Recombinant GITRL Protein Purification

The gene for mGITRL was cloned into the pEZT-BM expression vector (39 (link)) with a Strep tag added in frame at the N terminus. GITRL protein was produced in the same way as for the receptor protein except 10% (v/v) P2 virus was added when the suspension cells reached a density of 3.5 × 10⁶ cells/ml and cells were pelleted down at 96 hours after transduction. The whole purification was performed on ice or in the cold room at 4°C. The membrane pellet and the lysate supernatant were prepared the same way as for the receptor. The lysate supernatant was filtered through a 0.45-μm filter and loaded on a pre-equilibrated StrepTrap HP column (Cytiva) with purification buffer containing 20 mM tris (pH 8.0), 300 mM NaCl, and 0.5 mM DDM. The column was washed with 10 column volumes of purification buffer and eluted with 5 column volumes of purification buffer containing 20 mM desthiobiotin (IBA). The main elution fractions were concentrated to 700 μl using a centrifugal filter unit (10-kDa MWCO, Amicon) and injected on a Superose 6 Increase 10/300 GL column (Cytiva) equilibrated with purification buffer. The peak fractions were verified by SDS-PAGE gel and then pooled and concentrated.