Expressing and Purifying IEkMCC Protein

Plasmid construct for expressing the extracellular domain of IEk with covalently linked MCC peptide was a gift from John Kappler (National Jewish Health). For conjugation with polymers, the construct was modified by PCR to add a cysteine at the C-terminus of the β chain. Baculoviruses were generated with the construct and BaculoGold Linearized baculovirus DNA (BD Biosciences, San Diego, CA) in Sf9 insect cells (Invitrogen, Calsbad, CA). High titer stocks of cloned recombinant baculovirus were used to infect Hi5 insect cells (Invitrogen, Calsbad, CA) cultured in spinner flasks. IEkMCC protein was purified from the supernatant of infected Hi5 cell cultures using an affinity chromatography column conjugated with 14-4-4s antibody. The protein was further purified using a Superdex 200 10/300 GL gel filtration column (GE Healthcare, Piscataway, NJ) before conjugating with PEG linkers. The design and production of IEkMCC with a C-terminal AviTag was described previously [15] (link). The protein was biotinylated at the lysine residue of the AviTag using BirA enzyme as described previously [15] (link).