E. coli Inclusion Bodies

Liliana T. Yee, Research Associate, Purification Development, Genentech/Roche

During recombinant protein production in E. coli, the target protein may be associated with the insoluble phase following cell lysis and centrifugation. Protein purification from the insoluble phase can be challenging due to the presence of: aggregated species, misfolded proteins, and/or inclusion bodies (due to high levels of expression, cell induction, and/or growth conditions), as well as cell debris and DNA released in the cell lysis step. In this presentation, we will describe our recovery platform for the initial extraction and chromatography for his-tagged proteins that are expressed as inclusion bodies in E. coli. This procedure allows for the initial isolation of this of proteins independent of expression levels, allowing us the flexibility to scale this laboratory process to support a wide range of production needs.

ANTIBODY AFFINITY PURIFICATION 

2:30 Affinity Purification of a New Marker for Kidney Failure from Multiple Biological Sources

Kevin Rupprecht, Ph.D., Principal Research Scientist, Abbott Diagnostics Analytical Chemistry R&D, Abbott Laboratories

This talk will detail our use of a monoclonal antibody affinity column to purify NGAL (Neutrophil Gelatinase-Associated Lipocalin) from a variety of sources. We have isolated NGAL from neutrophils, lymphocytes, a leukocyte lysate, neutrophil granules and urine. This protein naturally binds iron siderophores. The siderophore co-purifies with the NGAL indicating the purified material is in a native conformation. The material isolated is very pure (>99%) and we have thoroughly characterized it. We present data comparing and contrasting the material from the various sources including carbohydrate content, siderophore content and dimer content.

3:00 From Quantitative Protein Complex Analysis to Disease Mechanism

Karsten Boldt, Ph.D., Professor, Medical Proteome Center, Centre for Ophthalmology, University of Tuebingen 

Using an affinity-based quantitative proteomic approach, we show that the Leber's Congenital Amaurosis (LCA) associated protein lebercilin specifically interacts with intraflagellar transport (IFT) complex A and complex B members. By quantitative complex comparison we demonstrate that LCA-associated nonsense mutations specifically disrupt the interaction with the IFT proteins. This implicates a disruption of IFT dependent protein transport in photoreceptors as the cause for LCA which was confirmed by inactivation of lebercilin in mice.

3:30 Networking Refreshment Break with Exhibit and Poster Viewing

4:15 Antibody Affinity Purification Using Nickel Particles

Zhiyu Li, Ph.D., Assistant Professor, Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences

Magnetic separation using functionalized magnetic adsorbent particles are emerging as reliable and convenient techniques in biomacromolecules (proteins and DNAs) purification and cells isolation. In this study, a novel method using Protein A -coated nickel ferromagnetic particles for affinity purification of antibody has been proposed and confirmed. Protein netting/caging method has been utilized to stabilize Protein A on nickel particle surface and limit protein leaching during affinity purification. IgG in mouse serum can be quickly isolated for as short as 5 minutes. In addition, the separation procedure is gentle, scalable, efficient and economical. Protein coated and netted nickel particles will not only be utilized in affinity purification but can also be further modified through functional groups of amino acids for other chromatographic applications.

4:45 Expanding the Repertoire of Split Proteins as Versatile Tools for Ttagging, Detection, and Purification

Geoffrey S. Waldo, Ph.D., Team Leader, Biosciences, Los Alamos National Laboratory

GFP, RFP and 'SNAP' are a convenient genetically encoded tags for labeling proteins, but are bulky and can perturb protein behavior or cause misfolding and aggregation. In 2005 we published a split GFP that has become widely used in protein production, cell-based assays, and library screening campaigns. We now report a new split fluorescent protein that is orthogonal to the original GFP. We describe its characterization and validation in protein applications. This unusually stable protein remains fluorescent in 7 M GndHCl when reconstituted from its two fragments. We also describe new color variants of our original split GFP. We describe the new applications that are possible using these orthogonal tags, including multiplex tagging, enhanced FRET experiments without causing protein misfolding, host-pathogen interactions, and tracking protein complex formation in living cells. These tools should also enable new TAP MS applications and library screens for proteins with improved solubility and stability. Mix and match tools for improving protein crystallization are another area hinted at.

5:15 End of Conference



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