Protein purification from Expi293 cells

The mEGFP‐GM130‐FLAG plasmid was transfected into Expi293 cells (Thermo Fisher) at 1 µg·mL⁻¹ culture, employing poly(ethylenimine) (µL) : DNA (µg) ratio of 3 : 1; 6 h post‐transfection, growth enhancers were added according to the instructions of the manufacturer. After 48 h, the cells were pelleted at 500 g for 15 min and washed with PBS. Typically, a pellet stemming from a 150 mL culture was resuspended in 15 mL buffer 1 [50 mm HEPES/KOH pH 7.3, 175 mm NaCl, 5 mm EDTA, 1 mm PMSF, 1 mm TCEP, protease inhibitor tablet (Roche, Basel, Switzerland)]. Next, 0.33% Triton X‐100 (v/v) was added and the lysate rotated at room temperature for 20 min. After adding buffer 1–50 mL and another 20 min of incubation, unlysed material was pelleted at 16 500 g for 15 min at 12 °C. Next, 4.5‐mL anti‐FLAG affinity resin was washed with 20 mL buffer 1, followed by a wash with 10 mL buffer 1 containing 0.1% (v/v) Triton X‐100. Next, the lysate was added to the washed beads and incubated for 3 h at room temperature. The suspension was settled on a column and drained, and washed with 10 mL buffer 1. Next, the beads were washed with 50 mL buffer 2 (buffer 1 plus 1 mm ATP, 1 mm MgCl₂). Recombinant GM130 was eluted from the beads in buffer 3 (buffer 1 plus 230 ng·mL⁻¹ FLAG peptide), 35 min per elution, six fractions total. Immediately after elution, the fractions were spun at 10 000 g for 6 min and the supernatant desalted on G25‐Sephadex (Thermo Fisher, NAP‐5) columns equilibrated with 5 mm HEPES/KOH pH 7.3. The concentration of recombinant mGFP‐GM130‐FLAG was determined by quantitative western blotting employing a recombinant GFP standard with known concentration (Abcam, Cambridge, UK). To determine the concentration of GM130 in cells, recombinant GFP‐GM130‐FLAG was blotted at increasing (known) protein amounts and compared to lysates of increasing amounts of EXPI293F cells that were subjected to automated cell counting (Bio‐Rad, TC20, Hercules, CA, USA). Statistical analysis was performed using graphpad prism 6 (GraphPad Software, San Diego, CA, USA) for unpaired, two‐tailed t‐tests. Differences were considered significant if P‐value < 0.05(∗), <0.01(∗∗), or < 0.001(∗∗∗).