Instruct Centre - United Kingdom > OPPF

The Oxford Protein Production Facility UK is a structural proteomics facility in the Division of Structural Biology, University of Oxford

OPPF

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Expression screening@OPPF, Harwell, UK

Homologous recombination in insect cells is used to construct recombinant baculoviruses which are amplified once and expression tested by infection of insect cells grown in suspension. Note the plasmids used in the E. coli expression platform are also baculovirus transfer vectors so that expression in both systems can be carried out in parallel.

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OPPF

Overview of OPPF platforms:

Cloning and expression screening

The OPPF has produced a series of vectors and protocols for expression screening in E. coli, mammalian and insect cells (the latter using the baculovirus system). Vector construction makes use of the pOPIN suite of expression vectors (Berrow NS et al. Nucleic Acids Res. 2007 ;35(6):e45; Bird Methods. 2011 Sep;55(1):29-37; Bird et al Methods Mol Biol. 2014 ;1116:209-34) .

The majority of the vectors allow expression of proteins from multiple hosts, i.e. a single vector capable of expression in E. coli, mammalian cell lines (e.g. HEK293T cells) and insect cell lines (e.g. Sf9 cells) using the baculovirus expression system. This enables parallel screening of constructs in multiple hosts.The process is simple to operate in HTP mode - the In-Fusion™ method has now been used to clone in excess of 2500 PCR products into pOPIN expression vectors with very high efficiency .All of the pOPIN vectors contain His-tags which are (with the exception of that from pOPINI) cleavable from the protein of interest, either with 3C protease, SUMO protease or Carboxypeptidase A.

The OPPF pOPIN vector suite is available to academic users from Addgene (www.addgene.org).

For more information, please go to the OPPF website (www.oppf.rc-harwell.ac.uk ).

Crystallization

The OPPF operates a high-throughput crystallization pipeline. Crystallization experiments are carried out in 96-well Greiner plates using nanolitre-scale droplets (typically 100 nL of protein + 100 nL of reagents). Crystallization plates are stored and imaged at regular intervals at either 4°C or 21°C in automated storage systems. Images are accessible remotely to the users via a web-interface using XtalPims.

Instruments available include:

Hydra instrument to transfer crystallisation solution from blocks to Greiner platesTwo Cartesian Microsys instruments to dispense nanolitre dropletsTwo Formulatrix Storage and Imaging System capable of storing 1000 plates at 21°CA Formulatrix Storage and Imaging System capable of storing 1000 plates at 4°CTwo stereo-microscopes with Pixera imaging cameras to view crystallization plates manually

The OPPF offers registered external and internal users access to the protein crystallization facilities:

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