Protein core facility
The Protein Core Facility offers services for the production of recombinant proteins for the Cancer Science Division.
It is located on level C in the Somers Building.
The PECF is aiming to help your research projects in several ways:
- Consulting for your own projects or to start a project with us
- Protein expression in bacteria
- Protein purification
The projects can be started at any stage:
- Cloning of the cDNA in the appropriate bacterial expression vector
- Induction of the protein, including tests for expression levels and solubility
- Purification of the recombinant protein by chromatography
Cloning
We routinely use the following bacterial expression vectors:
- pET for the addition of a small His tag
- pGEX for the fusion of the protein to GST
- pMAL for the fusion of the protein to MBP to increase the solubility of some proteins
We welcome any suggestions about vectors you have used and found useful.
Protein expression
The protein expression is carried out in BL21 bacteria strains. For the expression of mammalian proteins, we prefer the use of the strain BL21 CodonPlus which carries extra copies of tRNA genes encoding rarer bacterial codons
Protein Purification
The protein purifications are performed using two FPLC AKTA Prime systems. A range of columns are available for:
- affinity purification (Nickel for His-tagged proteins, glutathione sepharose for GST-fused proteins and amylose for MBP-fused proteins)
- ion exchange chromatography (Q-sepharose and SP-sepharose)
- gel filtration (size exclusion)
Examples
Here are a few examples of proteins we made or we are currently making:
- Annexin V – FITC for the detection of apoptotic cells by flow cytometry [available at £50 / mg]. Note that Annexin V can be labeled with other fluorochrome upon request.
- Preparation of tetramers for MHC class I loading
- Production of PCAF histone acetyl transferase and LSD1 histone demethylase enzymes
Here are a few examples of the use of the proteins we were asked to make:
- Production of antibodies
- ELISA
- Analysis of protein interactions (in vitro and NMR studies)
- Study of new chemical compounds on the enzymatic activity of histone modifying enzymes
- Microinjection of proteins into mammalian cells
- Use of tetramers to study immunological responses in mouse (H2Db, H2Kb) or human (HLA*0201)
A service designed to save you time…
We aim to run the service as smoothly as possible to respond quickly to new projects. In order to improve efficiency, we will include new technologies and tools as they become necessary and/or available.
We want to put the emphasis on communication. During the first meeting, we will define with you the best strategy to adopt according to the final use of the protein in terms of activity, amount and purity required. A timetable will be issued during a second meeting and updates on the project progression will be provided regularly.
Please note that we will need to know if your project is intended for clinical trials. All requests will be treated in confidentiality.
Dr. Patrick Duriez (pjd@soton.ac.uk)
Patrick studied Molecular and Cell Biology in France then did his PhD at Laval University in Quebec, Canada where he started to work on apoptosis. He continued working on the same subject for 7 years during his 2 post-docs in Vancouver Canada (Dr Ali Karsan), and Dublin, Ireland (Dr. Seamus Martin).
Patrick has an extensive expertise in molecular biology and over-expression studies in mammalian cells using transfection and retroviral infection systems during his postdocs. He started to make recombinant proteins in E. coli in 2004 and since then he has gained a strong experience in bacteria expression systems and protein purifications.
Patrick has worked in the Cancer Sciences Division since October 2006 for Prof. Graham Packham.
Pubmed: http://www.ncbi.nlm.nih.gov/pubmed?term=duriez+pj[au]
Citations: http://lib.bioinfo.pl/auth:Duriez,pj
