Purification and Characterization of Sulfotransferases

7-dehydrocholesterol (7-DH), 24-dehydrocholesterol (24-DH), dithiothreitol (DTT), ethylene-diamine-tetraacetic acid (EDTA), L-glutathione (reduced), 1-hydroxypyrene (1-HP), imidazole, isopropyl-thio-β-D-galacto-pyranoside (IPTG), lysozyme, 3-maleimido-PROXYL, N-cyclohexylmaleimide, pepstatin A, potassium phosphate, 2,2,2-trichloroethanol (TCE), and quercetin were the highest grades available from Sigma. Ampicillin, dimethyl sulfoxide (DMSO), KOH, LB media, MgCl₂, tris(hydroxymethyl)aminomethane (Tris) base, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Fisher Scientific. Glutathione- and nickel-chelating resins and Superdex 200 Increase, 10/300 GL columns were obtained from GE Healthcare. D₂O was purchased from Cambridge Isotope Laboratories. Competent E. coli (BL21(DE3)) was purchased from Novagen. SULT2B1a and SULT2B1b clones were obtained from IDT. The SULT2A1 clone and SULT2B1b pKK233 (32 (link)) vector were obtained from Dr. C. N. Falany. Synthesis and purification of PAPS and PAP is previously described (33 (link), 34 (link)) and anion-exchange HPLC analysis suggested they are ~99% pure.