Purification and Crystallization of SERT-Fab Complex

The hSERT constructs were expressed as C-terminal GFP fusions using baculovirus-mediated transduction of mammalian HEK-293S GnTI⁻ cells, as previously described^{25 (link),52 (link)}. Cells were subsequently solubilized in 50 mM Tris pH 8, 150 mM NaCl containing 20 mM DDM, 2.5 mM cholesteryl hemisuccinate (CHS), 0.5 mM dithiothreitol (DTT) in the presence of 1 μM inhibitor (paroxetine, (S)-citalopram, or Br-citalopram). The lysate was passed over 10 ml of Strep Tactin resin, washed with 18 column volumes of 1 mM DDM, 0.2 mM CHS, 5% glycerol, 25 μM lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol at a molar ratio of 1:1:1), and 1 μM ligand in TBS. SERT was eluted in the same buffer containing 5 mM desthiobiotin. The N- and C-terminus containing GFP and purification tags were removed by thrombin digestion and N-linked sugars were truncated using EndoH. SERT was mixed with recombinant 8B6 Fab at a 1:1.2 molar ratio. In the case of Br-citalopram complexed at the central site, Fab purified from hybridoma cells was used. The resulting complexes were further purified by size exclusion chromatography in TBS supplemented with 40 mM n-octyl β-D-maltoside, 0.5 mM CHS, 5% glycerol, 25 μM lipid, and 1 μM inhibitor. The purified SERT-8B6 complex was concentrated to 2 mg/ml⁻¹ and the transporter solution was spiked with 10 μM inhibitor and 1 μM 8B6 Fab, final concentrations, immediately prior to crystallization.