title: Expression and characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana.
abstract: The present study reports the recombinant expression, purification, and partial characterization of a typical aspartic proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris. The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47kDa consisting of heavy and light chain components, approx. 32 and 16kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant aspartic proteinases with activity in acid pH range, maximal activity at pH 4.0, K(m) of 44 microM, and k(cat) of 55 s(-1) using a synthetic substrate. The enzyme was inhibited by pepstatin A.
authors.affiliation: Department of Food Science, University of Guelph, Ontario, Canada N1G 2W1. mazorra@ciad.mx
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Mazorra-Manzano
authors.forename: Rickey Y
authors.initials: RY
authors.lastname: Yada
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004789:Enzyme Activation / Q000187:drug effects; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010436:Pepstatins / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D029681:Arabidopsis Proteins; D010436:Pepstatins; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin