磁性奈米複合材料應用於高效能蛋白質純化

Abstract

本實驗利用共沉澱法(Co-precipitation)製造出具有順磁性之四氧化三鐵奈米粒子，使用sol-gel的方式將Octadecyltrimethoxy silane與 Tetraethyl orthosilicate修飾於奈米粒子表面上。表面修飾過之奈米粒子於500 ℃高溫燒結下會形成孔洞大小均勻一致的孔洞性結構，利用此孔洞結構合成鎳奈米粒子於鐵奈米粒子的外圍。藉由傅立葉轉換紅外線光譜(FTIR)、EDX與SEM、XPS分析、超導量子干涉磁量儀(SQUID)等儀器進行性質鑑定後，確定合成鐵鎳奈米複合材料。

接著利用基因重組工程將前降鈣素(Procalcitonin)轉殖於大腸桿菌並大量表現含有組胺酸標籤(His tag)的粗粹取蛋白質，利用氧化鎳奈米粒子和His tag的親和性鍵結能將目標蛋白質從粗粹取液中分離，最後使用Imidazole將目標蛋白質置換於沖提液中。並利用一連串最佳化的實驗如:Imidazole濃度、NaCl添加的濃度等，試圖建立一個最適純化系統。

本研究所得的鐵鎳奈米複合材料不但成功純化PCT蛋白質且經過六次以上的重複使用後效率並無太大差別，即使與已商業化磁珠比較也在伯仲之間，甚至在蛋白質的純度上有更好的成效。此外也成功純化α-岩藻醣苷酶(α-fucosidase)活性測試之後證實其活性並不會受到奈米粒子的影響，最後利用MALDI-TOF證實所純化的蛋白質的確為PCT和α-fucosidase。

This dissertation is concern about how to synthesis a recyclable magnetic nanocomposite which is decorated by NiO nanoparticles for the purification of His-tagged protein. First, the non-modified Fe3O4 is synthesized via a co-precipitation method. Then the non-modified nanoparticles are coated with densly silica shell by a sol-gel reaction of tetraethylorthosilicate (TEOS). Next step, we employ the same sol-gel reaction to create a mesoporous shell by adding TEOS and OTMS (octadecyltrimethoxysilane) simultaneously. The nanocomposites are calcined at 500℃ to generate mesoporous by removing the long chain part of OTMS.Then we synthesize NiO nanoparticle in the mesoporous shell. Finally, by using FTIR、SEM、TEM and SQUID to detect the nanocomposites.

Then we choose procalcitonin and α-fucosidase as candidates for His-tagged purification process. After optimizing the purification process by changing the imidazole concentration or NaCl concentration we can set up a purification system successfully. By comparing our purification result with other commercial magnetic beads, we can realize that the efficiency of our beads is better than commercial ones either in purity nor recyclable use of purification.