Abstracts - Session 3 & 5

Day 1 Monday October 17th, 16.30-18.00

Session 3 - Methods for Challenging Proteins

3.01 iTAP - A Protein Complex Purification Strategy for Functional Proteomics	Matthias Wilm	EMBL, Heidelberg, Germany

3.02 A Generic Strategy for Membrane Protein Purification and Crystallization	Said Eshaghi, Marie Hedrén, and Pär Nordlund	Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
A simple and cost effective detergent screening strategy has been developed, by which 24 detergents were screened for their efficiency to extract the recombinant ammonium/ammonia channel, AmtB, from Escherichia coli, in order to select the most efficient detergents prior to large-scale protein production and crystallization. The method requires 1-ml cell culture and is a combination of immobilized metal ion affinity chromatography and filtration steps in 96-well plates. The method allows simultaneous tests, such as salt and imidazole concentrations as well as the type of chromatography medium, to improve purification. The quality of purified proteins was further analysed by size exclusion chromatography showing intact and homogenous AmtB. Large-scale protein purification and subsequent crystallization screening resulted in low resolution diffracting AmtB crystals with two detergents. This strategy may easily be applied to several proteins and detergents simultaneously, as a high-throughput platform.

3.03 Use of the Myosin Motor Domain as Large-Affinity Tag for the Expression and Purification of Proteins in Dictyostelium discoideum	Martin Kollmar	Department of NMR-Based Structural Biology, Max-Planck-Institut for Biophysical Chemistry, Am Fassberg 11, DE-37077 Göttingen, Germany.
The cellular slime mold Dictyostelium discoideum is increasingly used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches, can be grown easily in large quantities and contains a large variety of chaperones and folding enzymes, which are able to perform all kinds of post-translational modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species. Also, other proteins, including structural proteins, enzymes and regulatory proteins, from different species have successfully been expressed and purified.

3.04 Membrane Protein Crystallization Facilitated by Detergent “Fingerprint”	Lei Zheng and Xiao-Dan Li	Structural Biology Group, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland.
Membrane proteins count for 20–25% of all open reading frames in genomes and play central roles in wide range functions of cells. Structure determination of membrane protein is one of most challenging tasks. Besides the difficulties of membrane protein expression, most of membrane proteins function as heteroor homo-oligomers. Once membrane proteins are extracted from membrane bilayer with detergent, maintaining their homogenous oligomeric state is essential for successful crystallization. Detergent screening has been widely used for crystallization of membrane protein. Crystallization can be facilitated by effectively changing detergent type and concentration, but to identify crystallization-favorable detergent is still a tedious trial-and-error procedure. However, each membrane protein has its own profile to react with different detergents in solution; the oligomeric state of some membrane proteins can be easily disturbed by exchanging detergent. Such detergent “fingerprint” of each protein is important to determine the strategy for crystallization of individual membrane protein. We used the HPLC system equipped with autosampler, more than 14 detergents can be quickly screened in a few hours with as small amount as a few micrograms of protein. Furthermore, this method could also be used for the high-throughput screening of membrane proteins to select more stable targets suitable for crystallization. As a consequence, we were able to solve the crystal structure of an ammonia channel AmtB wild type from E. coli at 1.8 Ångstrom resolution in 9 months.

Day 2 Tuesday October 18th, 8.30-10.20

Session 5 - Purification of Proteins from Inclusion Bodies

5.01 Routes to Active Proteins	Johannes Buchner	Technische Universität München, Germany.
The tyrosine kinase receptor ErbB2 is implicated in cell proliferation and differentiation. No ligand responsible for its activation has ever In the past decades, the recombinant production of proteins has become a standard technique in research laboratories. In many cases, high level expression of the protein of interest has been achieved. Purification was simplified by the addition of tag sequences (especially the His-tag) which allowed the use of affinity separation. However, despite this progress, many attempts to obtain active proteins by recombinant expression in E. coli or S. cerevisiae failed. A big problem, observed already early on, was the formation of large granules of insoluble recombinant protein, the so-called inclusion bodies, inside the host cells. Interestingly, this aggregation reaction is also observed when cells cannot respond to stress conditions such as elevated temperatures by expressing a specific set of proteins. This strongly suggested that there is a cellular machinery which assists the folding process. Furthermore, a balance between the amount of nascent protein and the helper proteins seems required. In recent years, different strategies have been developed to overcome the bottle necks of recombinant protein production. These include the in vitro refolding of inclusion body proteins and the optimization of the cellular folding machinery for the production of soluble protein.

5.02 Expression, Purification and Refolding of the Protein Phosphatase 1 (Ppt1) from Saccharomyces cerevisiae	Sebastian K. Wandinger1, Michael H. Suhre1, Harald Wegele2, and Johannes Buchner1	1 Department Chemie, Technische Universität München, Lichtenbergstraße 4, DE-85747 Garching, Germany. 2 Pharma Development, Roche Diagnostics GmbH, Nonnenwald 2, DE-82377 Penzberg, Germany.
Here we report the recombinant expression of the catalytically active phosphatase domain of the Saccharomyces cerevisiae protein phosphatase 1 (Ppt1) in E. coli. Ppt1 consists of two domains: a 20 kDa TPR (tetratricopeptide repeat) domain, which mediates protein-protein interactions and directs Ppt1 to potential substrate proteins, e.g. the molecular chaperone Hsp90. The second, a 40 kDa phosphatase domain, exhibits catalytic activity and dephosphorylates phosphorylated serine/threonine residues of respective substrate proteins. The Ppt1 phosphatase domain was cloned and expressed in E. coli in unsoluble inclusion bodies. After isolating these, the aggregates were denatured and soluble protein was purified using affinity chromatography. Optimal renaturation conditions led to large amounts of the refolded phosphatase domain in high purity. Interestingly, further enzymatic studies revealed that the domain is correctly folded and shows higher catalytic activity compared to the full length protein.

5.03 Matrix-Assisted Refolding of Oligomeric Small Heat Shock Protein Hsp26	Titus M. Franzmann	Department Chemie, Technische Universität München, DE-85747 Garching, Germany.
Recombinant gene expression in the prokaryotic host Escherichia coli is of general interest for both biotechnology and basic research. Use of E. coli is inexpensive and advantageous due to the fully developed genetic accessibility. However, often accumulation of the insoluble target protein (inclusion body) is observed. Especially when over expressing eukaryotic or disulfide bond containing proteins inclusion body formation is likely to happen. Nonetheless, insolubly produced protein can be regained and refolded in vitro. Commonly, renaturation of proteins is accomplished by methods involving dilution or dialysis of the target protein. Another interesting alternative is matrix-assisted refolding in which the denatured protein is immobilized on a column. Refolding is initiated by buffer exchange while the target protein remains bound. We have used matrix-assisted refolding of a double cysteine variant of Hsp26, a small heat-shock protein from S. cerevisiae which is expressed quantitatively insoluble in E. coli BL21 (DE3) cells. We show that this oligomeric protein can be efficiently recovered from the insoluble fraction and refolded into its native oligomeric and chaperone active state using ion exchange and size exclusion chromatography.

5.04 Large-Scale Production, Bacterial Location Assessment and Two-Step Purification of Human Single-Chain Fv Antibodies against AlphaIIb-Beta3 Integrin	R. Robert1, G. Clofent-Sanchez1, A. Hocquellet2, M-J. Jacobin1, A. M. Noubhani2, and X. Santarelli2	1 UMR 5536 RMSB and 2ESTBB, Université V. Sagalen Bordeaux 2, 146 rue Léo Saignat, FR-33076 Bordeaux Cedex, France.
Human anti-alphaIIb-beta3 scFv fragments were previously isolated by phage-display using an immune library. They were further cloned in the pHOG21 vector for a soluble expression. Our objective was to investigate the compartmental E. coli localization in batch conditions of an anti-alphaIIb-beta3 scFv fragment referred to as scFv[EBB3]. Production in large-scale condition was recently optimized for another selected anti-alphaIIb-beta3 scFv fragment, scFv[TEG4]. We compare in this work different purification processes elaborated for cell compartments such as supernatant, cytoplasm and inclusion bodies for isolation of high quality antibody fragments. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) and a renaturation procedure performed on the column, a second step purification was further realized as a polishing step. The same two-step procedure was applied for sonicated cells or supernatant using streamline process. The purity of the product was assessed by silver stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The scFv activities were then tested by enzyme-linked immunosorbent assay (ELISA) and flow cytometry on human platelets. The final aim of our purification work was to couple highly purified scFv fragments with superparamagnetic or paramagnetic agents to be used as imaging agents of the thrombus by Magnetic Resonance Imaging.