Affinity Tag Protein Purification


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Wednesday, August 26, 2009

7:30am Registration and Morning Coffee


8:20 Chairperson’s Opening Remarks

8:25 Featured Presentation

Self-Cleaving Tags Based on Engineered Inteins: Recent Results and Future Prospects

David Wood, Ph.D., Assistant Professor, Chemical Engineering, Princeton University

Effectively any purification tag can be rendered self-cleaving by joining it to an engineered intein, which provides simple recovery of the target protein using any desired affinity strategy. Although some limitations still exist to this technology, it has the potential to become a critical platform in the production of recombinant proteins at virtually any scale. This talk will present our latest results with these methods in higher eukaryotic systems, and suggest their future role in industrial recombinant protein production.


9:00 Endo- and Exo-Proteolytic Removal of Affinity Tags

David S. Waugh, Ph.D., Protein Engineering Secti on, Macromolecular CrystallographyLaboratory, Center for Cancer Research, Nati onal Cancer Insti tute at Frederick

Methods and reagents for the removal of affinity tags will be described, with emphasis on the use of tobacco etch virus (TEV) protease and an A-type carboxypeptidase from Metarhizium anisopleae.

9:30 Advantages and Disadvantages of Using GST Tags for Protein Purification

David W. Speicher, Ph.D., Professor and Director, Center for Systems and Computational Biology; Director, Proteomics Laboratory, The Wistar Institute

Many recombinant proteins can be expressed at high yields in soluble form when fused to GST, including proteins that use alternative initiation sites or are not properly folded when expressed without the GST moiety.  In addition, the GST provides a high-affinity tag for facile purification or for pull-down experiments.  Potential disadvantages of GST-fusion proteins include oligomer formation via the GST moiety, potential steric hindrance caused by the large fusion tag, and increased costs associated with protease cleavage and removal of the affinity tag.

10:00 Networking Coffee Break with Exhibit and Poster Viewing


10:45 Improving TAP Tags

Marius Ueffing, Ph.D., Head, Protein Science, Helmholtz Center Munich, German Research Center for Environmental Health (GmbH)

11:15 Advances in Protein Complex Purification Approaches: GS-TAP and Beyond

Alexey Veraksa, Ph.D., Assistant Professor, Biology Department, University of Massachusetts 

We developed a system for efficient protein complex purification from Drosophila cultured cells and whole organisms, using the recently developed GS-TAP tag. This tag consists of two Protein G modules and a streptavidin binding peptide (SBP), separated by two TEV protease cleavage sites. We have compared the performance of this method to the “conventional” TAP tagging and found that the GS-TAP approach results in higher yields and reduced background. Our recent results from alternative tagging approaches will be presented as well.

11:45 Tandem Affinity Purification of PTP-Tagged Proteins

Arthur Günzl, Ph.D., Department of Genetics and Developmental Biology, University of Connecticut Health Center

The PTP tag combines two protein A domains, a TEV protease cleavage site and the protein C epitope.  PTP-tagged proteins can therefore be purified by IgG and anti-protein C affinity chromatography. The latter step is facilitated by the monoclonal antibody HPC4 which recognizes the protein C epitope with high affinity in a calcium ion-dependent manner.

12:15pm Sponsored Presentation (Opportunity Available – Please contact Suzanne Carroll at

12:30 Lunch on Your Own (Lunch Presentation Opportunity Available) 


1:55 Chairperson’s Remarks

2:00 Use of Affinity Tag Technology for Isotopic Labeling of Membrane Peptides and Proteins for NMR Analysis

Gianluigi Veglia, Ph.D., Associate Professor, Department of Chemistry & Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota

In this talk, I will describe the methods that we use in our lab and adapted to the expression of milligram amounts of isotopically labeled peptides and proteins to be studied by NMR spectroscopy.

2:30 Innovative Low Human Homology Affinity Tags

Fons Bosman, Ph.D., Formulation Expert, Innogenetics Biologicals

Numerous types of fusion tags have been developed during the last two decades in order to increase the expression levels of recombinant proteins or to facilitate their recovery by affinity purification. However, many tags have the drawback of causing enhanced false reactivity when used in diagnostic procedures. Furthermore, most of these affinity tags, even the commonly used His tag, are not suited for therapeutic applications for immunology-related reasons. We designed flexible small (multifunctional) low human homology affinity tags (LHH-tag) that maintain the benefit of single-step purification on commercially available resin, and that can resolve these general immunology-related disadvantages. The selection strategy of these LHH-tags, as well as purification- and immunology-related results will be presented and discussed.

3:00 Snags with Tags

Paul Ramage, Ph.D., CPC/Protease Platform, Senior Research Investigator 1, Novartis Pharma AG

Affinity tags are widely used for protein purification and expression. For some they are an essential tool, for others they are a necessary evil. In my presentation, I will describe our experiences with a number of different tags and cleavage sites, highlighting the advantages and disadvantages of the different combinations.

3:30 Ice Cream Refreshment Break with Exhibit and Poster Viewing

4:15 Element Coded Affinity Tags

Claude Meares, Ph.D., Professor, Department of Chemistry, University of California, Davis


Isotope-coded affinity tags (ICAT) represent an important new tool for the analysis of complex mixtures of proteins in living systems [Aebersold, R., and Mann, M. (2003) Nature, 422, 198-207]. We envisage an alternative protein-labeling technique based on tagging with different element-coded metal chelates, which affords affinity chromatography, quantification, and identification of a tagged peptide from a complex mixture.

4:45 Using the 15 Amino Acid FP mTag from a Novel Fluorescent Protein for Rapid in vivo and in vitro Protein 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. We introduce a new 15 amino acid tag, ‘FP microTag’, from a novel fluorescent protein. In living cells, or in the test tube in complex lysates, proteins tagged with FP microTag can be easily detected and quantified using the FP microTag Detector (the rest of the fluorescent protein). The fluorescent signal is stable even in 9 M urea, and is highly specific to the tag even in complex mixtures and cell lysates. Another version of FP microTag can be used both for detection and gentle purification of proteins from complex mixtures. We describe a number of novel applications in protein trafficking, protein interaction detection, high-throughput measurement of soluble protein in living cells, and engineering proteins for stability and solubility.

5:15 End of Day

For more information, please contact:
Margit Eder, Ph.D., Conference Director
Cambridge Healthtech Institute
Phone: 781-972-5478

For sponsorship information, please contact:
Suzanne Carroll, Manager, Business Development
Cambridge Healthtech Institute
Phone: 781-972- 5452

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