A final aim is that the PEF acts as a focal point for advice about all aspects of recombinant protein production and downstream applications.
Molecular Cloning Services...
The PEF offers molecular cloning services including construct design, DNA primer design and synthesis, and molecular cloning of constructs in the expression vector of choice. Also for certain complex cloning requirements, the PEF can advise on use of commercial cloning services. So if you want to generate recombinant expression constructs but don't have good molecular cloning skills within your group, or just want to kick-start a new project area, then the PEF will help you get your project started.
Protein Expression Services and Systems...
The PEF offers protein expression services focused on three recombinant expression systems, which collectively will enable production of a broad range of target proteins suitable for a wide variety of downstream applications. These are E.coli, mammalian cell expression, and insect cell expression. In each case, the PEF offers small scale test expression, expression optimisation, and large scale expression.
E.coli: A widely used expression system, E.coli production is quick, highly manipulable, low-cost in terms of reagents, and capable of generating high expression of the protein of interest. In addition, a diverse range of expression vectors and affinity tags/fusion partners are available, and efficient 2-H, 13-C, 15-N and Selenomethionine labeling approaches are established for downstream NMR and X-ray crystallographic applications. Limitations of E.coli expression systems include lack of post-translational modifications, and absence of suitable chaperones necessary for folding of some eukaryotic proteins.
Mammalian 293T: Recombinant expression in the Human Embryonic Kidney (HEK) 293T variant cell line is a widely used, quick and relatively cheap approach that is capable of generating high levels of a wide range of eukaryotic target proteins. Advantages include incorporation of human post-translational modifications, availability of glycosylation mutant strains, and the fact Selenomethionine labeling strategies for X-ray crystallographic approaches are established in this system. In addition, 293T expression is widely used for generation of immunogens for antibody production.
Insect: Stable transfection of Drosophila S2 cells is a well-established system for recombinant production of a wide range of proteins, including oligomeric complexes and cell surface proteins, and allows good expression levels and relatively homogenous glycosylation. One limitation is the length of time (~4 weeks) for selection of stable expression clones.
the PEF will offer advice concerning protein purification steps, and where possible, will offer protein purification services. Some purification services, such as GST or His-tag purifications, will be offered as a standard service, whereas less standard procedures will require discussion with PEF personnel and will be arranged on a case-by-case basis.
The protein expression systems used by the PEF will enable a wide range of downstream applications for recombinant proteins generated. Some of these are listed below, with relevant UofB research facility details provided.
1. Use as immunogens CMDS Antibody Production Facility Contact: Dr Margaret Goodhall, Dr Mark Cobbold
2. Protein-protein binding assays CMDS BIAcore Protein Interaction Facility Contact: Dr Steve Young
3. Cell staining of functional reagents CMDS Flow cytometry facility Contact:
4. Biophysical Studies (eg Circular Dichroism, Analytical Ultracentrifugation, Isothermal Titration Calorimetry) Birmingham Biophysical Characterization Facility (College of LES) Contact: Dr Timothy Dafforn, Dr Ben Willcox
5. Nuclear Magnetic Resonance Henry Wellcome Building for Biomolecular NMR Contact: Professor Michael Overduin, Professor Ulrich Gunther
6. X-ray crystallography Birmingham X-ray Diffraction Facility (College of LES) Contact: Dr Fiyaz Mohammed, Dr Klaus Futterer
The PEF can offer training to internal UoB staff and UoB Postgraduate/Postdoctoral colleagues.
We can provide training in various aspects of recombinant protein production as a charged service.
Areas covered include molecular cloning techniques, plasmid design, protein expression (in E.coli, 293T and Drosophila expression systems), refolding procedures, use of the AKTA purifier or pure and protein purification steps.
If you are interested in exploring training options, please contact Jamie Webster