Structure-function study on the activation mechanism of Endo T : Crystallization and biochemical characterization of the intact Endo T from Trichoderma reesei (Hypocrea jecorina)

University essay from SLU/Dept. of Molecular Biology (until 131231)

Abstract: Endo T is a deglycosylating enzyme secreted by the filamentous fungus Trichoderma reesei (Hypocrea jecorina). The active Endo T protein is both deglycosylated and C-terminal processed. Since the intact protein was never observed in the T. reesei culture medium, a construct of the intact Endo T was expressed in P. pastoris. Here, the glycosylated, intact and inactive protein was found in the medium but this form is slowly converted to the deglycosylated and proteolytic cleaved form. The structure of the active Endo T was already solved in previous work. We tried to solve the structure of the intact inactive Endo T to compare it with the active structure. Several structures of Endo T were solved, but none of them were structures of the intact Endo T. Different pH and T parameters were screened to find a condition where the C-terminal peptide was stable and the enzyme kept intact. A protease inhibitor cocktail was also added to limit the proteolytic process. We were able to control the proteolysis with the help of the protease inhibitor cocktail at pH 5 and higher, but we were not able to limit the proteolysis at pH 3, nor in any of the crystallization samples.

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