Recombinant expression of the Aryl Hydrocarbon Receptor.
PhD thesis, University of Nottingham.
Aryl Hydrocarbon Receptor (AhR) mediates drug and toxin action. The AhR proteins have been characterised in several mammalian species, and are soluble proteins found in various tissues. The AhR is normally found in the cytoplasm in a complex with 90 KDa heat shock protein (hsp90) and cellular chaperones such as ARA9 (AIP or XAP2) and p23. However, there has not been a systematic analysis of the proteins which chaperone the AhR ligand-binding domain (LBD). This work investigates the interaction between ligands and the AhR, the protein composition of the AhR ligand-binding domain (LBD) complex, by establishing translation of AhR LBD in reticulocyte lysate, which contains molecular chaperones such as hsp90, and p23 that stabilise the ligand binding form of AhR.
EGFP (Enhanced Green Fluorescent Protein) has been coupled to the mouse AhR b-1 LBD, to enable fluorescence analytical techniques of ligand-binding to the AhR. The Glutathione S-Transferase (GST) affinity tag was fused to EGFP (GST-EGFP), then fused to AhR.LBD with one or two EGFP (GST-EGFP-AhR.LBD and GST-EGFP-AhR.LBD-EGFP) to enable rapid one-step purification of AhR fusion proteins, and associated chaperone proteins. Proteins were expressed in E.coli (BL21(DE3)plysS). The GST protein is soluble, and not fluorescent, and GST-EGFP and GST-EGFP-AhR.LBD-EGFP was soluble and fluoresecent. The GST-EGFP-AhR.LBD was an insoluble fluorescent protein. Thus, the AhR proteins were purified from bacteria to test the specificity of the pulldown system under conditions which do not yield functional Ah Receptor.
The tagged AhR constructs were translated with [35S]-methionine in reticulocyte lysate and translation products were ~36, 66, 84 and 109 kDa on SDS-PAGE. Reticulocyte lysate programmed with GST-EGFP-AhR.LBD and GST-EGFP-AhR.LBD-EGFP both bound ~800 d.p.m 2,3,7,8-[1,6-3H]tetrachlorodibenzo-p-dioxin (a ligand for the AhR), while lysate programmed with GST.EGFP showed binding of ~15 d.p.m, indistinguishable from unprogrammed lysate. The AhR proteins were purified from reticulocyte lysate and subsequent pulldown experiments will enable proteomic analysis of the proteins associated with the AhR LBD.
Thesis (University of Nottingham only)
||QS-QZ Preclinical sciences (NLM Classification) > QU Biochemistry
||UK Campuses > Faculty of Medicine and Health Sciences > School of Biology > Former School of Life and Environmental Sciences
||23 Jan 2008
||14 Sep 2016 20:24
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