Determining drug:protein and protein:protein interaction sites


Lab rotation project description

Central to the long-term PhD proposal are skills in membrane protein expression, purification and functional validation. In the first rotation, you will be trained to do this for both bacterial expression systems (BmrA and Sav1866) and for a mammalian cell expression system (ABCG2). Following expression, protein functionality will be confirmed either whole cell fluorescence assays (flow cytometry). ABC multidrug pumps will then be extracted from the membrane using a novel, detergent-free method (styrene maleic acid and SMA). This will result in the extraction of ABC multidrug pumps in patches of native lipid membranes known as SMALPs (SMA lipid:protein particles).

Optimisation of protein purification using nickel affinity chromatography and subsequent protein concentration using centrifugal concentrators will be required to produce protein suitable to commence downstream analysis later in the main PhD.

Fact file

Research theme



School of Life Sciences



Main Supervisor: Ian Kerr

2nd supervisor

Robert Layfield and Neil Oldham

BBSRC Doctoral Training Partnerships

Linked PhD Project Outline

Drug export from cells is a major factor in the acquisition of cellular resistance to antimicrobial and cancer chemotherapy, and poses a significant threat to future clinical management of disease. Many of the proteins that catalyse drug efflux do so with remarkably low substrate specificity, a phenomenon known as multidrug transport. For these reasons we need a greater understanding of drug recognition and transport in multidrug pumps to inform research that attempts to circumvent their action.

One family of proteins that are capable of multidrug export is the ATP binding cassette (ABC) transporter family; bacterial, yeast, plant and mammalian ABC transporters can all contribute to multidrug (MDR) export. The standard model for ABC multidrug pumps is that the binding and hydrolysis of ATP at two intracellular nucleotide binding sites, results in the binding and transport of drug molecules from allosterically linked sites in the membrane spanning domains of these proteins. In this project, you will attempt to determine the drug binding sites on bacterial and human MDR pumps using a novel detergent-free, membrane protein purification technique known SMALPs, and a cutting edge mass spectrometric labelling technique, known as carbene footprinting.

Bacterial ABC transporters BmrA and Sav1866 will be expressed in E. coli C41.DE3 cells and human ABCB1 and ABCG2 will be expressed in mammalian HEK293T cells. The function of each transporter will be confirmed with drug export assays using flow cytometry (ABCG2 and ABCB1) or fluorescence spectroscopy (BmrA and Sav1866). Transporters will be isolated from the membrane using styrene maleic acid co-polymer (SMALP) solubilisation, which results in the extraction of the protein still in a patch of the native lipid bilayer. Given that the membrane bilayer is essential for the function of membrane proteins, SMALP confers significant advantages over conventional detergent solubilisation which removes the lipid bilayer. Purification of each transporter by Ni or Co-affinity chromatography will result in the production of purified ABC transporters suitable for mass spectrometric interrogation. The objectives for this project are as follows:

  • expression and purification of ABC transporters at concentrations and purities suitable for carbene footprinting
  • structural biology has already confirmed where nucleotides bind to ABC transporters and carbene footprinting of Sav1866, BmrA, ABCB1 and ABCG2 in the presence and absence of nucleotides will confirm that the technique can distinguish the binding sites of small molecules
  • the great unknown is precisely where on these transporters are the drug binding sites; determination of carbene footprinting sites masked by the interaction of known drug substrates of ABC multidrug transporters will determine the structural basis of MDR binding and transporter

Biotechnology and Biological Sciences Doctoral Training Programme

The University of Nottingham
University Park
Nottingham, NG7 2RD

Tel: +44 (0) 115 8466946