The following technologies have been developed within E-MeP.

Professor Richard Neutze developed

The Lipidic-Sponge Phase™ Screen - the latest tool for crystallising membrane proteins

Lipidic-Sponge Phase

A novel method for extending the lipidic cubic phase crystallisation concept to a lipidic sponge phase (Wadsten, P., Wohri, A.B., Snijder, A. et al. 2006, J. Mol. Biol., 364: 44–53).The lipidic-sponge phase screen is a new innovative protein crystallisation screen, which can be used for all types of bacterial and eukaryotic membrane proteins. The new screen accommodates larger membrane proteins and it can be used in an ordinary hanging- or sitting- drop vapour diffusion experiments. It also has broader usage and is compatible with high-throughput crystallisation approaches. The screen can also be optimised to suit particular crystallisation experiments.

The figure shows a cartoon representation of the a sponge phase, showing the large (aqueous) pores able to accommodate memnbrane proteins with extra- and/or intracellular domains (100-150 A)

Professor Neutze and Molecular Dimensions Ltd have successfully collaborated to take to the market and develop the new crystallisation screen into a commercial product. More details can be found on the Molecular Dimensions website.

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In 2006 Professor Horst Vogel presented

Cell membranes suspended across nanoaperture arrays for functional analysis of membrane proteins

Cell membranes suspended across nanoaperture arrays

We present a method for spreading large (>100 µm2) cell membrane fragments across nanoapertures in planar supports. Electron-beam and focused-ion-beam lithography were used to fabricate arrays of 50-600 nm diameter holes in free-standing silicon nitride (SiN) solid films 100-500 nm thick. By pressing adhering live cells onto the nanostructured SiN surface and then removing them, planar cell membrane sheets (CMSs) were transferred in a well-defined orientation onto the SiN support.

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Professor Bert Poolman developed

High-throughput cloning and expression in recalcitrant bacteria

High-throughput cloning and expression in recalcitrant bacteria

Presented here is a generic method for high-throughput cloning in bacteria less amenable to conventional DNA manipulations. The method employs ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector backbone exchange (VBEx) into a bona fide, organism-specific plasmid ready for high-efficiency transformation. Here, we describe the VBEx procedure for Lactococcus lactis.

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In 2006 Dr Edmund Kunji presented

Determination of the dimensions and mass of small membrane proteins in detergents by size exclusion chromatography

Determination of the dimensions and mass of small membrane proteins in detergents by size exclusion chromatography

When size exclusion chromatography is used to establish the molecular mass of a small membrane protein, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate values for the protein.

In a detergent series, in which the detergents differ only by the length of the alkyl chain, the molecular mass of the protein can be determined accurately by plotting the molecular mass of the protein-detergent micelle against the detergent-protein ratio.

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Dr Edmund Kunji developed

Functional expression of eukaryotic membrane proteins in the Gram-positive bacterium Lactococcus lactis

Functional expression of eukaryotic membrane proteins in the Gram-positive bacterium Lactococcus lactis

Structural and functional studies of eukaryotic membrane proteins have been hampered by the large number of difficulties that are encountered in their overproduction. Among the complications are the untimely death of the host cell, incorrect targeting and the production of dysfunctional material due to faulty insertion and folding. In order to tackle this important bottleneck new approaches in membrane protein over-expression have to be developed.

Here we show that the nisin expression system of the Gram-positive bacterium Lactococcus lactis can be used for the functional expression of mitochondrial carriers from yeast.

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Further information:

Eukaryotic membrane protein overproduction in Lactococcus lactis

Lactococcus lactis as host for overproduction of functional membrane proteins

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Dr Edmund Kunji developed

On-line measurements of microbial cultures in fully aerated fermenters

On-line measurements of microbial cultures in fully aerated fermenters

Growth measurements in a fully aerated fermenter of strains of Saccharomyces cerevisiae expressing constitutively the mitochondrial ADP/ATP carrier AAC3 with either an N-terminal six histidine or a nine-histidine tag.

Continuous measurements (shown at one minute time intervals) were carried out with a fibre-optic probe fitted with an air bubble excluder (view international patent).

The WB12 strain containing plasmid pYES3-Paac2-N6His-aac3 had a doubling time of 3.53 h, whereas the strain with plasmid pYES3-Paac2-N9His-aac3 had a doubling time of 3.98 h, showing the effect on growth of the introduction of three more histidine residues to the fusion protein.

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In 2008 Dr Roslyn Bill developed

Protein expression system

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

Tuning BMS1 transcript levels in yeast in a doxycycline-dependent manner results in optimized yields of functional membrane and soluble protein targets. Polysome profiling shows that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis.

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Further information:

UK Patent Application Number GB0813253.2 filed on 18th July, 2008

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The team of Dr Nicolas Le Novère developed

E-MeP infrastructure

E-MeP computing infrastructure

In order to support the E-MeP project, the Computational Neurobiology group of the EBI developed an authenticated knowledge infrastructure reusable in structural genomics projects.

The platform allows monitoring of the project progression at the level of an experimental task applied to a target, and permits to reconstruct workflows from target selection to structure determination.


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