PROTEIN PRODUCTION PLATFORM (P-CUBE)

The Oxford Protein Production Facility (www.oppf.ox.ac.uk) is a structural proteomics facility in the Division of Structural Biology of the Department of Medicine, Oxford University. The OPPF has developed and implemented a high throughput pipeline for the production of proteins in E. coli which encompasses: (i) multi-construct design (ii) ligation-free cloning into customized expression vectors (iii) semi-automated small-scale screening for soluble expression (iv) scale-up and semi-automated purification (Berrow et al. Nucleic Acids Res. 2007;35(6):e45).  The use of the OPPF pipeline both significantly reduces the lead time for the identification of soluble proteins and enables the parallel processing of multiple protein targets. Recent examples of the application of the pipeline which have led to the determination of new protein structures include the methyl transferase and helicase from Murray Valley Encephalitis virus (Assenberg et al. J Gen Virol. 2007 88 :2228-36; Mancini et al. Protein Sci. 2007 16:2294-300) the matrix protein of Lagos bat virus ( Graham et al. 2008 Dec;4(12):e1000251) and a number of transcription factors from Neisseria meninigitidis (Sainsbury et al. Nucleic Acids Res. 2009 May 27. [Epub ahead of print] Nichols et al.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 65:204-9. Ren et al. J Biol Chem. 2007 282:14655-64. Nichols CE et al.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 62:494-7).

Modality of access and work

The OPPF offers access to the E. coli production pipeline through two modes of access:

1. External users would be hosted by the OPPF for a working visit of up to 2 weeks to construct vectors and carry out an expression screen of these in E. coli. It would be possible to carry out vector construction and expression trials as separate activities. The working unit would be a set of 48 expression plasmids and the deliverables would be the vectors and associated expression data.
2. Vector construction and supply to external users would operated as service by the OPPF. Constructs would designed interactively with the user or user group and the work carried out by a scientist dedicated to the infrastructure project.

The OPPF facilities would enable one external user to be accommodated at a time under the P3  funding 9 external user projects could be carried each year under either access mode [1] or [2].

Support offered

Training and supervision would be provided by the OPPF including advice on construct and primer design as appropriate. Accommodation for visitors would be arranged locally (cost is not covered by the proposal).

Figure 1: Automation of molecular cloning in the OPPF.