Targets Expression systems Crystallisation Structure

The E-MeP consortium selects membrane protein targets, which are expressed, refolded or solubilised, purified and delivered for crystallisation trials, to generate new 3D structures of membrane proteins.


workflow

Targets

Selection of targets is based on bioinformatic analysis (multiple sequence alignments, clustering by similarity, structure prediction), literature-based data mining, and expert analysis for each of the main protein families, based on existing databases, know-how and previous work of the consortium.

E-MeP's targets consist of ~200 eukaryotic, GPCRs, ion channels, transporters and pumps, representative of major classes of membrane proteins in human health and diseases and ~100 prokaryotic targets, relevant homologues of eukaryotic targets (e.g. ABC transporters and secondary active transporters, glutamate receptors) and membrane targets from pathogenic bacteria, for which milligramme quantities are already available.

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Expression systems

E-MeP is focused on protein production of prokaryotic proteins, for which efficient protocols are already available, and of eukaryotic proteins, for which production systems need substantial improvement. The expression systems used are: bacterial systems (E. coli, L. lactis) for X-ray crystallography, EM & NMR; yeast systems (P. pastoris, S. cerevisiae); animal cells (Semliki Forest Virus, Baculovirus).

Once membrane proteins are produced the next steps are solubilisation, stabilisation and purification for crystallisation purposes.

Purification approaches are applied to a large variety of proteins, with emphasis on GPCRs, channels, secondary transporters and ABC transporters. Purification schemes include gel filtration, ion-exchange, immobilised-metal and hydrophobic interaction chromatography, isoelectric-focusing, and affinity chromatography using immobilised ligands, calmodulin, hemagglutinin, monomeric avidin (for biotinylated proteins) or streptavidin columns (with Strep-tag I/II) and immobilized G-proteins. E-MeP makes extensive use of automated procedures such as those implemented on the Äkta purification system and rapid scouting of chromatography conditions with on-line detection of protein quality by light scattering.

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Crystallisation

Pure, stable and functional membrane proteins are then used for crystallisation, crystal optimisation and structure determination. The key objectives are discovery of initial crystal leads during the screening; and optimisation of crystals to diffraction quality. Optimisation experiments include the use of additives, inhibitors, amphiphiles, varying protein concentration, improvements in protein preparation, addition of lipids, varying freezing conditions, simple procedures for testing unfrozen crystals, thermal annealing etc. These studies benefit from using small quantities of sample, and frequent access to European synchrotron X-ray sources.

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Structure

When well diffracting 3D membrane protein crystals have been recovered, Molecular Replacement or Multiple Anomalous Dispersion is used to recover phases. X-ray diffraction data is collected at dedicated protein crystallography and MAD beamlines at synchrotrons throughout Europe. All X-ray laboratories have regular access to these user facilities. As soon as diffracting membrane protein crystals are recovered, heavy-atom derivatives and seleno-methionine labelling are pursued in parallel.

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