标题: | 醯亚胺水解酵素之序列分析与表现 Sequence Analysis and Bacterial Expression of Human Liver Dihydropyrimidinase cDNA |
作者: | 邱美惠 Mei-Hui Chiu 楊裕雄 Yuh-Shyong Yang 生物科技學系 |
关键字: | 醯亚胺水解酵素;Dihydropyrimidinase |
公开日期: | 1998 |
摘要: | 近年来,许多与醯亚胺水解酵素(dihydropyrimidinase;DHPase)有关的基因陆续被发现,它们的胺基酸序列与此酵素有50 ~ 60 %的高相同性(identity),本实验欲以分子演化(molecular evolution)的角度,应用生物资讯(bioinformatics)的方法,研究类似基因间彼此可能的关系,并设计实验加以验证。以BLAST软体搜寻资料库(library)内相同性高的蛋白质序列,经整理后归为六大类。再分序列相似性及功能相似性两主题,进一步分别比较其详细之胺基酸,寻找、比对其序列上相似与相异之区域,由此来推测与蛋白质功能与胺基酸序列之相对关系。经比对后发觉位于靠近N端区域一段长约30个胺基酸的保留区域(conserved region) 推测可能与醯亚胺水解酵素的催化特性有相关。因此本实验设计合成人类肝脏中的醯亚胺水解酵素 cDNA全长及缺乏N端约100个胺基酸的蛋白质并放入原核细胞内表现,以印证比对所得之推论。现阶段表现出缺乏N端的蛋白,初步结果显示,蛋白质形成了包含体(inclusion body),经调整其生长及表现条件来改善其状况;而所表现的产物并未测到活性。根据上述比对不同类型的蛋白质序列及实验结果,初步推论N端之蛋白序列可能与DHPase活性有关。 Recently, many genes related to dihydropyrimidinase (DHPase) have been discovered and revealed 50 ~ 60 %identity to DHPase. We used methods of bioinformatics to study the molecular evolution of these related proteins, and then designed experiment to study the structure/function relationship of DHPase and its related proteins. BLAST was used to search protein with high sequence identity ( > 40 %) with DHPase from DDBJ/EMBL/GenBank. The proteins were classified into six families by two approaches, through comparison of sequence homology and functionally related proteins. DHPase was compared with 8 sequence homologous proteins and 10 functionally related protein. We found that a region near N-terminal is conserved among functionally related proteins, but varied between DHPase and DHPase related proteins which do not have known catalytic function. We suggest that this region might be important for the catalytic function of DHPase. In order to investigate the significance of this region, we prepared and expressed an N terminal truncated protein in Escherichia coli. Conditions for the expression of the truncated DHPase were investigated to improve the production of soluble protein. No DHPase activity can be detected. Preliminary data indicates that N terminal of DHPase may be important for catalytic function. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#NT870111005 http://hdl.handle.net/11536/63849 |
显示于类别: | Thesis |