P. pastoris Secrete <i>Ac-</i>APR-1 Zymogen that Autoactivates at Low pH and Degrades Canine Hb

<div><p>SDS-PAGE gel stained with Coomassie Brilliant Blue showing purification of recombinant APR-1 zymogen from <i>P. pastoris</i> culture supernatant.</p> <p>(A) Lane 1, molecular weight markers; lane 2, concentrated culture supernatant; lane 3, flow-through from a nickel-IDA column; lane 4, 5 mM imidazole wash; lane 5, 20 mM imidazole column eluate; lane 6, 60 mM imidazole eluate; and lane 7, 1 M imidazole eluate. Purified recombinant APR-1 zymogen was activated by buffer exchange into 0.1 M sodium formate/0.1 M NaCl (pH 3.6).</p> <p>(B) Lane 1, molecular weight markers; lane 2, 5.0 μg of canine Hb (pH 3.6); and lane 3, 5.0 μg of canine Hb (pH 3.6) incubated with 0.2 μg of recombinant APR-1.</p>