標題: 利用奈米生物科技研究自組裝蛋白質
Application of bionanotechnology to study self-assembled proteins
作者: 林志杰
ChieChieh Lin
黃國華
G. Steven Huang
材料科學與工程學系奈米科技碩博士班
關鍵字: 奈米生物科技;自組裝;蛋白質;高效液相西層析儀;核醣體;bionanotechnology;self-assembled;protein;HPLC;ribosome
公開日期: 2006
摘要: 第一部份: 科學家對蜘蛛絲有極大的興趣,因其具有相對人造絲更良好的機械特性。本實驗取出人面蜘蛛絲主絲囊的蜘蛛絲蛋白質,並研究出可穩定保存蜘蛛絲蛋白質的保存液。更進一步利用蜘蛛絲蛋白質混合有機溶液來達成自組裝成絲狀及網狀的結構。 第二部份: 我們利用高效能液相層析儀 [ high-performance liquid chromatography (HPLC) ] 配合膠體過濾法來純化並分析核醣體。粗粹取物利用大腸桿菌 XL1-Blue經由超高速離心來得到核醣體。大腸桿菌粗粹取物70S 核醣體於高效能液相層析儀分析出單一峰值,核醣體是由粗粹取物經高速離心純化而得到。而高效能液相層析儀純化之核醣體之雜質較高速離心純化得到核醣體之雜質為少。經由高效能液相層析儀純化之核醣體吸光值比值 ( 260nm/280nm )可達2.31,而高速離心純化得到核醣體之只有1.92。當環境溫度為42℃時,經由高效能液相層析儀純化之核醣體較高速離心純化得到核醣體來的穩定。高效能液相層析儀也偵測50S和30S的結合,也經由穿透式電子顯微鏡證實,而這些變化應為缺少鎂離子所造成。目前的研究提供了一個核醣體純化和結構變化分析的平台。
Part I: Spider silks have extraordinary mechanical properties. Even man-made fibers cannot achieve them. In this study we collected major silk gland from Nephila pilipes. Found out the stable buffer to storage spidroin. Application of spidroin with organic solvents to induce self-assembled silk-like and net-like structures. Part II: We established a high-performance liquid chromatographic (HPLC) system with gel filtration column for the purification and analysis of ribosomes. Crude extract was prepared from Escherichia coli XL1-Blue and went through ultra-centrifugal procedure to obtain ribosomes. 70S ribosome appeared as single peak in the HPLC chromatogram analyzing E. coli crude extract. Ribosomes were purified from crude extract and from ultra-centrifugation prep. HPLC-purified ribosomes contained fewer impurities than ribosomes prepared from ultra-centrifugal procedure. OD 260nm/280nm ratios of HPLC-purified ribosomes achieved 2.31, while the best ultra-centrifugal prep was only 1.92. When incubated at 42 °C, HPLC-purified ribosomes presented improved stability compared to conventionally purified ribosomes. This HPLC system was also capable of detecting conformational change of ribosomes. In the absence of magnesium ion, HPLC detected aggregation of 50S and 30S subunits which were confirmed by electron microscopy. A higher order of conformational reorganization might have occurred in the absence of magnesium ions. The current study provided an alternative platform for the purification and conformational analysis of ribosomes.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT009452512
http://hdl.handle.net/11536/82019
Appears in Collections:Thesis


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