A codon-optimised human RETECD (hRETECD) cDNA encoding residues 1-635 followed by a TEV-cleavable Avi and C-tag was cloned into a pExpreS2.1 vector (ExpreS2ion Biotechnologies, Hørsholm, Denmark) with Zeocin resistance. A stable pool of S2 cells, secreting hRETECD, was generated by transfecting 25 ml of S2 cells grown in Ex-Cell420 medium (Sigma) with 10 % (v/v) FBS at a density of 5×106 cells/ml using 12.5 μg of DNA and 50 μl of ExpresS2-Insect TR (5×). Stably transfected cells were selected with 2 mg/ml Zeocin with repeated medium exchange. The culture was expanded to 1 litre in a 5L glass-flask and the supernatants collected after 7 days.
For purification, 1 ml of C-tag capture resin (ThermoFisher) was added to a cleared and filtered S2 supernatant and incubated for 18 hrs at 4°C. The resin was pelleted and washed several times with PBS before eluting bound hRETECD by competition with PBS containing 200 μg/ml SEPEA peptide. At this point, the affinity and biotinylation tags were removed by digestion with TEV (a 1:10 ratio of TEV protease:RET). The purified hRETECD was further purified by size-exclusion using a Superdex200 10/300 with a 50 mM Tris (pH 7.5), 100 mM NaCl buffer.
For purification, 1 ml of C-tag capture resin (ThermoFisher) was added to a cleared and filtered S2 supernatant and incubated for 18 hrs at 4°C. The resin was pelleted and washed several times with PBS before eluting bound hRETECD by competition with PBS containing 200 μg/ml SEPEA peptide. At this point, the affinity and biotinylation tags were removed by digestion with TEV (a 1:10 ratio of TEV protease:RET). The purified hRETECD was further purified by size-exclusion using a Superdex200 10/300 with a 50 mM Tris (pH 7.5), 100 mM NaCl buffer.