Purification of SPIN1/SPIN4 Fusion Proteins

WT SPIN1, WT SPIN4, or mutant SPIN4 coding sequence were cloned into pSecTag plasmid containing a secretary peptide sequence so that the expressed proteins are secreted into the culture media. An antibody Fc region was inserted at the carboxyl terminus, connected with a G4S linker (38 (link)) to allow protein purification using protein A resin (GenScript) as previously described (39 (link)). Briefly, protein A resin slurry (1–5 mL) was packed into a glass Econo-column (Bio-Rad) and equilibrated with 50 mL of binding/washing buffer (0.15M NaCl, 20 mM Na₂HPO₄ [pH 8.0]). Four to 6 days after transfection, Expi293 cells were spun down, and the culture media containing the expressed protein was loaded onto the column. After unbound proteins were washed away with binding/washing buffer, SPIN1 or SPIN4-Fc fusion proteins were eluted with 30 mL of elution buffer (100 mM acetic acid [pH 3.0]), neutralized by 1/10 volume of neutralization buffer (1M Tris-HCl [pH 9.0]), concentrated, and buffer-exchanged to PBS using Amicon Ultra-15 filter unit (MilliporeSigma). The purity of the antibodies was checked by SDS-PAGE, and the concentration was determined by Nanodrop (Thermo Fisher Scientific).