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標題: 十字花科黑腐病菌 XpsE 蛋白特性之研究 : 1. 利用分析式超高速離心分析 XpsE 之四級結構, 2. 利用高效能逆相液相層析分析與 XpsE 結合之腺核苷酸
Characterizations of XpsE protein of the Xanthomonas campestris pv. campestris type II secretion apparatus : 1. Analytical ultracentrifugation analysis of quaternary structure, 2. RP-HPLC analysis of protein-bound adenine nucleotides
作者: 郭柏村
Kuo, po-tsun
關鍵字: type II secretion apparatus;第二型分泌機制
出版社: 生物化學研究所
十字花科黑腐病菌中第二型分泌機制由 12 個蛋白所組成,XpsE 蛋白為成員之一。XpsE 蛋白位於細胞質中,共有 567 個胺基酸。與其他第二型分泌系統中的同源蛋白 (GspE) 胺基酸序列比對,歸類屬於 ”GspE-VirB11 NTPase” 一大家族,具有核苷酸結合區域,推測可能有 ATPase 酵素活性。本論文第一部分中利用分析式超高速離心 (analytical ultracentrifugation) 實驗偵測 XpsE 蛋白的沉降係數,結果顯示 XpsE 蛋白在試管內呈現單倍體、雙倍體、三倍體、四倍體、五倍體與六倍體的四級結構,其中單倍體佔總量的一半。若以分子篩管柱層析分析由 XpsE 的 N 端第 1~152 個胺基酸組成的,XpsEN 蛋白,在沒有 His6-tag 的情況下 XpsEN 蛋白是以單倍體型式存在。但是,若是融合有 His6-tag 的 hXpsEN 蛋白能夠以多倍體與單倍體的型式共同存在。暗示 hXpsEN 蛋白可能因 N 端具有 His6-tag 而形成多倍體的條件。本論文第二部分利用核苷酸萃取與高效能液相逆向層析分析結合於 XpsE 蛋白上的核苷酸。結果顯示,XpsE 蛋白上可能結合有微量的 AMP、ADP 與 ATP 核苷酸。

The type II secretion apparatus of Xanthomonas campestris pv. campestri is constituted of 12 proteins components. One of them XpsE is a cytoplasmic protein composed of 567 amino acid residues. Sequence alignment of XpsE protein and its homologues of other type II secretion systems (GspE) revealed that they belong to a “GspE-VirB11 NTPase” superfamily. All members of the protein family posses nucleotide-binding motifs, and proposed to be putative ATPases. In the first part of this study, I determined sedimentation coefficient of XpsE protein by using analytical ultracentrifugation. The results showed that XpsE appears as monomer, dimer, trimer, tetramer, pentamer and hexamer in vitro. Half of them are monomeric. Gel filtration analysis of the XpsEN protein, which is constituted of the N-terminal 1~152 residues of XpsE, indicated that untagged XpsEN exists as solely monomer. In contrast, His6-tagged hXpsEN appeared as monomer and multimer. This observation suggests that multimerization of hXpsEN may be due to its N-terminal His6-tag. In the second part, by using nucleotide extraction followed by reverse phase high performance liquid chromatography (HPLC), I analyzed the XpsE-bound adenine nucleotides. Minute amounts of AMP, ADP and ATP were detectable in XpsE-bound state.
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