- 學位論文
共表現與純化酵母菌monopolin蛋白複合體及分析Csm1/Lrs4蛋白複合體分子組成
Coexpression,copurification of Sacchromyces cerevisiae monopolin complex and analysis of Csm1/Lrs4 complex stoichiometry
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摘要
Monopolin 蛋白質複合體為酵母菌(Saccharomyces cerevisiae)在第一次減數分裂時期所形成的蛋白質複合體。Csm1與Lrs4此時會從核仁位置移動至姊妹染色體著絲點附近與只在減數分裂時才表現的Mam1結合，形成由Mam1/Csm1/Lrs4 所組成的monoplin蛋白複合體，功能在於促使姊妹染色體的著絲粒結合於同方向性的紡綞絲，幫助同源染色體的異向分離及促使姊妹染色體的同向移動，使得第一次減數分裂得以順利進行，並確保染色體的正確分離。 在此我們利用高效能選殖的方式，得到能產生大量溶解蛋白的Mam1、Csm1與Lrs4融合蛋白殖株，利用單獨表現方式與Ni-NTA親合管柱，大量純化His6-Csm1蛋白。此外，我們成功在BL21-CodonPlus(DE3)大腸桿菌中同時轉型兩個帶有不同融合蛋白的基因質體以共同誘導表現的方式表達Csm1/Lrs4及Mam1/Csm1/Lrs4蛋白複合體並利用接於目標蛋白上的融合蛋白共同純化Csm1/Lrs4及Mam1/Csm1/Lrs4 蛋白複合體。本實驗證實Csm1與Lrs4及Mam1、Csm1與Lrs4分別可在in vitro的狀態下形成蛋白質複合體。另一方面，利用分子篩管柱層析以及蔗糖梯度溶液高速離心沉降實驗，我們測定Csm1單獨以三聚體的形式存在，而其與Lrs4則可能會利用超捲曲螺旋的區塊交互作用而結合，形成Csm1與Lrs4為三對三結合的異六聚體蛋白複合體。
並列摘要
In budding yeast, Saccharomyces cerevisiae, Mam1, Csm1 and Lrs4 form monopolin complex around sister centromeres during meiosis I for sister chromatids cosegregation. Sister kinetochores can be clamped togethor by monopolin complex to attach the same oriented poles during metaphase and the sister chromatids will be pulled to the same cell after the first cell division of meiosis. For studying the protein complex, we used the high-throughput screening of soluble recombinant proteins method to obtain the clones which could overexpress the target fusion protein. By transforming two hetero-locus plasmids to E. coli., BL21-CodonPlus(DE3), Csm1/Lrs4 or Mam1/Csm1/Lrs4 complex was coexpressed in the double antibiotic selected strains. Using Ni-NTA or amylase affinity column, we have purified His6-Csm1, His6-Csm1/Lrs4 and MBP-Mam1/His6-Csm1/Lrs4 protein complex indepedently. Futhermore, we also determined the stoichiometry of Csm1/Lrs4 complex by combining the gel filtration and the sucrose gradient velocity sedimentation assay. In conclusion, we confirmed Mam1, Csm1 and Lrs4 can form monopolin complex in vitro and Csm1 can form homotrimer alone and heterohexamer with Lrs4 via the interaction of their coiled coil domains.
參考文獻
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延伸閱讀
- 莊博為（2005）。裂殖性酵母菌 S. pombe 2476 鎘誘導蛋白質之純化與性質檢定〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2005.01041
- 夏維志（2009）。I.酵母菌假設性甲基轉移酶YJR129c蛋白質受質之鑑定分析II.重組酵母菌醛類去氫酶ALD6p之非降解表現與一步純化〔碩士論文，淡江大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0002-2707200908534000
- Fang, C. Y. (2007). 探討酵母菌核醣核酸蛋白Rbp1p之後轉譯修飾及蛋白交互作用 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2007.10309
- 羅旻玉（2008）。利用酵母菌雙雜合系統尋找與FLJ11252結合的蛋白質〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2008.00036
- 林郁文（2022）。Molecular cloning and characterization of cyclic trypsin inhibitor peptide from Momordica cochinchinensis〔碩士論文，國立屏東科技大學〕。華藝線上圖書館。https://doi.org/10.6346/NPUST202200420