Application of bionanotechnology to study self-assembled proteins
G. Steven Huang
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.
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