University of Oxford Facility
This Strubi facility 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, Acta Cryst. D. 2006);
- technologies for controlling glycosylation in secreted proteins (Chang et al, Structure 2007) and SeMet labelling (e.g. Aricescu et al, EMBO J. 2006);
- co-expression to produce oligomeric protein complexes for structure determination.
Development work includes membrane protein expression in mammalian cells. Our expression facilities feed into well-equipped protein purification and crystallization laboratories. All platform components are in place; however, we anticipate increasing user numbers over the four-year period as the infrastructure matures.
- The user will generate the constructs in their home laboratory.
- After construct QA (sequencing) small-scale screening of up to 12 constructs (four days) will be carried out.
- Based on the results of the screen up to four constructs will be selected for scale-up, each construct typically using four roller bottles or the equivalent number of hyperflasks if the automation robot is used for the work.
Users will typically stay approximately one week. 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 Instruct (as a useful option for many non-UK users, screen results can be monitored remotely).
Charges for using the crystallisation facility will be handled separately and will require a new proposal submission.
|University of Oxford is an Instruct Centre|