Expression and characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana |
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Authors: | Mazorra-Manzano Miguel A Yada Rickey Y |
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Institution: | Department of Food Science, University of Guelph, Ontario, Canada N1G 2W1 |
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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 47 kDa consisting of heavy and light chain components, approx. 32 and 16 kDa, 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, Km of 44 μM, and kcat of 55 s−1 using a synthetic substrate. The enzyme was inhibited by pepstatin A. |
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Keywords: | Arabidopsis thaliana Cruciferae Aspartic proteinase Pichia pastoris Recombinant expression Plant APs Protein processing Glycosylation |
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