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