Figure 1  Bacterial production of N-terminally acetylated Tα1-PAS. (a) Expression vector for Tα1-PAS and the N-acetyltransferase RimJ from a bicistronic operon under control of the inducible tetp/o. The gene cassette cloned on pASK75 comprises the T7 phage ribosome binding site (T7 RBS) followed by the coding sequence for human Tα1 fused to a 601-residue PAS#1 sequence, the second RBS, the coding sequence for RimJ, and the lipoprotein terminator (tlpp). (b) Western blot analysis after 4–12% SDS–PAGE of Tα1-PAS in the E. coli whole cell lysate using an antibody directed against the PAS#1 sequence. M, protein size standard; lane 1, whole cell extract before induction; lane 2, whole cell extract 15 h after induction. (c) 10% SDS-PAGE of purified Tα1-PAS. M, protein size standard; lane 1, Tα1-PAS after ammonium sulfate precipitation, subtractive anion exchange (AEX), cation exchange (CEX), and final AEX chromatography.