建構以磁性粒子為基礎之修飾方式及其在蛋白質純化之效率探討

Abstract

蛋白質純化為生物科技領域中重要的一環，儘管至今已經發展出多種純化蛋白質與前處理的方式。但透過管柱純化會稀釋蛋白質濃度及增加溶液體積，故本研究以矽氧烷(-NCO基團或maleimide基團)及鄰苯二酚基團在Fe3O4上修飾NTA ，另製備鎳鐵粒子，得到具有結合his-tagged蛋白質能力之磁性粒子來純化蛋白質，並嘗試以His-EAAAR序列一鍋純化成不具任何修飾之蛋白質。 本研究藉由IR光譜上1630 cm-1附近(C=O stretching)的穿透度變化來判斷修飾Fe3O4是否成功。再以動態光散射儀測量Fe3O4及鎳鐵粒子之平均粒徑：得知矽氧烷修飾Fe3O4平均粒徑約50 nm； dopa修飾Fe3O4平均粒徑則約750 nm；鎳鐵粒子平均粒徑為770 nm。最後透過XRD鑑定鎳鐵粒子之成分，與資料庫比對後發現其為Ni•Fe3O4。 使用his-tagged eGFP乃因其在波長488 nm有易於觀察之吸收峰，可用比爾定律測量濃度定量。最初我們以磁珠釋放/收集設備作為自動化純化工具，但發現會造成磁珠大量耗損且有最小體積的限制，決定改以人工進行純化程序來克服這些問題。從結果發現透過矽氧烷修飾之粒子較有競爭力，NTA-NCO-SiO2@Fe3O4重複使用四次時能維持約83% 的回收率，而NTA-maleimide-SiO2@Fe3O4擁有最大洗脫量(39.3 mg/g粒子)。NTA-dopa@Fe3O4因為NTA過密集或鍵結不夠穩定導致回收率低及容載量衰退快。而鎳鐵粒子與his-tagged鍵結穩定，無法洗脫出目標蛋白，但仍可用來作為酵素固定化之平台。 我們選擇NTA-NCO-SiO2@Fe3O4純化eGFP以外的蛋白質，分別是cAmp scfv(14 kDa)、myostatin(28 kDa)及Anti-ubiquitination protein(74 kDa)，皆有十分卓越的表現，與商品化之NTA管柱相比，大分子量蛋白質不會影響其純化純度。 同時我們結合了NTA磁珠與自斷裂胜肽建構一鍋自斷裂純化系統。令eGFP固定在磁珠上後發生自斷裂，使eGFP游離。因NTA-maleimide-SiO2@Fe3O4具最大容載量，故做為自斷裂純化平台。最終取得純度95%之eGFP。

Protein purification is an important issue for the life science development. There are many methods have been developed to purify protein, such as ammonium sulfate precipitation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, etc. However, all these approaches will dilute protein into a lower concentration and increase the volume of sample. That is not ideal for protein preparation. To solve those disadvantages, we design a new approach to purify His-tag protein with magnetic nano-particles. In our studies, we use NTA as a ligand to modify magnetic ferrite nanoparticles surface by silane and catechol methods. In addition, we also prepare nickel-ferrite particles for other chooses. Consequently, these particles (NTA-SiO2@Fe3O4 and NTA-maleimide-SiO2@Fe3O4,) can be utilized to purify and concentrate His-tag protein by selective bind. This efficient protein purification process reveals a highly recoveries rate (>80%) and the maximum binding capacity of all particles is around 39.3 mg/g (GFP-His6 protein/particles). In addition, we found that the binding between nickel-ferrite particles (Ni/Fe3O4) and His-tag protein is irreversible. That indicates a nickel-ferrite particle is potentially a good platform for enzyme immobilization.