| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術研究所 === 91 === Many major allergens from mites and moulds have been shown to be hydrolases,and the enzymatic activity may be related to the allergenicity.However,few pollen allergens display hydrolytic activities.The relationship between the enzymatic activity,protein structure and allergenicity still remains unknown.There are controversial arguments whether the group I pollen allergens possess the protease activity. In this thesis, the group I allergen,Phl p 1 from Phleum pratense is isolated and characterized.
First,the recombinant Phl p 1 protein in a Pichia pastoris system was expressed and purified.Rapid degradation of isolated proteins implies that Phl p 1 may contain protease activity.The zymogram experiments showed that Phl p 1 could cleave gelatin,suggesting that Phl p 1 possesses the enzymatic activity.
Second,the recombinant Phl p 1 protein in a E.coli system was also expressed and purified by a Ni-chlelating column.After screening and refinement of protein crystallization,single crystals are obtained,which diffract x-ray up to 7 Å resolution. Secondary structural analysis by circular dichorism measurement reveals that Phl p 1 is mainly composed of β-strands.
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