title: [Production and properties of recombinant glutenin-cleaving proteinases from Eurygaster integriceps Put].
abstract: cDNAs coding for a mature form of glutenin-cleaving trypsin-like proteinase (referred to as glutenin-hydrolyzing proteinase 3 or GHP3) from the insect pest-Eurygaster integriceps Put. and a zymogen of this proteinase containing a signal peptide required for protein secretion were cloned into vectors pPIC9 and pPIC3.5, respectively. The constructs were used for protein expression in cells of the methylotrophic yeast Pichia pastoris. The recombinant protein corresponding to the mature form of the proteinase was secreted into the culture medium and possessed proteolytic activity, while the zymogen acquired activity after trypsin, treatment. Both recombinant enzymes hydrolyzed high-molecular weight glutenin subunits from wheat of the variety Ege-88 and a range of other soft and durum wheat varieties. Chymotrypsin inhibitor I from potatoes and related inhibitors from seeds of plants of the subclass Asteridae, the Kunitz-type trypsin inhibitor from soybeans, and bovine aprotinin had a weak inhibitory effect on the recombinant proteinases, while the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans did not interact with these enzymes:
authors.forename: V V
authors.initials: VV
authors.lastname: Dolgikh
authors.forename: I V
authors.initials: IV
authors.lastname: Senderskiĭ
authors.forename: A V
authors.initials: AV
authors.lastname: Konarev
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000737:chemistry; D002417:Cattle; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000737:chemistry*; D015870:Gene Expression; D005983:Glutens / Q000737:chemistry*; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007313:Insecta / Q000737:chemistry / Q000201:enzymology; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D059748:Proteolysis; D014908:Triticum / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry
chemical_list: D004792:Enzyme Precursors; D019476:Insect Proteins; C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D021382:Protein Sorting Signals; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; C023578:proteinase inhibitor I (plants); D005983:Glutens; D007611:Aprotinin; D010447:Peptide Hydrolases; D014357:Trypsin; C037302:glutenin