The facility at the University of Oxford (UOXF) is dedicated to protein expression in mammalian cells for structural biology. It includes

  • Streamlined methodologies for transient expression in 293T cells, first for rapid small scale screening of multiple constructs, and then for scale up of selected constructs (Aricescu et al, 2006 Acta Cryst D),
  • Technologies for controlling glycosylation in secreted proteins (Chang et al, 2007, Structure) and SeMet labelling (e.g. Aricescu et al, 2006, EMBO J) and
  • Co-expression to produce heterodimeric protein complexes for structure determination.

Development work includes membrane protein expression in mammalian cells. Our expression facilities feed into well-equipped protein purification and crystallisation laboratories. All platform components are in-place, however, we anticipate ramping up user numbers over the 4-year period as the infrastructure matures.

EMBL HD: The facility in EMBL HD offer expertise and support to use modern fluorescence/light microscopy techniques. EMBL HD platform combines the expertise in protein expression, purification and biophysical characterisation of the EMBL HDG Protein Expression and Purification Core Facility (PEPcore) with the expertise of the Advanced Light Microscope Facility (ALMF). EMBL HD can provide expression vectors for different fluorescent proteins, fluorescence labels, and chemical reagents to produce fluorescently labelled biomolecules for light microscopy analysis in vitro and in vivo. Users can collect fluorescence data, process and evaluate them using optimal equipment and software under the guidance of highly experienced light microscopists. The combination of protein expression and advanced light microscopy facilities in the vibrant scientific environment of EMBL HD is a unique resource in Europe. The project aims to make this resource available. So far PEPcore has served in-house users but also provides reagents and expertise to scientists outside EMBL, ALMF is closely linked to the EMBL Cell Biology Unit and is an internationally recognised centre for light microscopy.

 

Modality of access and work

UOXF: The user will generate the constructs in their home laboratory. After construct QA (sequencing) small scale screening (4 days) will be carried out by the infrastructure personnel before the user arrives at the facility. Based on the results of the screen up to 4 constructs will be selected for scale-up (2 weeks) each construct typically using 4 roller bottles (a total of 16 roller bottles will be available). A typical duration of stay of users will be approximately 1 to 2 weeks. The user will be given experience and support in all aspects of the production run (including SeMet labelling and manipulation of N-linked glycosylation if required). After protein purification (affinity purification followed by gel filtration) users will either return to their home laboratory to set up crystallisation trials or make use of the UOXF OPPF crystallisation facility through the infrastructure 3 (as a useful option for many non-UK users, screen results can be monitored remotely). Charges for using the crystallisation facility will be handled as part of the infrastructure 3.


EMBL HD: Users and staff will plan the experiments (e.g. microscope access) and required reagents prior to the visit. At the facility users will be helped to produce material (e.g. protein expression in different cell lines for in vivo analysis, recombinant protein production for in vitro studies, chemical labelling of protein). Subsequently, they will use the appropriate microscopes (fluorescent-correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence energy transfer (FRET), particle tracking and co-localisation). EMBL HD plan to receive and support around 8 users/year and offer a total of at least 8 months of visitor time. An average user will stay 4 weeks (2-8 weeks depending on the problem) to prepare sample, do the light microscopy analysis and process data. For expert users with more demanding projects (stay up to three months, repeat visits, 1-2 users per year) the expected outcome will be defined for each visit and is less predictable.

Support offered

UOXF: Design stage advice. Small-scale expression screening and support for scale-up by dedicated (Pcube) tissue culture technician. Support for purification and crystallisation trials.

EMBL HD: Expert advice for planning labelling and light microscopy experiments by the two facility heads. Technical support and advice by a dedicated (Pcube) technician specialised in fluorescence labelling and light-microscopy technologies. Logistic support at the interface of the Structural and Cell Biology Units.