followed by an Avi-Tag and restriction sites for BamHI and XhoI. The sequence encoding TLX followed by two stop codons was cloned in frame between these restriction sites. From this construct, a fusion protein is expressed with N-terminal maltose-binding protein (MBP) followed by an Asn10 linker, a His8-Tag, a cleavage site for TEV protease, an Avi-Tag, and the TLX LBD with unmodified C-terminus. For expression, E. coli T7 express cells (NEB) were co-transformed with pGro7 (TAKARA Bio Inc., Kusatsu, Japan) and the aforementioned expression construct, and selected overnight at 37°C on LB (Luria Broth) agar containing 34 µg/ml chloramphenicol and either 100 µg/ml ampicillin (for pMal) or 35 µg/ml kanamycin (for pET). Culture in liquid LB was inoculated and grown at 37°C with constant shaking at 180 rpm until optical density at 600 nm (OD600) reached 0. Purification was achieved by immobilized metal affinity chromatography (IMAC) using columns packed with Ni Sepharose 6 Fast Flow resin on an ÄKTApurifier FPLC system (GE Healthcare, Chicago, IL, USA). After washing with buffer supplemented with 50 mM imidazole, the protein was eluted with 300 mM imidazole. Afterwards, GFP fusion proteins were processed with His tagged TEV protease overnight while imidazole content was reduced to 10 mM by dialysis against buffer A in order to allow for reverse IMAC. The flow through was concentrated and applied to size exclusion chromatography using a 16/60 Superdex200™ column equilibrated and run in HTRF assay buffer [25 mM HEPES pH 7.5, 150 mM KF, 10% (w/v) glycerol, 5 mM DTT]. Following the initial IMAC purification step, the MBP fusion protein for generation of biotin labeled TLX LBD was processed with MBP-tagged TEV protease during overnight dialysis against buffer A. Afterwards, uncleaved fusion protein, free MBP-Tag, and TEV protease were removed by passaging through a gravity flow column packed with Amylose High Flow resin (NEB). The flow through was then supplemented with 0.5 mM biotin, 0.5 mM ATP, 5 mM MgCl2, and E. coli biotin ligase birA at a molar ratio of approx. 1:10 for enzymatic conjugation of biotin to the lysine residue in the avitag.