博碩士論文 992203038 詳細資訊




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姓名 陳盈安(Ying-an Chen)  查詢紙本館藏   畢業系所 化學學系
論文名稱 人體突變生長激素受質膜內區段與半乳醣凝集素-12的表現、純化與結晶
(Expression, Purification and Crystallization of Cytoplasmic Region of Human Growth Hormone Receptor Mutant (hGHRm)and Galectin-12 (Gal-12))
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摘要(中) 本研究將針對兩種結構尚未被解出之蛋白質,膜內生長激素受質突變蛋白與半乳糖凝集素-12蛋白,進行純化並結晶,希冀藉此得其結構並推測作用機制,最終運用於生物研究上,以探討此蛋白質對人體的影響。
  人體的生長與GH和GHR的結合有著密不可分的關係。當GHR與GH結合同時JAK2將被磷酸化,此訊息的傳導控制著細胞增生與葡萄糖傳遞,進而影響人體的生長。倘若將DNA基因段1319處的G突變為T,會導致胺基酸序列中第440的位置由Cys轉變為Phe,研究指出此處的突變將會使得侏儒症患者對GH的需求量較低,且由於這個位置與膜內GHR和JAK2的作用位置Tyr469相近,則當Cys轉變為Phe時勢必會造成結合程度的降低,影響STATs的磷酸化傳遞。因此希望能了解在Cys440Phe位置上的變異是否會對於其結構本身造成很大影響進而影響到生長訊息的傳遞。本實驗藉由兩步驟純化方法,固定化金屬親和層析法與膠體過濾層析法進行蛋白質純化,能夠得到最終純度高達95%的目標蛋白質,並將如此高純度的蛋白質繼續進行結晶實驗。
  Galectin-12對於細胞週期以及脂肪分化作用皆具有相當影響力。研究指出當表現量增加會造成細胞週期停止於G1 phase或G1/S之間,導致細胞停止生長。galectin-12基因的缺乏能使老鼠具較高脂肪降解能力和粒線體呼吸作用,進而降低全身油脂量與胰島素抗性。因此galectin-12可望作為抑制腫瘤細胞以及減肥的藥物。本次實驗成功地利用硫酸銨沉澱法進行galectin-12的純化,得到初步純度之蛋白質,接著將此結果經過離子交換法與膠體過濾法,以獲得更高純度,並做後續結晶與生物實驗。
摘要(英) For this study, our goal is trying to purify two unknown structural region of protein:cytoplasmic region of human growth hormone receptor mutant(GHR)and galectin-12(gal-12). In order to understand proteins related information, our experiments are focusing on protein structure determination by the use of high purity proteins crystallization.
Because the wild-type GHR had been successfully purified in previous study, this experiment continues to work on GHR related mutant protein. According to research report regarding to GHR Cys440Phe mutant, it may cause Dwarfism with lower GH demanded. Due to this mutant site located on around active site of cytoplasmic region, point mutation on the site of Cys440 will affect binding affinity of SOCS2 as well as it results in a higher rate of STAT5 phosphorylation. Therefore, we are interested in the investigation of function and structure of GHR Cys440Phe mutant. In order to obtain high purity protein for study, this study used two-step methods, immobilized metal affinity chromatography and gel filtration chromatography, for purification. After two steps protein purification performances, finally purity of GHR mutant is able to reach up to 95% based on densitometry measurement. Interestingly, mostly dimer conformation of GHR bands were found in denature SDS PAGE gel.
In addition, we also perform protein purification of the Galectin-12 (Gal-12)which is considerable influence on the cell cycle and on lipolysis. Recent report pointed out that the Gal-12 with high expression will cause cell cycle arrested in G1 phase or between G1/S phase so as to stop adipocytes growth. As with a potential function for inhibition of tumor cells and regulation of adipocytes. Galectin-12 is good target for the drug development of cancer and anti-obesity. In this study, we are successfully using ammonium sulfate precipitation method for preliminary purification of the Gal-12, and get an apparent purity of the Gal-12. In the future, resultant of ammonium sulfate purification will be applied to gel filtration chromatography column, in order to obtain a higher purity of galectin-12 so that protein crystallization and other biological experiments are able to be performed.
關鍵字(中) ★ 半乳糖凝集素
★ 生長激素
關鍵字(英) ★ galectin
★ galectin-12
★ GHR
★ growth hormone receptor
論文目次 中文摘要 i
英文摘要 ii
謝誌 iv
目錄 v
圖目錄 viii
表目錄 x
第一章 緒論 1
1-1 生長激素受質(Growth Hormone Receptor, GHR)1
1-1-1 認識生長激素 1
1-1-2 生長激素受質的介紹 2
1-1-3 生長激素受質的訊息傳遞路徑 3
1-2 半乳糖凝集素-12(Galectin-12) 5
1-2-1 認識凝集素 5
1-2-2 動物性凝集素的分類 5
1-2-3 Galectins的介紹 7
1-2-4 Galetin-12的介紹 9
1-2-5 細胞週期(Cell Cycle) 9
1-2-6 脂肪細胞的培養與分化 12
第二章 研究動機與目標 17
2-1 膜內生長激素受質突變蛋白 17
2-2 半乳糖凝集素-12蛋白 18
第三章 GHR的表現及純化分離實驗 19
3-1 實驗藥品與儀器 19
3-2 轉形作用(Transformation) 20
3-3 菌株篩選與蛋白質表達(E. coli Cloning and Protein Expression) 21
3-4 蛋白質製備(Cell Lysate Preparation) 22
3-5 蛋白質純化(Protein Purification) 22
3-5-1 固定化金屬親和層析(Immobilized Metal Ion Affinity Chromatog raphy, IMAC) 22
3-5-2 快速蛋白液相層析(Fast Protein Liquid Chromatography) 24
3-6 純化結果分析 26
3-6-1 十二烷基磺酸鈉-聚丙烯醯胺膠體電泳(Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, SDS-PAGE) 26
3-6-2 西方墨點法(Western Blot) 31
3-6-3 圓二色光譜(Circular Dichroism Spectroscopy, CD) 31
3-7 蛋白質結晶(Protein Crystallization) 32
3-8 蛋白質濃度定量 33
第四章 胞內GHR突變蛋白之實驗結果 35
4-1表現結果 35
4-2 純化結果 36
4-2-1 固定化金屬親和層析 36
4-2-2 膠體過濾層析法 37
4-3 蛋白質濃度測定結果 39
4-4 圓二色光譜分析結果 40
第五章 Galectin-12的表現及純化分離實驗 42
5-1 實驗藥品與儀器 42
5-2 轉形作用(Transformation) 43
5-3 菌株篩選與蛋白質表達(E. coli Cloning and Protein Expression) 44
5-4 蛋白質製備(Cell Lysate Preparation) 45
5-5 蛋白質純化 46
硫酸銨沉澱法(Ammonium Sulfate Precipitation) 46
第六章 Galectin-12蛋白之實驗結果 48
6-1 Galectin-12的表現及萃取 48
6-2 純化結果 51
硫酸銨沉澱法 51
第七章 結論 54
7-1 胞內GHR突變蛋白質 54
7-2 半乳糖凝集素-12蛋白質 54
第八章 參考資料 56
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指導教授 謝發坤(Fa-Kuen Shieh) 審核日期 2012-8-27
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