The Protein Expression Core Facility provides researchers with high-throughput (HTP) cloning and expression screening activities in which many variations of a protein can be performed in parallel. Alternatively, the HTP activities may be directed at the genome of a particular organism or family of organisms to clone and expression screen many different proteins from a single organism.

In addition, the facility has the expertise and equipment necessary to produce and purify milligram amounts of protein from both prokaryotic and eukaryotic expression hosts-currently E. coli, Sf9 (insect cells) and HEK293, HEK293A or 293-6E (mammalian cells). Many of the protocols are automated, with the facility making full use of liquid-handling robotics for HTP plate-handling for small-scale (μg) expression screening and automated purification systems (Äkta Xpress) for larger (mg) scale protein purification. The facility also offers many high quality reagents for cloning and protein expression such as competent bacteriophage-resistant E. coli strains, specialized expression media, and recombinant enzymes.

Detailed services

• Custom HTP cloning to generate expression vectors. The In-FusionTM ligation and restriction enzyme-independent cloning technique allows the precise production of user- defined constructs, including the production of mutant, chimaeric, and bi-cistronic (E. coli constructs. There are many pOPIN or pPEU In-FusionTM - ready vectors available The majority of these vectors enable expression in E. coli (T7-based promoters), mammalian cells (CMV immediate early enhancer fused to the chicken ß-actin promoter) or insect cells (p10 promoter and in-cell recombination of ORFs 603 and 1629 with baculoviral genome). Generally the researcher will be expected to provide the ‘template’ for cloning operations but the facility can also source plasmids or cDNAs if required.
• Expression screening in E. coli. A micro-titre plate of 96 (facility or user-derived) expression clones can be screened in E. coli in approximately one week. The screen currently consists of the use of two expression strains, with expression in each strain being tested using both IPTG and auto-induction methods. Additional (DE3) E. coli strains can be incorporated into the screening process if required.
• HTP plasmid mini-preparation: 96 mini-preps from E. coli pellets in MTP format in approximately two hours
• Custom protein expression and purification of intracellular or secreted proteins at the 2 milligram scale . A minimum of two purification steps generally gives us protein purities in excess of 95% for most proteins. The hosts currently available for large-scale expression cultures are E. coli, HEK293 and 293-6E cells and we are currently introducing large-scale insect cell culture to our list of services. The HEK293 and 296-6E cells system has been used to successfully produce milligram quantities of secreted glycosylated proteins.
• Production of seleno-methionine-labelled proteins in auxotrophic or prototrophic E.coli strains for crystallographic structure determination. Our bulk seleno-methionine purchasing produces very competitive pricing for our labelled proteins from E. coli.
• Production of 15N-labelled proteins in auxotrophic or prototrophic E. coli strains for NMR.
• Expression screening in mammalian (eg HEK293) cells. A microtitre plate of 96 (facility- or user-derived) expression clones can be screened in cells in 1-2 weeks.
• Production of recombinant baculo-viruses either via the pOPIN or pPEU vector suites or from existing (eg pFastBac) constructs. The in-cell (Sf9) recombination of the pOPIN and pPEU vectors with the KO1629 baculoviral genome is rapid and without background.
• Recombinant his-tagged 3C(PreScission), TEV and SUMO proteases are available for the removal of fusion ‘partners’ from expressed proteins.

We also hope to make the recombinant glycosidases PNGase and EndoF1 available for the removal of sugar moieties from glycosylated recombinant proteins prior to crystallisation. The facility also sources many high quality reagents, ranging from specialized E. coli-competent cell strains and reagents for protein expression to labelling and cloning reagents for use by individual researchers. Purchasing reagents through the facility is generally very cost-effective, please note that these reagents are only available to IRB research groups.