IDs were expressed by transfection using the FreeStyle 293T and GnT1− 293T Mammalian Expression System (Invitrogen) for functional and structural analysis, respectively, and according to the protocol provided by the manufacturer. IDs were purified using an N5-i5 IgG affinity column. N5-i5 IgG was chemically crosslinked to protein A resin using the Pierce Protein A IgG Plus orientation kit (Thermo Fisher Scientific). Protein bound to the N5-i5 IgG affinity column was eluted with 0.1 M glycine (pH 3.0) and immediately diluted 10:1 with 1 M Tris-HCl (pH 8.5).
ID2 for co-crystallization studies was grown in the Origami (DE) Escherichia coli strain (Novagen). ID2 sequence was cloned into the pMal-c5e expression vector (New England Biolabs) with an N-terminal maltose-binding protein (MBP)-thioredoxin tag followed by a six-histidine tag and a thrombin cleavage sequence. The cell lysate (after 2–3 min of sonication and centrifugation at 12,000 × g for 30 min) was loaded onto a HiTrap nickel column (GE Healthcare). MBP-thioredoxin-ID2 was eluted with 25 mM Tris-HCl (pH 8.0) and 500 mM imidazole (pH 8.0). The MBP-thioredoxin tag was removed by digestion overnight at 4°C with agarose-linked bovine thrombin (Sigma) and passing the lysate over an amylose column. Flow-through fractions were concentrated and purified further using an N5-i5 IgG affinity column as described above.
ID2 for co-crystallization studies was grown in the Origami (DE) Escherichia coli strain (Novagen). ID2 sequence was cloned into the pMal-c5e expression vector (New England Biolabs) with an N-terminal maltose-binding protein (MBP)-thioredoxin tag followed by a six-histidine tag and a thrombin cleavage sequence. The cell lysate (after 2–3 min of sonication and centrifugation at 12,000 × g for 30 min) was loaded onto a HiTrap nickel column (GE Healthcare). MBP-thioredoxin-ID2 was eluted with 25 mM Tris-HCl (pH 8.0) and 500 mM imidazole (pH 8.0). The MBP-thioredoxin tag was removed by digestion overnight at 4°C with agarose-linked bovine thrombin (Sigma) and passing the lysate over an amylose column. Flow-through fractions were concentrated and purified further using an N5-i5 IgG affinity column as described above.