Rosenkilde, Anne Lind3; Dionisio, Giuseppe3; Holm, Preben Bach3; Brinch-Pedersen, Henrik3
1 Department of Molecular Biology and Genetics - Afgrødegenetik og Bioteknologi, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University2 Molekylær Genetik og Bioteknologi, Faculty of Agricultural Sciences, Aarhus University, Aarhus University3 Department of Molecular Biology and Genetics - Afgrødegenetik og Bioteknologi, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University
During germination of barley seeds, mobilization of protein is essential and cysteine proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins. Cysteine proteases exist as pro-enzyme and is activated through reduction of the active site cysteines and by removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. A cDNA clone of the barley key cysteine endoprotease B2 (HvEPB2) was ligated into the Pichia pastoris expression vector pPICZ Aα and electrotransformed into Pichia pastoris strain KM71H. Heterologous protein production was induced with 2% MeOH and maximum yield were obtained after 4 days where the supernatant was harvested. Purification of HvEPB2 from the supernatant were performed by Ni2+-affininty chromatography. The purified fractions were analyzed via SDS-PAGE, western blotting for confirming the presence of HvEPB2 and via activity assaying. Incubation of purified HvEPB2 with Osborne fractionated barley seed storage proteins for 12 hrs revealed after SDS-PAGE a significant degradation of the storage proteins.
1st International Conference on Plant Proteases 2011. From Biology To Biotechnology, 2011
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The 1st International conference on PLANT PROTEASES 2011. From biology to biotechnology