Vivaspin 6 ultracentrifugation spin columns

Soluble NPC1-C containing an N-terminal FLAG and 6X His tag were produced by transfection in FreeStyle 293-F cells (Thermo Fisher, Waltham, MA, USA) with polyethyleneimine (PEI MAX, Polysciences, Warrington, PA, USA). Six days post-transfection, cell culture supernatant containing soluble protein was harvested and purified using PerfectPro Ni-NTA Agarose beads (PRIME GmbH, Neuss, Germany). Purified NPC1-C was dialyzed 20 mM Tris-HCl, 100 mM NaCl, 2 mM 2-mercaptoethanol, and 10% glycerol and concentrated in Vivaspin 6 ultracentrifugation spin columns (Sartorius, Gottingen, Germany). NPC1-C was stored at −80 °C prior to use. KZ52 was produced by transfection of plasmids encoding heavy and light chains into FreeStyle 293-F cells with PEI. At 3 days post-transfection, the cell culture supernatant was collected and replaced with fresh medium. The supernatant was collected again at 6 days post-transfection. The 3- and 6-day supernatant fractions were pooled, and KZ52 antibody was extracted using standard protein A affinity chromatography. After eluting from protein A in 0.2 M citric acid pH3, KZ52 was dialyzed into PBS and stored at −80 °C prior to use. GPΔTM was produced as described below.

GPΔTM was produced by transfection in FreeStyle 293-F cells (Thermo Fisher, Waltham, MA, USA) with polyethyleneimine (PEI MAX, Polysciences, Warrington, PA, USA). Wild-type GPΔTM and GPΔTM 32-A1/501-A4 expression constructs were co-transfected at a ratio of 2:1. At 6 days post-transfection, cell culture supernatant containing soluble protein was harvested and purified using PerfectPro Ni-NTA Agarose beads (PRIME GmbH, Neuss, Germany). Purified GPΔTM was exchanged into PBS supplemented with 10mM MgOAc using Vivaspin 6 ultracentrifugation spin columns (Sartorius, Gottingen, Germany) and then incubated overnight at room temperature with 5 µM each of fluorophores LD550 and LD650 (Lumidyne Technologies, New York, NY, USA) conjugated to coenzyme A (CoA; Millipore Sigma, Burlington, MA, USA) and 5µM of the labeling enzyme Acyl carrier protein synthase (AcpS). Labeled protein was purified away from free dye and enzyme via size exclusion chromatography, on a Superdex 200 Increase 10/300 GL column (GE Healthcare, Chicago, IL, USA) in PBS. Fractions containing GPΔTM were pooled and concentrated to 1–2 mg/mL. Aliquots were flash-frozen in liquid nitrogen and stored at −80 °C until use.