Expression And Purification Of The Multidrug Resistance Protein P – Glycoprotein And High – Throughput Assay Development For Drug Discovery.

  • Swathi Lingam

Student thesis: Phd


P – glycoprotein is an ATP binding cassette transporter that is involved in multidrug resistance in diseases such as cancer. It is a protein that can bind and transport a wide variety of unrelated hydrophobic compounds. Several polymorphisms of human P – glycoprotein have been associated with altered expression and drug resistance in vivo, but there is no consensus on their effect on the function of the protein. In order to study the effect of polymorphisms on the thermal stability and activity of human P – glycoprotein (ABCB1) in a purified, lipid reconstituted protein system, wild type human P – glycoprotein and two single nucleotide polymorphisms R669C and S1141T were expressed in the yeast Saccharomyces cerevisiae. A C–terminal GFP tag was used for rapid detection of the expressed protein. All three constructs appeared to be localised to the plasma membrane / endoplasmic reticulum of the cells. All three constructs were able to transport Hoechst 33342 out of yeast cells. R669C and S1141T polymorphisms appeared to be more active than the wild type protein in yeast. All three constructs were subsequently purified from detergent-solubilised yeast membranes. In vitro, R669C was significantly more thermally stable than the wild type protein as judged by a fluorescence assay. After reconstitution into porcine brain polar lipids, purified R669C basal ATPase activity was significantly higher than that of the reconstituted wild type protein, although the activity could not be significantly stimulated by a well-characterised allocrite (verapamil). The Hoechst 33342 transport activity of R669C was similar to that of the wild type protein, but unlike the wild type protein, its transport activity could not be inhibited by the addition of the drugs tariquidar, nicardipine and ivacaftor. In vitro, the stability and function of purified S1141T appeared to be compromised. After reconstitution into brain polar lipids, the ATPase activity of S1141T could not be stimulated by verapamil and S1141T could not transport Hoechst 33342. The purified protein and lipidreconstituted systems were employed to develop drug binding and drug transport assays. To this end, a fluorescence-based thermal stability readout was investigated as a drug binding assay to screen potential allocrites of wild type human P – glycoprotein. This was set up in a medium – throughput fashion and was found to be a good assay to determine if drugs could interact with wild type human P – glycoprotein, although it could not be used to differentiate between substrates and inhibitors. Alternatively, the Hoechst 33342 transport assay was tested as a way to screen inhibitors of P – glycoprotein. It was adapted to a 96 well plate format to make it high – throughput, but this was not as effective as the previously developed fluorimeter assay due to the limitations of the plate reader and the transient nature of the transport readout. More work needs to be done to make this assay robust for a high – throughput format.
Date of Award31 Dec 2017
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorRobert Ford (Supervisor) & Eileithyia Swanton (Supervisor)

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