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Heterologous Expression, Purification, Refolding, and Structural-Functional Characterization of EP-B2, a Self-Activating Barley Cysteine Endoprotease

By Michael T. Bethune, Pavel Strop, Yinyan Tang, Ludvig M. Sollid and Chaitan Khosla

Abstract

SummaryWe describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, α2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 Å resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue

Publisher: Elsevier Ltd.
Year: 2006
DOI identifier: 10.1016/j.chembiol.2006.04.008
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