| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術研究所 === 90 === Many major allergens from mites and moulds were shown to be enzymes. However, few pollen allergens display enzymatic activities. There are controversial arguments whether the group I pollen allergen posses the protease activity. In this thesis, the group I allergen (Cyn d 1) from Cynodon dactylon was isolated and characterized.
Cyn d 1 can be divided into acidic and basic groups. By using a monoclone antibody affinity column and from Pichia pastoris expression system both native and recombinant Cyn d 1 were isolated from Cynodon dactylon. Rapid degradation of isolated proteins implies that Cyn d 1 may contain protease activity. The substrate digestion experiment also reveals that Cyn d 1 can cleavage the C-terminal of arginine. Furthermore, the zymogram experiments showed that both basic and acidic Cyn d 1 could cleave Casein and Gelatin not BSA. Only EDTA can inhibit the protease activity when incubation with various inhibitors. Therefore, Cyn d 1 might be a metallo-proteinase.
Structural prediction by 3D-PSSM suggested that the N-terminal domain of Cyn d 1 (144 amino acids) is similar to endoglucanase, and the C-terminal domain (100 amino acids) is similar to group 2 allergens. Both endoglucanase and group 2 allergens are β proteins. Consistent with the prediction, the CD measurement revealed β structure. Both N- and C-terminal domains were expressed in Pichia pastoris expression system and shown to contain protease activity by the zymogram experiments.
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