Expression and purification of the mammalian translocator protein for structural studies
(A) Small-scale expression test: in-gel fluorescence showing the expression levels of C-terminal GFP-10xHis constructs expressed in HEK293T cells and solubilised in 1% DM (1 human, 2 mouse, 3 rat, 4 dog, 5 bovine, 6 pig, 7 sheep, 8 chicken, 9 Xenopus laevis, 10 danio rerio, 11 danaus plexippus, 12 drosophila melanogaster). (B) Radioligand binding assay with [3H]PK11195 performed with all 12 C-terminally GFP-tagged TSPO homologues. (C) Thermostability of nine eukaryotic TSPO homologues assessed by incubating the in DDM solubilised proteins for one hour at a range of temperatures and subsequent analysis by FSEC. Control sample was incubated at 4°C (D) Small-micelle detergent screen of dog, bovine, pig and sheep TSPO. DDM-solubilised TSPO was diluted 10-fold into a buffer containing a secondary detergent, analysed by FSEC and the peak height was compared to the DDM-solubilised control sample.