Isopropyl β d thiogalactoside

To optimize the expression conditions for GST-ALR (the fusion protein), Escherichia. coli cells carrying the human GST-ALR expression plasmid (pGEX-4 T-1, with N-terminal GST) were grown in fresh Luria-Bertani liquid medium containing 0.1 mg/mL ampicillin and 0.05 mg/mL chloramphenicol overnight at 37°C in a shaking incubator. The production of protein was induced with 1.0 mM isopropyl-β-D-thiogalactoside (Beyotime, Shanghai, China) for another 4 h, until the optical density at 600 nm was between 0.6 and 0.8. Bacteria were centrifuged and resuspended in 1× PBS. Bacterial cell walls were broken by ultrasonication. Supernatants and precipitates were collected and processed for 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Supplementary Fig. 2A). Purified GST-ALR was obtained using a GST-tag Protein Purification Column (Beyotime), followed by washing with 20 mM reduced L-glutathione (Sigma-Aldrich, St. Louis, MO, USA) and purified with ÄKTA (GE Healthcare) overnight at 4°C to obtain the purified GST-ALR (Supplementary Fig. 2B). After dialysis against PBS for 2 days, GST-ALR was diluted to 1 mg/mL with PBS and processed for 15% SDS-PAGE (Supplementary Fig. 2C). The peptide sequence was NH₂-MRTQQKRDTKFREDCPPDREELGRHSWAVLHTLAAYYPDLPTPE QQQDAQFIHLFSKFYPCEECAEDLRKRLCRNHPDTRTRACFTQWLCHLHNEVNRKLGKPDFDCSKVDERWRDGWKDGSCD-COOH.