Figure 3  Preparation of an N-terminally PASylated version of the Tα1 peptide and analysis of its integrity, purity, and hydrodynamic volume. (a) Expression vector for the bacterial production of PAS-Tα1. The gene cassette cloned on pASK75 comprises the T7 RBS, the coding region for a 601-residue PAS#1 sequence preceded by a start methionine and a proline codon, the human Tα1 sequence, and, finally, tlpp. (b) The deconvoluted ESI-MS spectrum of PAS-Tα1 purified from the E. coli cell extract by subtractive AEX and CEX (see text) reveals a molecular mass of 52,789.69 Da, which matches the calculated mass for PAS-Tα1 with a fully processed start methionine (52,789.66 Da). (c) Reverse-phase chromatography indicates a purity > 96% for PAS-Tα1. (d) Analytical SEC of the purified PAS-Tα1 in PBS results in a single peak with an elution volume of 13.2 mL. Comparison with a half-logarithmic calibration curve (inset) indicates a strongly expanded hydrodynamic volume with an apparent molecular size of 665 kDa.