Abstracts - Session 1 & 2

Day 1 Monday October 17th 13.15

1.01 A Bacterial Factory for the Production of Membrane Proteins	Philip D. Laible	Protein Engineering Group, Argonne National Laboratory, 9700 South Cass Ave., Argonne, IL 60439, USA.
The determination of the structures and functions of all proteins encoded by genomes is a focal point of biological research in the 21st century. Current structural and functional genomics efforts are biased strongly against the investigation of proteins that are particularly challenging, namely membrane proteins, protein complexes, and large multi-domain, single-chain assemblies of eukaryotic origin. Most unfortunate for the community is the fact that the majority of genes encoded by complex organisms encode proteins that belong to one, or more, of the challenging classes. It is, thus, clear that novel approaches and innovative strategies will be required in order to begin to study efficiently these numerous and vital biological macromolecules. Whatever technologies are pursued, they must allow for intelligent and effective approaches to the more demanding, intricate, and functionally diverse sets of challenging polypeptides. An example of progress towards such efforts with membrane proteins will be presented as the type of groundbreaking inroads that will need to be made in order to start studying these types proteins at rates that only remotely approach the rate at which soluble proteins are studied today. Membrane proteins perform a variety of tasks that are ultra-critical for the viability of healthy cells. As such, they play pivotal roles in disease and represent the majority of drug targets. A system is presented which seeks to overcome some of the current membrane protein hurdles by exploiting the unique physiology of an extensively studied species of photosynthetic bacteria - Rhodobacter - for the heterologous expression of membrane proteins. This Rhodobacter expression system is one example of a developing technology for a particular class of challenging proteins that we hope can be widely adopted in order to exert a major impact upon structural and functional genomics efforts.

14.20: Session 2 - Purification of Membrane Proteins

2.01 Introduction to Topic: The Challenge of Membrane Proteins	Roslyn Bill	Aston Academy of Life Sciences, Aston University, Birmingham, UK.
The importance of membrane proteins is undisputed within the academic and pharmaceutical industry communities. In the field of basic science, understanding how cells and networks of cells function together in integrated ways in tissues, organs and whole organisms is a key driver in elucidating membrane protein structure-activity relationships. For these reasons, membrane proteins already constitute approximately 70% of pharmacological targets, while 55-85% of all new drug targets for the next decade will be membrane proteins. Astonishingly, no drug against a membrane protein target has been rationally designed with the benefit of the target’s structure - no suitable structures are available. As a consequence of this, much focus has been placed on solving membrane protein structures, and several international initiatives have arisen with this goal in mind (e.g. E-MeP, MePNet, SWEGENE, ProAMPand MPSI). It is perhaps surprising then, that although at least 30% of the genome is estimated to encode membrane proteins, they account for less than 0.003% of known protein structures or approximately 3% of all unique structures. Inevitably, the development of new tools and technologies to accelerate progress in this area has already reaped benefits and new membrane protein structures are being deposited in the public databases. Despite these successes, however, high-throughout membrane protein structure determination is still far from possible, with the rate of progress still being significantly slower than that required to address structure-activity relationships in the membrane proteome. This is acknowledged to be because a number of bottlenecks exist from gene to structure, the most significant being that of the production of purified, functional, recombinant eukaryotic membrane proteins suitable for crystallization trials.

2.02 Generation of GPI-linked CCL5 Based Chemokine Receptor Antagonists for Suppression of Acute Vascular Damage in Transplantation	Mike Notohamiprodjo	Medizinische Poliklinik, Schillerstrasse 42, DE-80336, Ludwig-Maximilians-University of Munich, Germany.
Limiting the acute vascular damage associated with leukocyte infiltration is a central issue in solid organ transplantation. The family of chemotactic cytokines (chemokines) help to regulate leukocyte recruitment. Systemic treatment with the chemokine ligand-5 (CCL5) based antagonist Met-RANTES, was previously shown to suppress acute damage to transplanted kidneys by blocking effector cell recruitment. To address problems associated with systemic-long term administration of protein antagonists, a reagent was designed that could be integrated into endothelial surfaces of the organ just prior to transplantation. Proteins anchored by glycosylphosphatidylinositol (GPI), when purified and added to cells, are efficiently incorporated into their cell surface membranes. A series of modifications were introduced into the CCL5 protein to generate a functional antagonist. These included the addition of an N-terminal methionine group, a mutation to render the protein a dimer, and a GPI signal sequence for surface expression. The resultant protein was stably expressed in CHO cells, GPI anchorage was confirmed, and the protein purified by FPLC. Exogenously administered Met-CCL5(dimer)-GPI efficiently inserted into the membrane of microvascular endothelial cells. The reagent is being tested in murine models of renal transplantation. The effect on subsequent immune induced damage will be assessed.

2.03 Production and Characterisation of Recombinant Forms of Human Pulmonary Surfactant Protein C (SP-C) Preserving its Structure and Surface Activity	Dunja Lukovic	Departament de Bioquímica i Biologia Molecular, Universitat de València, ES-46 100 Burjassot, Spain.
Surfactant protein C (SP-C) is an essential component for the surface-tension-lowering activity of the pulmonary surfactant system. It is considered to be one of the most hydrophobic proteins known so far, containing a valine-rich a helix that spans the surfactant phospholipid bilayers. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material, and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C to be included in new therapeutic surfactant preparations. We describe herein a protocol for recombinant SP-C production in bacterial cultures. We overexpressed SP-C as fusion to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimera was partially purified by affinity chromatography and subsequently subjected to protease digestion. SP-C was recovered from the digestion mixture by organic extraction and further purified by size exclusion chromatography. CD spectroscopy showed spectra typical for a-helical proteins, SDS-PAGE analysis disclosed a single band, and optimal biophysical activity measured by surface spreading of lipid/protein suspensions was obtained with the recombinant protein. By this method we can produce up to 0.5 mg of highly pure SP-C per litre of culture. Moreover, this work opens new possibilities to develop improved therapeutic preparations.

2.04 Structural Characterization of the Integral Membrane ErbB-2 Tyrosine Kinase Receptor	Miro Venturi	Pharmacia Italia S.p.A., Viale Pasteur 10, IT-20014 Nerviano, (MI), Italy.
The tyrosine kinase receptor ErbB2 is implicated in cell proliferation and differentiation. No ligand responsible for its activation has ever been identified. We tested different extraction procedures to isolate the receptor from the ErbB2-over-expressing SKOV3 cell line and probed different antibodies for their ability to bind the receptor under native conditions. The high affinity monoclonal antibody trastuzumab is capable of purifying the receptor with a yield equal to 0.2 mg/107 cells. The pure receptor preparation has been further characterized by CD spectroscopy and analytical ultra-centrifugation analysis. The purified ErbB2 protein can be used for studies of ligand screening and structural characterization.

2.05 Deglycosylation to Obtain Stable and Homogeneous Pichia pastoris - Expressed N-A1 Domains of Carcinoembryonic Antigen	Noelia Sainz-Pastor	Department of Oncology, Royal Free and University College Medical School, University College London, Rowland Hill Street, London, UK.
Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-A1 to test this hypothesis. The N-A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His6-tag) in the yeast Pichia pastoris. His6-tagged N-A1 was captured from the supernatant by batch purification with copper-loaded Streamline™ Chelating, an immobilized metal affinity chromatography (IMAC) matrix usually utilized in expanded bed techniques. Purified N-A1 was heterogeneous with a molecular weight range from 38-188kDa. Deglycosylation with endoglycosydase H resulted in three discrete molecular weight forms of N-A1. These were separated by concanavalin A chromatography followed by HiTrap™ IMAC. The procedure resulted in single-band-purity, mannose-free N-A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from Pichia pastoris.