Purification Procedure of Unknown Biomolecules from Analyte

隨著後基因體時代的來臨，蛋白質成為大家共同極力研究的目標之一。到目前為止，純化與分離蛋白多是使用薄膜分離法、層析法與磁性分離法。蛋白質體學主旨在於製作高親合力之配體或探針能與蛋白質有特異性結合，用於檢測與獲取感興趣之蛋白質。配適體具有極大的潛力作為與目標分子有高度親合力之探針，配適體是一小段單股DNA或RNA序列，透過體外篩選得到與目標物有高親合力之結果，配適體比起抗體好的優點在於其容易生產合成，可重複利用和穩定性佳。 本實驗使用磁性輔助篩選配適體平台(MARAS)，利用旋轉磁場作用簡單且快速的得到配適體，並證明了配適體與未知目標蛋白具有高度的親合力與特異性，顯示可將其應用於未知蛋白純化上。選擇磁性奈米粒子作為磁性載體並固定上經由MARAS所篩選獲得配適體作為生物探針純化未知目標蛋白，並再次利用MARAS作為把未知目標蛋白從磁性載體脫附的工具，固定旋轉磁場強度為14 Gauss、調控旋轉磁場頻率 (5~27 KHz)，並收集從各頻率條件下脫附的未知目標蛋白，經聚丙烯醯胺凝膠電泳分析證明非特異性結合之蛋白可以在低頻率旋轉磁場條件下被移除，而在高頻率 (12.5 KHz) 轉磁場條件下得到未知目標蛋白。最後，將未知目標蛋白進行質譜儀分析，並確認未知目標蛋白質身分為chaperonin。 我們建立了從複雜蛋白質體中純化出未知目標蛋白質的方法，以便日後質譜儀的鑑定。利用此程序可以快速篩選出與未知目標分子具有高度親和力與特異性之配適體，將配適體固定於磁性載體上作為配適體探針，逆向純化出具有潛力且新穎的未知生物分子。With a rapid approach of post-genome era, proteomics becomes one of the focus points in commonly-interested biomedical researches. Nowadays, the characterization of protein expression has been relied on membrane separation, chromatography, and magnetic separation for isolation, purification and separation techniques. Proteomic initiatives is to produce high affinity ligands or probes that specifically recognize protein targets in assays which can be used to detect and purify proteins of interest. Particularly versatile probes with tremendous potential for use as affinity molecules are aptamers. Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on affinity toward a target molecule. Aptamers offer advantages over traditional antibody-based affinity molecules in their ease of production, regeneration and stability. In this study, Magnetic-Assisted Rapid Aptamer Selection (MARAS) was used for ssDNA aptamer selection. MARAS using a rotating magnetic field is relatively easy and scalable without the need for precious time and skill. The ability of aptamers generated by MARAS to bind their target protein with high affinity and specificity was demonstrated. The aptamers were shown to be useful for affinity purification. Aptamers, generated via MARAS, immobilized to magnetic beads were utilized for unknown target protein purification. After incubating the aptamer-magnetic bead and analyte, MARAS was again used for unknown protein purification. The bound mixtures were sequentially subjected to a constant magnetic field strength (14 Gauss) and a series of increasing rotating field frequencies (5, 10, 15, 20, 25 K, and 27 KHz) and the supernatants were collected via magnetic separation. Analyzing collected supernatants using SDS-PAGE, it shows that the unknown target protein can be purified under a rotating magnetic field of 12.5 KHz/14 Gauss. Finally, the identification of the unknown protein was analyzed by MALDI-TOF mass spectrometer. The result shows that the purified unknown protein is chaperonin protein. A novel protocol for the purification of unknown target protein from analyte containing a complex protein composition was developed and demonstrated. This procedure generated aptamers capable of binding the unknown target protein with high affinity and specificity, and the generated aptamer immobilize to magnetic beads can be utilized for the purification of potential and novel unknown biomolecules.誌謝辭-I 摘要-II Abstract-III 目錄-V 圖目錄-VIII 表目錄-X 第一章 緒論-1 1.1 前言-1 1.1.1 蛋白質純化技術-1 1.1.2指數式擴增配體的系統進化技術 (Systematic Evolution of Ligands by EXponential enrichment: SELEX)-2 1.1.3磁性輔助篩選配適體平台 (Magnetic-Assisted Rapid Aptamer Selection，MARAS)-3 1.2 文獻回顧-5 1.2.1 磁性奈米粒子純化分離上的應用-5 1.2.2 Aptamer於純化生物分子之應用-9 1.2.3 核酸配適體蛋白質純化技術與層析、抗體的比較-11 1.3 研究動機-13 第二章 實驗設備、方法及步驟-14 2.1 實驗使用之蛋白來源 (FBS culture medium、FBS medium及No FBS culture medium)-14 2.2 濃縮細胞培養基蛋白-14 2.3製備生物功能性磁性流體試劑-14 2.4 MARAS篩選配飾體-15 2.5 核酸配適體基因轉殖、序列定序及二級結構預測-17 2.5.1聚合酶連鎖反應 (Polymerase Chain Reaction, PCR)-17 2.5.2 DNA 接合反應 (DNA ligation)-18 2.5.3 細胞轉殖 (Transformation)-18 2.5.4序列定序 (Sequencing)-18 2.5.5二級結構預測 (Secondary ssDNA structure prediction)-18 2.6 核酸配適體單股純化-18 2.7以即時定量PCR ( Real-time quantitative PCR) 反向驗證核酸配適體與實驗控制組之結合-19 2.8 製備配適體探針與純化目標蛋白質-20 2.9 聚丙烯醯胺凝膠電泳-21 第三章 實驗結果與討論-24 3.1 使用MARAS從檢體篩選與未知標的蛋白結合之配適體-24 3.2 反向驗證核酸配適體與未知標的蛋白之結合-27 3.3 純化蛋白電泳結果-30 3.4 質譜儀定序分析-32 3.4.1 蛋白質資料庫MASCOT database比對鑑定蛋白質身分-32 第四章 結論與未來研究方向-34 4.1 結論-34 4.2 未來研究方向-34 參考文獻-35 附錄-3