Generation of recombinant human PANKs

Recombinant human PANK1, PANK2, and PANK3 were cloned and expressed using a bacterial expression system. The gene encoding human PANK1β was mutagenized using the QuikChange site-directed mutagenesis kit (Stratagene) with primers that added a Nhe I restriction site to the NH2-terminus and a Xho I restriction site to the COOH-terminus. The PCR product was cloned into pET28a to obtain an NH₂-terminal His₆-tag fusion protein, and the resulting plasmid was transformed into the E. coli BL21(DE3) (Stratagene) expression strain. The cells were grown to mid-log phase, and the protein was induced with 1.0 mM isopropyl-β-d-thiogalactopyranoside for 18 hours at 16°C. The PANK1β protein was purified using a nickel–nitrilotriacetic acid affinity column and eluted with 50 mM tris (pH 7.9), 500 mM NaCl, 10% glycerol, and 300 mM imidazole. The purified protein was dialyzed overnight at 4°C in 20 mM tris (pH 7.5) and 300 mM NaCl and stored at −80°C in equal volume glycerol until further use. The genes encoding the mature form of human PANK2 (residues 141 to 570) and the catalytic core domain of human PANK3 (residues 12 to 368) were similarly cloned and purified as previously reported (56, 57).