利用突變策略探討CadA蛋白功能區域

Using mutagenesis strategy to disclose the functional domains of CadA Cd2+ transporting ATPase

李宗建(Tsung-Chien Lee)

指導教授 ： 蔡淦仁

中山醫學大學/醫學院/醫學研究所/碩士(2007年)

摘要

CadA是金黃葡萄球菌細胞膜上一個具有排鎘功能的膜蛋白，它本身是一種Cd2+-ATPase，並被歸類為P-type ATPase中的CPx-ATPase家族。過去研究發現，像CadA這一類的細胞膜蛋白會藉由透過水解ATP取得能量以主動運輸的方式，而將細胞質內外來的金屬離子排出細胞外。本研究係利用酵母菌雙雜合系統，來進一步瞭解CadA轉運金屬離子功能，與其蛋白結構之間的關聯，並且使用化學致突變劑Hydroxylamine做CadA基因隨意突變，試著找出CadA膜蛋白上與功能有關的重要胺基酸分佈。雖然實驗結果不如預期一樣，並沒有發現CadA細胞膜內蛋白之間結合的現象，也沒有幸運地得到CadA功能缺陷的突變株，但是我們歸納出幾項使用酵母菌雙雜合系統研究CadA蛋白有關的實驗瓶頸、與需改進的地方，並且檢討隨意突變實驗中，實驗設計不周全導致偽陽性的變因，同時找出可以改良或替代的研究方法，以提供未來要探討CadA蛋白結構、與抗鎘機轉相關研究一些重要的參考資訊。

關鍵字

金黃葡萄球菌 ； 酵母雙雜合 ； 任意突變

並列摘要

Staphylococcal CadA Cd2+-ATPase mediates cadmium resistance through an active transporter to exclude the toxic cations across the membrane. The Cd2+-ATPase, the CadA protein is characterized as a member of CPx-type ATPases. Previous studies have shown that CadA utilized the energy from ATP hydrolysis to drive the cation-transporting, however the functionally important residues have yet to be systemically identified. In this thesis study, researches were designed to determined if those intracellular domains of the CadA interacts with each other using the yeast two-hybrid system. Furthermore, we also take the advantage of using the chemical mutagen, hydroxylamine, to generate random mutants throughout the cadA gene. However, our data did not support any interaction between these intracellular domains of CadA protein, neither had we acquired a series of random mutations from the chemical treatments. In this thesis research, we have pointed out the potential obstacles for these experiments, and the alternatives might be taken to overcome these problems were also discussed. Hopefully, it would be valuable for the future mutagenesis study of this cadmium resistant protein.

並列關鍵字

Staphylococcus aureus ； Yeast two-hybrid ； random mutagenesis

參考文獻

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Endo, G. and S. Silver (1995). "CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258." J Bacteriol 177(15): 4437-41.