Schematic and electrophoretic representation of proteins and peptides employed. Polypeptides comprising portions of API M358R (hatched bar, with residue numbers above) with or without N-terminal extensions (white) derived from HCII (symbols inset, and residue number below) or HV3 (symbol inset, and residue numbers below) are represented schematically in Panel A. Each protein contains an N-terminal MGSH6 tag (at left of each schematic representation, thin grey bar). Fusion proteins contain a spacer peptide (thin black bar) following the N-terminal extension of either 6 or 8 residues identified above the bar. Reactive centre loop (RCL) sequences are either API M358R (open box, with exploded sequence below) or M358R with additional F352A/L353V/E354V/A355I/I356A/I460L substitutions (RCL5, with open box inset “5”, and exploded sequence below). Recombinant proteins are identified to the right of each bar diagram. 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 HV3API M358R 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; DEAE, final preparation following ion exchange on DEAE-Sepharose) are shown. Panel C shows two replicas of a reduced SDS gel loaded with 100 ng of each of the purified proteins identified above the lanes (GST; glutathione sulfotransferase, negative control) immunoblotted with either anti-API (upper panel) or anti-hexahistidine (anti-H6, lower panel). Panel D shows a reduced 12% SDS gel stained with Coomassie Brilliant Blue, containing 250 ng of each of the purified proteins identified above the lanes electrophoresed. The positions of molecular mass markers are labelled, in kDa, to the left of Panels B-D.