Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35992
標題: 水稻結鈣激活酵素OSCK1融合蛋白之純化與活性分析
Protein Purification and Activity Assay of a Recombinant Calcium-dependent Calmodulin-independent Protein Kinase (OSCK1) from Rice
作者: 楊中翰
Yang, Chung-Han
關鍵字: 水稻;Recombinant Calcium-dependent Calmodulin-independent Protein Kinase;結鈣激活酵素;cdpk
出版社: 農業生物科技學研究所
摘要: 
Calcium-dependent calmodulin-independent protein kinase (CDPK)為一種富含serine及threonine之激活酵素,目前於許多植物中發現以多基因家族形式存在。CDPK於植物細胞內部的位置呈多樣性,且專一地表現於的特定時期及組織,因此推測CDPK在多種訊息傳遞的途徑中扮演重要角色。本實驗室藉由差異性表現基因選殖法由水稻中篩選出一命名為OSCK1之基因,由胺基酸序列推測出其具有典型CDPK的蛋白質結構,亦即N端至C端依序為歧異性區、激活酵素區、自我抑制連結區及C端之鈣離子結合區。藉由序列比對發現OSCK1與玉米花粉專一表現的CDPK具有高度相似性 (85﹪),為了分析OSCK1之生化活性,本實驗利用聚合酵素連鎖反應增幅出各蛋白質區之DNA,並選殖入pET系列載體內,藉由大腸桿菌表現系統表現出各融合蛋白質,並經由金屬親和性層析法純化後進行生化活性測試。測試結果發現含鈣離子結合區之融合蛋白質,可於SDS-PAGE上產生結鈣蛋白質結鈣後所特有之條帶變動現象。此外還發現,全長OSCK1可將加入之histone III-S受質磷酸化,在hemoglobin及α-casein二種受質則無法遭此磷酸化現象。而於磷酸化及自我磷酸化活性測試中,則發現OSCK1之激活酵素活性分別可受鈣離子的活化及EGTA的抑制,因此推測OSCK1可受鈣離子的調控。

The calcium-dependent calmoldulin-independent protein kinases (CDPKs) are the most abundant serine/threonine kinases in plants that are encoded by a multigene family. CDPKs have been found in various subcellular localizations and are expressed with distinct profiles, which indicate they were involved in multiple signal transduction pathways. By differential screens, we have isolated a CDPK gene from rice and designated it as OSCK1. The predicted OSCK1 protein includes an N-terminal highly variable region, followed by a protein kinase domain, an auto-inhibitory junction and a C-terminal calcium-binding domain. Based on sequence comparison and overall gene structure analysis, OSCK1 is highly homologous to a pollen-specific CDPK gene in maize. To characterize the biochemical activities of OSCK1, DNA fragments encoding various domains of OSCK1 were amplified by polymerase chain reaction and subcloned into pET vectors. Using E. coli protein expression system, the soluble form of recombinant proteins were partially purified by metal-affinity chromatography. The recombinant protein contain calcium-binding domain showed a Ca2+-dependent electrophoretic mobility shift in SDS-PAGE. Furthermore, the recombinant full-length OSCK1 protein could efficiently phosphorylate artificial substrate histone III-S, but not hemoglobin or α-casein. Both the phosphorylation and autophosphorylation activities of the recombinant OSCK1 could be stimulated by Ca2+ and inhibited by EGTA, indicate that they are regulated by Ca2+.
URI: http://hdl.handle.net/11455/35992
Appears in Collections:生物科技學研究所

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