Bl21 de3 cells

Mammalian expression plasmid of HSA was obtained from Addgene. An E400R point mutation was introduced to the HSA sequence by the Q5 site-directed mutagenesis kit using the primer HSA-F and HSA-R. After sequence verification by Sanger Sequencing, plasmids bearing wild type HSA and the mutant were transfected to HeLa cells using Lipofectamine 3000 transfection kit (Invitrogen) and Opti-MEM (Gibco) according to the manufacturer's protocol. The cells were cultured overnight before the change of medium to DMEM without FBS supplements to remove BSA. After a 48 h culture at 37°C, 5% CO₂, the media expressing HSA were collected and stored at −20°C. The media were analyzed by SDS-PAGE and Western Blotting to confirm protein expression. The PDZ domain (in the pGEX6p-1 vector) was obtained from the General Biosystems. A double point mutant of PDZ (i.e., R46E: K48D) was introduced by the Q5 Site-directed mutagenesis kit using specific primers of PDZ-F and PDZ-R. After verification by Sanger Sequencing, the mutant vector was transformed into BL21(DE3) cells (Thermo Scientific) for expression. The GST fusion PDZ mutant protein was purified by GSH resin as previously described.

The expression plasmid (Genscript, Piscataway, NJ, USA) containing human KAT II (hKAT II) as C-terminal intein (chitin binding domain) fusion, in pTXB1 vector, was transformed into BL21 (DE3) cells (Thermo Fisher Scientific). Cells were grown in 2YT media (Thermo Fisher Scientific) at 37°C until the optical density reached 0.6 (measured at 600 nm), and induced with 300 μM isopropyl 2-D-1-thiogalactopyranoside for 18-20 h at 18°C. The cells were then harvested by centrifugation (2,700 x g, 20 min), and the pellet was re-suspended in chitin column buffer, pH 8.5, containing 20 mM Tris, 500 mM NaCl and 1 mM EDTA in 10% glycerol and 5 mM 2-mercaptoethanol. hKAT II was purified by affinity chromatography using a chitin column (New England Biolabs, Ipswich, MA, USA). Intein was cleaved on the column by washing with the chitin column buffer containing 50 mM dithiothreitol. This was followed by ion exchange chromatography using a DEAE column (GE Healthcare Life Sciences, Marlborough, MA, USA). Pure hKAT II was obtained after a final round of purification by size exclusion chromatography, using a S200 column (GE Healthcare Life Sciences). Protein (in 20 mM Tris, 50 mM NaCl and 40 μM pyridoxal-5′-phosphate, pH 8.5) was concentrated to 10.2 mg/ml, flash-frozen in liquid nitrogen and stored at −80°C.