Cancer Sciences Protein Facility

Technical specification

Molecular biology

• Cloning with standard restriction digestion and ligation techniques 
• Cloning with sequence ligation-independent cloning (SLIC) techniques 
• Design of synthetic DNA with sequence optimisation; PCR, Inverse-PCR, mutagenesis 
• Making of high competent bacteria for cloning (JM109 and others) 
• Bacterial vectors: pET (His6), pGEX (GST), pOPIN (His6, GST, MBP, SUMO, TG, NusA, Thioredoxin) 
• Insect vectors: pMT-Puro, pFastBac, pOPIN 
• Mammalian vectors: pcDNA3, pDSG (His6, TwinStrep), pCI-Puro, pEE6.4

Expression systems

Bacteria

Pros: High levels of protein, quick, low cost, scalable, simple culture conditions.

Cons: Difficult with large proteins, proteins can be misfolded, lack of post-translational modifications.

• E. coli BL21 Strains DE3, pLys, pLacI, Rosetta2 
• Culture in LB and Terrific Broth media 
• IPTG induction and auto-induction

Insect cells  

Pros: Closer to mammalian protein processing.

Cons: More demanding culture conditions than bacteria, production of baculovirus can be time consuming.

• Stable transfection of S2 cells with copper-inducible system 
• Baculovirus system with Sf9 and High5 cells

Mammalian cells  

Pros: Natural folding, post-translational modifications, pyrogen-free

Cons: Expensive 

• HEK MEXi293E cells - transient expression 
• ExpiCHO cells - transient expression  

Protein purification

• Refolding of insoluble proteins expressed in bacteria 
• Affinity chromatography for tagged proteins (His6, GST, MBP, TwinStrep), antibodies and Fabs 
• Ion exchange chromatography
• Size exclusion chromatography

Protein modification

• Tag removal: TEV, H3C, SUMO proteases 
• Labelling of proteins and antibodies  
• Endotoxin removal from protein sample

Specific equipment

• MaxQ 6000 Shaker incubators for bacterial expression (room temperature – 37C) 
• MaxQ 6000R Refrigerated shaker incubator for bacterial expression (4C – 37C) 
• Infors HT Multitron shaker incubators for mammalian transient expression 
• Infors HT Minitron shaker incubator for insect expression 
• Cytiva Akta PrimePlus protein purification systems 
• Bio-Rad NGC Protein purification system 
• Amicon stirred cell concentrator

Applications

• Flow cytometry 
• Fluorescent microscopy 
• Protein-protein interaction – BiaCore, ELISA 
• Enzymatic assays 
• Cell culture 
• Mouse work 
• Protein Structure in collaboration with IfLS groups

Examples of recombinant proteins produced by the Protein Facility

• Tag removal: His6-H3C / GST-H3C, His6-TEV and SUMO proteases  
• Biotinylation ligase Bir A enzyme 
• Annexin V-FITC and Annexin V-APC  
• MHC I tetramers  
• Cytokines and interleukins 
• Antibodies, Fabs and ScFv 
• Tailored proteins