Rosenkilde, Anne Lind4; Dionisio, Giuseppe4; Holm, Preben Bach4; Brinch-Pedersen, Henrik4
1 Molekylær Genetik og Bioteknologi, Faculty of Agricultural Sciences, Aarhus University, Aarhus University2 Department of Genetics and Biotechnology, 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 University4 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, the 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 until activated through reduction of the active site cysteines and via 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. One of the key cysteine proteases in Barley, (Hordeum vulgare) endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ Aα and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH and the protein expression were monitered during induction by collecting 1 ml samples every hr for 24 hrs. After 4 days, the supernatant were harvested and analyzed by SDS-PAGE, activity assay and Western blot. A significant amount of functional, heterologous protein was produced and the protein production was highest after 4 days and the expression in the C-terminal mutant was slightly higher than for the full length protease.
Main Research Area:
Technologies in Protein Sciences. 4th Workshop in Protein, 2010