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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題名:	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)
作者:	陳盈安;Chen,Ying-an
貢獻者:	化學研究所
關鍵詞:	半乳糖凝集素;生長激素;galectin;galectin-12;GHR;growth hormone receptor
日期:	2012-08-27
上傳時間:	2012-09-11 18:38:04 (UTC+8)
出版者:	國立中央大學
摘要:	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。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.
顯示於類別:	[化學研究所] 博碩士論文

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