AP-HSA fusion protein design, expression, and characterization. (A) Schematic diagram of proteins. Relevant polypeptides are shown in linear form, with important residues identified above each bar. White bars correspond to the N-terminal dodecapeptide sequence removed from Met-α2AP to form Asn-α2AP, shown in grey; HSA is shown in black, and C-terminal hexahistidines are shown as stippled bars. (B) Electrophoretic profile of conditioned media samples taken from P. pastoris cultures at the times indicated above the lanes, post-induction with methanol. A Coomassie Blue-stained 10% SDS-polyacrylamide gel is shown. Cell lines were transformed with plasmid constructs directing the synthesis of the proteins identified above the horizontal lines. Markers on the leftmost lane of the gel are, in kDa: 160; 140; 120; 100; 90; 80; 70; 60; 50; 40; 30; and 25. (C) A stained gel similar that in panel B is shown, except that 5 μg of the purified proteins identified above the lanes were electrophoresed. M, markers, same as in panel B. (D) N-termini found in purified α2AP(13-42)-HSA by amino acid sequencing of the preparation shown in the leftmost lane of panel C. Numbers above the box identify every fifth amino acid residue in α2AP(13-42)-HSA.