Schematic and electrophoretic representation of proteins and peptides employed in this study. Polypeptides comprising portions of HCII (solid black bar, with residue numbers inset in white) or hirudin (Hir) (primary sequence shown) are represented schematically in panel A. Each HCII-derived protein or peptide contains an N-terminal MGSH6 tag (shown at left of each schematic representation): Ac, acetyl; SO3H, sulfate group on Hir Tyr63. Panel B shows a reduced 12% SDS polyacrylamide gel stained with Coomassie Brilliant Blue. Ara- and Ara+ lanes show total bacterial lysates from cultures expressing HCII 1-75 grown in the presence or absence of 0.002% (w/vol) arabinose. Aliquots of bacterial lysates purified by nickel chelate chromatography (FT, flow-through; Ni, imidazole-eluted peak fractions; QS, final preparation polished by ion exchange on Q-Sepharose) are shown. Approximately 0.75 μg of purified HCII 1-75 (QS), glutathione sulfotransferase (GST) and HAPI M358R (HCII 1-75 α1-PI M358R fusion protein) were electrophoresed in the last three lanes at right. Panel C shows the reaction products of purified HAPI M358R or HAPI T345R/M358R incubated with (+) or without (-) thrombin (IIa) electrophoresed on a reduced 12% SDS polyacrylamide gel stained with Coomassie Brilliant Blue. The positions of molecular mass markers are labeled, in kDa, to the left of panels B and C; positions that are not labeled due to insufficient space correspond to 160, 120, 100, 90, and 80 kDa.
Boyle et al. BMC Biochemistry 2013 14:6 doi:10.1186/1471-2091-14-6