人類生長激素受體細胞膜內部份的純化與結構探討

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池兆為(Chao-wei Chih) 查詢紙本館藏

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化學學系

論文名稱

人類生長激素受體細胞膜內部份的純化與結構探討
(Purification and crystallization and structure invesigation of growth hormone receptor cytoplasmid domain)

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摘要(中)

因應少子化的發展，人們對於子女的生長發育愈趨重視，與生長發育相關的各種罕見疾病的治療，也漸漸發展起來。然而促進細胞生長與癌症息息相關，如何在其中取得平衡變成一個重要的課題。
人類體內有一種名為生長激素受體(growth hormone receptor)的物質，是影響生長發育的一種重要的蛋白質，主要接受賀爾蒙生長激素(growth hormone)的訊息，並進一步影響細胞的增殖和生長。雖然近年來對生長激素受體的研究頗豐，但大部分都集中在細胞膜外部分的研究，追根究底，這是由於細胞膜內的空間結構尚未被科學家解開，要知道蛋白質結構決定了蛋白質的功能，無法解出其結構就很難更深入研究該蛋白質的功能。
有鑒於此，本實驗室研究的對象為人類生長激素受體細胞膜(cytoplasmic domain)內的區段。透過對對GHR細胞膜內的部分進行表現以及純化，進而解出其蛋白質結構，同時進一步了解GHR細胞膜內區段與Janus kinase 2的作用以及GHR自身降解的機制。藉此與細胞膜外的結果相對照，找出人類生長激素完整的訊息傳遞機制。
由於研究蛋白質首先必須先將將蛋白質表現及純化，本實驗室利用基因工程的方法將細胞膜內生長激素受體的基因C端修飾六個組胺酸(histidine)，稱為his-tag，利用含鈷(Co2+)離子的樹脂與his-tag形成配位鍵，以達到與其他蛋白質分離的效果，再利用膠體過濾管柱(gel filtration column)做第二階段的純化分離，達到最終純化的效果，且蛋白純度高達98%以上。
本實驗最終想透過蛋白質結晶，從而得到蛋白質的立體空間的結構，以便提供對蛋白質酵素功能與活性的更完整的資訊。此研究結果初步發現結晶的條件，此結果有利於後續蛋白質單晶成長條件的建立，同時幫助解開細胞膜內生長激素受體結構，使我們對於生長激素受體的研究能有進一步的發展。

摘要(英)

In recent years, the research combined protein function and structure analysis has become a famous area in the proteomics field. Through the protein crystallization method, high resolution of protein structure is able to be determined so as to provide important information relative to function and activity of enzyme. Currently, by the use of genetic deletion, insertion and point mutation methods to manipulate protein function is the major way to understand the relationship between protein function and structure in enzymology science. However, how to obtain a good quality of protein crystal is the most difficult issue in the protein structure determination.
Herein, our study is investigating the cytoplasmic region of human growth hormone receptor, and this part contains three important structural and functional sequence motifs. First one is proline-rich motif called Box 1, which is critical for association with JAK2. Second one is six tyrosine resides region playing important signaling transduction pathway through GH-induced phosphorylation; Last one is UbE (ubiquitin-dependent endocytosis) motif and it mediates GH-induced GHR ubiquitination.
Since the outer membrane region structure of growth hormone receptor had been reported, interactions between the outside structure of growth hormone receptor and hormone have been well known. In order to establish the signal transduction pathway of growth hormone receptor function from outer to inner membrane, therefore our experiment is focusing on structure determination of the cytoplasmic region of Growth receptor hormone through high quality of protein crystals and X-ray crystallography methods. According to protein structure information, the details of growth hormone induced signal transduction pathway in inner membrane are able to be completely understood. eg, GHR ubiquitinated mechanism, proliferation and and cell differentiation and diseases related to GHR disorder. .
Here we report that the cytoplasmic region peptide of Growth hormone receptor with over 95% purity by using Co2+ metal affinity resin interacting with six histidines on C-terminal of GHR and then applying to Gel filtration column filtering by different molecular weight. Interestingly, the result SDS-PAGE gel of gel filtration column shows that most growth hormone receptor forms dimer and tetramer complexes.
In addition, the result of crystallization is a good start to determine protein structure of the human growth hormone receptor cytoplasmic region, although crystal forms are not single crystals s. In the future, our studies will concentrate on finding better growth conditions for single protein crystallizations so that the determination of GHR protein structure can be achieved.

關鍵字(中)

★ 快速蛋白質液相層析儀
★ 生長激素
★ 金屬親和性樹脂
★ 生長激素受體
★ 萊倫氏症候群

關鍵字(英)

★ IPTG
★ IMAC
★ growth hormone receptor
★ visceromegaly
★ Box 1
★ growth hormone
★ FPLC

論文目次

中文摘要-------------------------------------------------------------------------------------------i
英文摘要-------------------------------------------------------------------------------------------ii
目錄-----------------------------------------------------------------------------------------------iv
圖目錄---------------------------------------------------------------------------------------------vi
第一章緒論-------------------------------------------------------------------------------------1
1-1 何謂生長激素---------------------------------------------------------------------------1
1-2 GHR的發現------------------------------------------------------------------------------2
1-3 GHR序列-----------------------------------------------------------------------------------2
1-4 GHR病理角色-------------------------------------------------------------------------------3
1-5 GHR的訊息傳遞路徑-------------------------------------------------------------------------3
1-6 研究重點----------------------------------------------------------------------------------5
第二章研究目的---------------------------------------------------------------------------------7
第三章實驗原理方法與設備材料-------------------------------------------------------------------9
3-1 細胞轉化----------------------------------------------------------------------------------9
3-2 蛋白質的表現與放大------------------------------------------------------------------------12
3-3 利用IMAC(Immobilized Metal Affinity Chromatography)純化蛋白-----------------14
3-4 凝膠電泳(Gel Electrophoresis)-------------------------------------------------------------16
3-5 利用快速蛋白質液相層析儀(FPLC)搭配膠體過濾管柱(Gel Filtration)純
化蛋白--------------------------------------------------------------------------------19
3-6 蛋白質膠內酵素水解(In Gel Digestion:LC-MS/MS前處理)-------------------------------------21
3-7 液相層析串連式質譜儀(Liquid Chromatography-Mass Spectrometry /
Mass Spectrometry，LC-MS/MS)----------------------------------------------------------22
3-8 西方點墨法(West Blotting)-----------------------------------------------------------------25
3-9 二維膠體電泳---------------------------------------------------------------------------25
3-10 蛋白質結晶原理與步驟---------------------------------------------------------------27
第四章實驗結果---------------------------------------------------------------------------------30
4-1 第一階段純化------------------------------------------------------------------------------30
4-2 LC-MS/MS鑑定結果-----------------------------------------------------------------------30
4-3 第二階段純化(Gel Filtration Column)-------------------------------------------------------32
4-4 生長激素受體加入DTT測試-------------------------------------------------------------------34
4-5 生長激素受體結晶結果----------------------------------------------------------------------35
第五章總結--------------------------------------------------------------------------------------37
第六章參考文獻----------------------------------------------------------------------------------38
第七章附錄--------------------------------------------------------------------------------------41

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