臺灣博碩士論文加值系統

SUMO的全名為 small ubiquitin-like modifier，雖然在序列上只和泛素擁有約18%的相似度，其摺疊完成後之立體構形卻和泛素極度相似。SUMO亦藉由和泛素類似的作用機制與目標蛋白質進行共價結合，即 SUMO化 (SUMOylation) 過程也是經由E1活化酶、E2銜接酶和E3黏合酶三種酵素依序參與活化、銜接與黏合的步驟；但泛素化與SUMO化間存有三項顯著的差異：(1)在脊椎動物的系統中，目前已知參與泛素化作用的酵素有許多種類，但SUMO化僅有一種E1 (SAE1/SAE2 複合體) 和一種E2 (Ubc9) 參與；(2) 在許多in vitro研究中顯示，SUMO化可能不須要E3的參與，而僅藉由E1及E2的活性完成；(3) SUMO化是一個可逆的反應，可經由一類去SUMO化酵素破壞SUMO與目標蛋白質間的異胜肽鍵。此類SUMO蛋白酶亦負責SUMO蛋白質的成熟化，能將C端末尾的片段經水解去除，使SUMO露出雙甘胺酸序列 (di-glycine motif) 而能和目標蛋白質進行鍵結。相較於絕大多數經泛素修飾的蛋白質所導向的蛋白酶體降解途徑，SUMO化在細胞中已被發現有更多樣且範圍廣泛的生理功能，如訊息傳遞、蛋白質穩定性、酵素活性調控，以及細胞內分子運送等，因而使其相關的研究受到高度的重視。
相較於只有一種形式的泛素，目前發現的SUMO在不同生物間有許多種類，其中脊椎動物共擁有三種型式的SUMO (SUMO1-3)；其中SUMO-2和SUMO-3享有95%的序列相同性，卻僅和SUMO-1有約50%的序列相同，因此在目前的研究中，多把SUMO-2及SUMO-3總稱為 SUMO-2/3一類，而將SUMO-1獨立為另一類。特別的是，由於近年來發現SUMO-2/3的序列中具有一個SUMO化偏好發生的保守序列VKTE，因能夠像多泛素般產生聚合長鏈對目標蛋白質進行修飾，而SUMO-1由於缺乏此序列而僅能進行單一SUMO化；此項發現使單一SUMO化或多SUMO化各自帶有的生理意義受到極度重視，並引發廣泛的研究。
本論文實驗藉由點突變方法製造出帶有SUMO化共有基序的SUMO-1 (D15V)、SUMO-1 (D15V/K17T) 及SUMO-1 (K25E) 三種SUMO-1突變株，以及失去SUMO化共有基序的SUMO-2突變株SUMO-2 (K11A)，並建立在in vitro或在大腸桿菌中進行的SUMO化系統，以及能表現帶有tag之SUMO的動物穩定細胞株，作為研究SUMO化作用的工具，並探討SUMO化共有基序對SUMO聚合作用造成的影響。

Although SUMO (small ubiquitin-like modifier) shares only 18% sequence identity with ubiquitin, its folding structure highly resembles that of ubiquitin except for the N terminal extension. In addition, SUMO conjugates to its substrates through a mechanism similar to that of ubiquitination. That is, SUMOylation is also a sequential enzyme catalytic cascade, which includes E1 activation, E2 conjugation and E3 ligation. However, there are three major differences between these two post-translational modifications: (1) in the mammalian system, there is only one SUMO E1 and one SUMO E2 compared to the many known ubiquitination enzymes; (2) many in vitro studies have shown that SUMOylation may not need the participation of E3 ligase; (3) SUMOylation can be reversed by de-SUMOylation enzymes. These enzymes are not only responsible for de-conjugation of SUMO but are also in charge of its C-terminal maturation. In contrast to ubiquitination, sumoylation does not target modified proteins for degradation, but can affect their signal transduction, stability, enzyme activity and localization showing its importance.
Higher eukaryotes express three SUMO family members, SUMO1-3. Mature SUMO-2 and SUMO-3 (referred to as SUMO2/3) are 97% identical but differ substantially from SUMO1 (~50% identity). Interestingly, SUMO2/3 contains an internal consensus motif that is missing in SUMO1 which is responsible for polychain formation. This gives rise to vast research on the physiological consequences of mono- and poly-SUMOylation.
To investigate the importance of SUMOylation consensus motif on SUMO polymerization, we have generated three different SUMO-1 mutants, SUMO-1(D15V), SUMO-1(D15V/K17T) and SUMO-1(K25E), which bear a SUMOylation site in their sequences. We also produced a SUMO-2 mutant losing the Lys11 SUMOylation residue by the same site-directed mutagenesis method. We have conducted several experiments with these SUMO mutants in our in vivo or in vitro SUMOylation systems, and successfully obtained some interesting findings among different types of poly-SUMOylation events.

目錄 i
縮寫表 v
摘要 vi
Abstract vii
第一章 緒論 1
1.1類泛素蛋白質家族 1
1.2 SUMO化修飾系統 1
1.2.1 SUMO簡介 1
1.2.2 SUMO化與去SUMO化 2
1.2.3 SUMO化參與之生理作用 4
1.3 SUMO之聚合反應 6
1.3.1 SUMO化共有基序 6
1.3.2 SUMO-2/3 之聚合反應與生理作用 7
1.4 研究動機與方向 7
第二章 材料與方法 11
2.1 實驗材料 11
2.1.1 大腸桿菌菌株 11
2.1.2 動物細胞 11
2.2 各目標基因表現載體之建構 12
2.2.1 真核表現系統載體 12
2.2.2 原核表現系統載體 12
2.2.3 核酸引子設計 13
2.2.4 聚合酶鏈鎖反應 14
2.2.5 限制酶切反應 16
2.2.6 接合反應 17
2.2.7 點突變聚合酶鏈鎖反應 18
2.3 原核宿主細胞表現系統 22
2.3.1 化學法勝任細胞製備 22
2.3.2 大腸桿菌細胞轉形 23
2.3.3 重組蛋白質誘導表現 24
2.4 重組蛋白質之純化方法 25
2.4.1 6xHis重組蛋白質親和性層析法 25
2.4.2 GST重組蛋白質親和性層析法 26
2.4.3 離子交換法 27
2.4.4 蛋白質脫鹽與濃縮 28
2.5 真核宿主細胞表現系統 29
2.5.1 動物細胞培養 29
2.5.2 真核細胞轉染 31
2.5.3 細胞冷凍保存 32
2.6 DNA相關基本操作方法 33
2.6.1 小量質體DNA製備 33
2.6.2 大量質體 DNA 製備 35
2.6.3 洋菜膠體電泳 36
2.6.4 核酸定量 37
2.6.5 核酸純化 38
2.7 蛋白質相關基本操作方法 39
2.7.1 蛋白質定量 39
2.7.2 蛋白質電泳檢定 40
2.7.3 蛋白質電泳膠片染色法 43
2.7.4 蛋白質轉印法 45
2.7.5 酵素免疫染色 46
2.7.6 蛋白質免疫沉澱 47
2.8 單株抗體製備 48
2.8.1 抗原製備 48
2.8.2 小鼠免疫與採血 48
2.8.3 細胞融合 50
2.8.4 篩選有效價之細胞株 52
2.8.5 細胞單株化 54
2.8.6 單株抗體篩選 55
2.8.7 單株抗體生產 57
2.8.8 單株抗體純化 58
2.9 胞外SUMO化修飾 59
第三章 結果 60
3.1 In vitro SUMO化與去SUMO化系統之建立 60
3.1.1 SUMO1、SUMO2 表現質體之建構及確認 60
3.1.2 SUMO與SUMO化酵素之蛋白質表現 61
3.1.3 In vitro SUMO化系統之建立 63
3.1.4 SENP1蛋白質表現與活性分析 63
3.2 In vitro多SUMO聚合反應系統之建立與分析 64
3.2.1 SUMO 化共有基因序列之點突變與表現質體建構 65
3.2.2SUMO突變株蛋白質表現 67
3.2.3 野生型與突變型SUMO之in vitro聚合反應分析 67
3.2.4 SENP1對野生型與突變型多SUMO聚合鏈之活性分析 69
3.3 大腸桿菌內SUMO化修飾系統之建立與分析 70
3.3.1 大腸桿菌宿主細胞之轉形 70
3.3.2 SUMO化系統於大腸桿菌之誘導表現 71
3.4 真核細胞胞內 SUMO 化修飾目標蛋白質之分析 72
3.4.1 野生型SUMO1與SUMO2動物細胞表現質體之建立 72
3.4.2 SUMO突變株蛋白質表現質體之建立 73
3.4.3 動物細胞轉染與穩定細胞株之建立 74
3.4.4 動物細胞內SUMO化現象之分析 75
3.5 SUMO突變株對目標蛋白質之修飾作用 76
3.5.1 大腸桿菌轉形勝任細胞製備 76
3.5.2 野生型與突變型SUMO對p53之修飾作用 77
3.6 SUMO 單株抗體製備 78
3.6.1 抗原準備與小鼠免疫 78
3.6.2 融合瘤細胞株建立與單株抗體篩選 79
3.6.3 抗體純化與效價測試 80
第四章 討論 81
4.1 In vitro SUMO化與去SUMO化系統能作為有用之研究工具 81
4.2 SUMO-1能於In vitro SUMO化系統中產生聚合現象 82
4.3 SUMO化共有基序對SUMO聚合反應之影響 83
4.4 SENP1對不同種類SUMO之選擇性 85
4.5細胞中SUMO-1與SUMO-2對目標蛋白質之共同修飾現象 86
第五章 未來展望 88
圖與表 90
參考文獻 119

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