Protein expression

Protein expression

We provide a comprehensive resource for the high level expression and scale-up production of recombinant proteins. Currently we offer a choice of four expression systems: bacteria, pichia, insect and mammalian cells. Depending on particular needs we are able to provide either small scale production facilities for biochemical analysis and antibody production or larger scale production for structural studies. We can offer:

  • High level expression and scale-up production of recombinant proteins
  • Protein purification (Native or His/GST/MBP tagged proteins. Second stage polishing can be carried out using ion exchange or gel filtration chromatography). Tag removal using thrombin or other selective proteases (eg TEV/ Precission)
  • Cloning service
  • Troubleshooting & advice service
  • In-house R&D vector design
  • Training courses

Enquiries

Dr Eddie McKenzie | edward.a.mckenzie@manchester.ac.uk | Tel: +44 (0)61 306 4170

Highlights

The facility collaborates extensively across departments in the University that includes: Michael Smith building, AV Hill, Stopford Building, St Mary’s Hospital, Patterson Institute and MIB. External collaborators have been growing over the last two years now and some examples are Sheffield University, Abcam, Syngenta, Takeda & Heptares.

We have recently been named on two successful grants (£250,000 ‘EURenOmics EU FP7 collaborative project & £157,000 from Kidney Research UK) with Professor Paul Brenchley at St Mary’s Hospital to study the role of the protein PLA2R in the disease membranous nephropathy.

Selected recent publications

Simmons SC, McKenzie EA, Harris LK, Aplin JD, Brenchley PE, Velasco-Garcia MN, Missailidis S. Development of Novel Single-Stranded Nucleic Acid Aptamers against the Pro-Angiogenic and Metastatic Enzyme Heparanase (HPSE1). PLoS One. 2012;7(6):e37938. Epub 2012 Jun 15.

Weston R, Peeters H, Ahel D. ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response. Genes Dev. 2012 Jul 15;26(14):1558-72. Epub 2012 Jul 3.

Hamilton NM, Dawson M, Fairweather EE, Hamilton NS, Hitchin JR, James DI, Jones SD, Jordan AM, Lyons AJ, Small HF, Thomson GJ, Waddell ID, Ogilvie DJ. Novel steroid inhibitors of glucose 6-phosphate dehydrogenase. J Med Chem. 2012 May 10;55(9):4431-45. Epub 2012 Apr 27.

Equipment

  • ÄKTAxpress Protein Purification systems
  • Biomek FXp liquid handling robot
  • Syngene chemiluminiscence imaging system
  • 10 litre wavebag (GE Healthcare) for insect cell scale up

Services

Following an initial meeting to discuss the project the facility will draft a workplan with timelines tailored to your needs. A full report write up with scanned gels and methodology used will be submitted after completion.

  • Cloning (conventional restriction enzyme cloning or in –fusion methods) with ready switching between proprietary vectors.
  • Pilot protein expression projects with all systems are first carried out to optimise conditions prior to scale up.
  • New bacterial expression host screen (use of 10 selected cell types to help improve protein folding, medias and molecular chaperones) to optimise expression and solubility. Bulk up expression and purification to X-ray crystallography grade (affinity purification followed by IE or GF).
  • Pichia cell expression using a variety of cell hosts (Invitrogen pPicZ system).
  • Insect cell expression using protease deficient baculovirus DNA and choice of cell lines (Sf9 or Hi5). Proteins can be targeted for either intracellular or secretion.
  • Mammalian expression using HEK293 EBNA cells (either grown adherently or in suspension using fibre cell disks) for intracellular expression or secretion.

“The protein expression facility has helped my lab by doing our first cloning on a new project. My PhD student shadowed the staff in the protein expression facility for this service, and thus we have achieved technology transfer, as well as a boost to start to the project more quickly. The PhD student has subsequently cloned two more constructs independently, and accurately, as confirmed by sequencing. I would highly recommend this service and tailored mini-course to others.”

Dr Alan Roseman, Michael Smith Building

“At the DDU we have worked with Dr Eddie McKenzie and his group at the Protein Expression Facility, MIB for over 3 years. During that time Eddie and his team have produced over 11 proteins and 9 mammalian expression constructs for us, affecting at least 7 drug discovery programmes. In each case the proteins have been of a consistently high standard in terms of both quantity and quality and have been delivered expediently to match our portfolio needs. Depending on our individual programme needs these proteins have been used for in vitro assay development (primary and secondary), selectivity assays, binding assays such as SPR and BioCore and crystallography.

As with all protein production occasionally technical issues have arisen. At all times Eddie and his team have kept us appraised of the situation and have worked proactively with us to quickly resolve the problem and ensure delivery according to our needs.”

Dr Ian Waddell, Paterson Drug Discovery Unit