title: Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease.
abstract: We 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, alpha2-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 A 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.
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Bethune
authors.forename: Pavel
authors.initials: P
authors.lastname: Strop
authors.forename: Yinyan
authors.initials: Y
authors.lastname: Tang
authors.forename: Ludvig M
authors.initials: LM
authors.lastname: Sollid
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli; D015870:Gene Expression; D005983:Glutens / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007976:Leupeptins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D007976:Leupeptins; D011498:Protein Precursors; D011994:Recombinant Proteins; D005983:Glutens; D014357:Trypsin; D003546:Cysteine Endopeptidases; D010434:Pepsin A; C032854:leupeptin