Protein expression/HTX description for EMBL Grenoble
ESPRIT and HTX are currently available for funded access though the EU-FP7 P-CUBE project (http://www.p-cube.eu/). They will be available through BioStruct-X from 1st April 2013.
MultiBac expression of protein complexes
The MultiBac platform at the EEF is the lead technology for eukaryotic multiprotein complex production (Berger et al, Nat. Biotechnol. 2004; Fitzgerald et al, Nat. Methods 2006; Bieniossek et al, Curr. Protocols 2008). MultiBac has been developed by the Berger laboratory, and is an advanced baculovirus expression vector system (BEVS) particularly tailored for protein complex production. Since its introduction it has been distributed to more than 400 laboratories (academic and industrial) worldwide and has catalysed research in many areas of the life sciences (see, for example, Imasaki et al, Nature 2011; Yamada et al, Nature 2011; Schreiber et al, Nature 2011; Leonard et al, Cell 2011; Zeqiraj et al, Science 2009).
MultiBac is installed at the Eukaryotic Expression Facility (EEF) at EMBL Grenoble. The EEF consists of a fully equipped cell culture facility including sterile benches, light microscopy, fluorescence microscopy, shaker platforms, incubators and tissue culture equipment. Protein purification equipment and facilities for protein analysis including SDS-PAGE, immuno-blot and state-of-the-art biophysical techniques are also provided. All MultiBac reagents, protocols and cell strains required are available at the host laboratory.
MultiBac ultilizes an optimized baculovirus that has been engineered in the Berger lab for high-quality protein production. A fluorescent marker has been integrated into the viral backbone to enable virus performance tracking and protein production. This virus is accessed by multigene constructs containing the DNA encoding for the protein complex produced. This multigene construct is generated from small synthetic precursor plasmid modules into which the genes of interest are inserted.
The MultiBac platform has been part of consortia in FP6 and FP7, and has been accessed by many scientists from Europe and world-wide for MultiBac training and for protein production. Visitors are supported in project planning and construct preparation prior to their visit, and intensively trained on a one-to-one basis during their stay at the EEF (usually 10 working days).
ESPRIT: Screening of tens of thousands of constructs of a single gene to identify well-behaving soluble constructs
Academic structural biologists often work on proteins that lack accurate domain annotations. When the full-length protein cannot be expressed and a domain-focused approach is necessary, problems arise since it is unclear how to design high yielding, soluble expression constructs. Some proteins have little or no sequence similarity to others and this prevents domain identification using multiple sequence alignments. More often, some functional annotation exists e.g. from mutagenesis or deletion studies, but these regions do not define well the structural boundaries. Even when a soluble construct is obtained, disordered extensions may confound crystallisation attempts. We are all familiar with these situations; in many cases they are what keep our proteins “hot” and out of the PDB.
The ESPRIT technology has been developed in the Hart lab at EMBL to express proteins whose domain boundaries are difficult to predict. It does not aim to replace the initial PCR cloning experiments based upon careful inspection of the protein sequence, but provides a rescue strategy when this fails - as it often does. ESPRIT, which stands for “expression of soluble proteins by random incremental truncation”, is a directed evolution-type process combining random deletion mutagenesis with high throughput solubility screening. We use exonuclease to truncate the ends of the target gene sequence in a sequential manner, thereby generating all possible construct termini for downstream testing. Up to 28,000 constructs per gene are isolated using colony picking robots and gridded out to form high density colony arrays for protein expression testing. For detection of soluble constructs in the library, the efficiency of in vivo biotinylation of a fused C-ter peptide is measured using fluorescent probes. Recent developments include adaptation to protein complexes using a coexpression system (An et al., 2011a) and incorporation of a genetic selection to eliminate out-of-frame constructs from the library, a step that greatly enhances the screening power of ESPRIT and its application to more challenging systems (An et al., 2011b).
Over the last seven years, the ESPRIT platform has been visited by European scientists who have brought their problematic targets for screening. This has now been formalised by an EU grant, P-CUBE, which funds their travel and experimentation costs. Despite all the failures preceding the visit (typically over one or two years), about half of these users have returned home with soluble, purified proteins for further study. A recent highlight was the identification and soluble expression of the terminase domain from HCMV that resulted in its crystal structure (Nadal et al., 2010). Applications beyond structural biology have included vaccination of animals with domains from pathogen proteins and use of the soluble constructs for raising monoclonal antibodies.
See publications tab for some papers on the ESPRIT technology and examples of use.
High - Throughput Crystallisation
The High Throughput crystallization platform at the EMBL Grenoble Outstation ( the HTX Lab), offers automated crystallization services to local researchers in the context of the Grenoble Partnership for Structural Biology (PSB) and to European researchers through the EC-funded project P-CUBE. It is also actively involved in the development of new concepts and methods in macromolecular crystallization. The HTX lab is one of the largest crystallization facilities in Europe and is located next to the European Synchrotron Radiation Facility (ESRF) and the Institute Laue Langevin (ILL).
We offer automated vapour diffusion crystallization screening in hanging and sitting drop set up at two temperatures ( 20 deg and 5 deg ). Access to the results is provided through the web-based Crystallization Information Management System (CRIMS), which includes fast and convenient interfaces for the evaluation and annotation of experiments. Crystallization plates containing crystals are made available to users upon requests (typically during their next synchrotron visit) and facilities for crystal mounting and freezing are provided. Since the beginning of operations in 2003, the HTX lab has provided crystallisation screening services to over 400 scientists resulting in more than 4 million individual crystallization experiments.
The process at the HTX lab is divided in three major areas: (1) preparation of crystallization screens, (2) set up of crystallization experiments and (3) imaging:
- The preparation of crystallization screens is performed at a TECAN station designed to cope with the large volume of screens required for the operation of the platform and requiring minimal manpower. In addition to the standard pipettingand plate handling systems it integrates a 96-line pipetting station, a storage carrousel with capacity for 200 microplates and an automated plate sealer. These three components are controlled by a single software. This station is used to reformat commercial crystallization screens into deep-well blocks, and crystallization plates as well as to generate custom and refinement screens;
- The crystallization experiments are set up with the aid of a Cartesian PixSys crystallization system (genomic Solutions) using volumes of 100 + 100 nl. In this system it is possible to process up to three plates in parallel, each plate holding up to three samples.
- Currently we have three automated imaging systems each one with a capacity for 500 plates. These systems operate at 20 and 5 deg.
Our standard crystallization protocol involves the use of the vapor diffusion sitting drops. However we have developed a number of protocols to handle samples with special requirements, such as membrane proteins. We can also set up crystallization experiments at low temperature. After the experiments are set up, users receive a report with experimental details, quality control information and information on any possible incidence. Access to results is provided in real-time through the CRIMS interface. Plates in the storage systems are made available to users on-site upon request, typically at the time of a synchrotron visit. A laboratory with microscopes and equipment for crystal mounting and freezing is at their disposal.
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