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研究生:李俊逸
研究生(外文):Chun-Yi Lee
論文名稱:利用NMR的PRE技術研究半乳糖凝集素-3蛋白之N與C端之間的交互作用
論文名稱(外文):Characterizing the Interaction Between N- and C-termini of Galectin-3 by NMR Paramagnetic Relaxation Enhancement (PRE)
指導教授:黃介嶸
指導教授(外文):Jie-rong Huang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:91
中文關鍵詞:核磁共振順磁弛豫半乳糖凝集素-3
外文關鍵詞:NMRParamagnetic Relaxation Enhancementgalectin-3
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半乳糖凝集素–3是一個由250個胺基酸組成的蛋白質,包含前端一段長112個胺基酸的無結構胺基酸多肽鏈以及後端138個胺基酸的有結構碳水化合物辨認位。半乳糖凝集素–3會廣泛的分布在哺乳動物的體內;不論是循環系統中或者是細胞內、外,都可以發現半乳糖凝集素–3的存在。而半乳糖凝集素–3的生物功能也同樣廣泛,已知半乳糖凝集素–3能夠調控細胞轉化、調控細胞凋亡以及調控細胞的生命週期。而前人的研究指出,半乳糖凝集素–3這些功能的調控並不僅僅是受控於有結構的碳水化合物辨認位。研究指出位於無結構的胺基酸多肽鏈位置的突變會影響到半乳糖凝集素–3與Ras蛋白的結合,而導致細胞轉化的持續發生。而同樣在無結構的胺基酸序列的部分胺基酸的缺失會影響半乳糖凝集素–3進出細胞核的能力。還有研究指出位於無結構的胺基酸序列有突變發生時,會影響到半乳糖凝集素–3辨認乳糖的能力。這代表半乳糖凝集素–3的無結構的胺基酸序列用某種方式影響著整個半乳糖凝集素–3的功能,但關於無結構的胺基酸序列如何影響整個蛋白質的相關研究卻很少。於是我們在完整的半乳糖凝集素–3蛋白質上的無結構胺基酸序列上選定三個點(10、75、100)。因為A10靠近S6以及S12這兩個與功能有密切關係的磷酸化位點。而A100則是靠近Y107與碳水化合物辨認位辨認能力有關連。A75則是因為靠近N端無結構的中段,希望可以藉由這個點來探討N端無結構蛋白的中段結構變化。藉由核磁共振的順磁弛豫增強實驗來研究這三個點與半乳糖凝集素–3的碳水化合物辨認位是否存在交互作用。我們的研究結果顯示半乳糖凝集素–3的無結構胺基酸序列部分存在有與碳水化合物辨認位交互作用的傾向;以及無結構的胺基酸序列本身可能也存在互相靠近的傾向。
Galectin-3 is the protein consisted of 112 disordered amino acids sequence of N-termini and 138 structural amino acids sequence of C-terminal carbohydrate recognition domain (CRD). Galectin-3 is widespread on mammalians body; including nuclear, plasma and circulatory system. Galectin-3 also has the wide biofunctions. We know that galectin-3 may involve the cell transformation, regulation of cell apoptosis and regulation of cell cycle. Some papers reported that disordered N-termini of galectin-3 may affect biofunctions of galectin-3. The papers showed that mutations on galectin-3 disordered N-termini avoid binding of galectin-3 and K-Ras protein, it will lead the cell continuously transform. And deletions on disordered N-termini of galectin-3 make the galectin-3 loss ability of transportation from nuclear to plasma. On the other research said that the lactose binging affinity of galectin-3 decreased as mutations on N-termini of galectin-3. It shows that N-termini of galectin-3 affects the protein functions by some interactions, but there are little research about N- and C- interaction. We want to know how the interaction between N- and C-terminus. We make three cysteines to alanines mutantion on disordered N-terminal of galectin-3, galetin-3-A10C, -A75C and -A100C. We made the NMR paramagnetic relaxation enhancement on three mutants. On this research, we found that disordered N-terminal of galectin-3 may interact with CRD and disordered N-terminal of galectin-3 may have the special attention of folding.
致謝 i
英文摘要 ii
中文摘要 iii
目錄 iv
圖目錄 v
表目錄 vi
Chapter 1. 背景介紹 1
前言 1
1.1 半乳糖凝集素的介紹 4
1.2 半乳糖凝集素的生物功能與疾病 9
1.3 半乳糖凝集素–3的結構與結構對功能的影響 17
目的 22
Chapter 2. 材料與方法 23
2.1 試劑 23
2.2 儀器 29
2.3 方法與步驟 30
Chapter 3. 實驗原理與結果 42
3.1建立半乳糖凝集素–3與其突變株的質體 42
3.2 測試並建立培養突變半乳糖凝集素–3之表現標準流程 55
3.3 建立檢驗MTSL標定標準流程 67
3.4 順磁弛豫增強(paramagnetic relaxation enhancement,PRE)實驗 72
Chapter 4. 討論與結論 81
附錄 87
參考文獻 88

圖 1. 半乳糖凝集素在生物體中的重要功能示意圖 8
圖 2. 半乳糖凝集素家族參與癌細胞的轉化(transformation) 12
圖 3. 半乳糖凝集素家族對於癌細胞中的細胞凋亡的調控 13
圖 4. 半乳糖凝集素家族對於癌細胞的細胞週期的調控 16
圖 5. 半乳糖凝集素–3的結構 20
圖 6. 蛋白質SDS膠體電泳之預染色蛋白標定(marker)參考 28
圖 7. 蛋白質多肽鏈與MTSL反應以及標定 43
圖 8. 半乳糖凝集素–3–C173A以及A10C、A75C、A100C的突變株的胺基酸序列示意圖 46
圖 9. 融合蛋白SUMO-gal3-A75C-C173A之質體建構以及SUMO專一性蛋白酶ulp1c作用示意圖 49
圖 10. 以基因重組將Gal3–A75C–C173A接在pET32a–SUMO系統中 53
圖 11. 分別以Rosetta、BL–21勝任細胞表現半乳糖凝集素–3–C173A的蛋白質SDS電泳膠圖 57
圖 12. 以lactosyl – Sepharose 4B®管柱純化半乳糖凝集素–3–C173A 59
圖 13. 比較不同溫條件下之半乳糖凝集素–3–C173A之表達量 61
圖 14. 半乳糖凝集素–3–A75C–C173A在pET25b系統以及pET32a–SUMO系統下的表現 62
圖 15. 分別表現半乳糖凝集素–3–A10C∕A75C–C173A 63
圖 16. 純化SUMO–Gal3–A75C–C173A 65
圖 17. 以DTNB來辨識R–SH的原理 68
圖 18. 以DTNB檢測半乳糖凝集素–3–C173A–A10C被MTSL標定 71
圖 19. 常用於順磁弛豫增強實驗的化學藥物 76
圖 20. 半乳糖凝集素–3的順磁弛豫增強實驗結果 79
圖 21. 檢證A10C、A100C的NMR實驗樣品是否發生降解 80
圖 22. 預測的半乳糖凝集素–3之N端與C端交互作用的模型 86

表 1. 半乳糖凝集素家族的分類 6
表 2. 化學藥品 23
表 3. 同位素標記藥品 27
表 4. 試劑套組及標準樣品 27
表 5. 使用KAPA PCR kit的反應混和液配置 34
表 6. 使用DpnI酵素的反應混和液配置 34
表 7. 大腸桿菌(Escherichia coli,E. coli)之基因使用頻率表(gene codon usage) 43
表 8. 量化100毫升 LB培養液培養之半乳糖凝集素–3–C173A 61


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