臺灣博碩士論文加值系統

重组蛋白的分離與纯化一直是生物化學研究中所面臨的一大困難，利用常規的蛋白質純化技術，例如：親和層析(Affinity chromatography)，不僅成本高，耗時長，純化效率又不高，因此，發展一套簡單易操作之蛋白質純化之技術，為本研究之首要目標。
類彈性蛋白(elastin-like polypeptide, ELP)標籤，被視為最具有前瞻性的重组蛋白纯化標籤。而類彈性蛋白主要是由五肽重複序列單元(VPGXG)串聯组成，具有溫度誘導的可逆相變特性。不僅ELP本身具有這個特性，當ELP與目的蛋白融合後，也會赋予重组蛋白有相類似的可逆相變性質。因此，藉由多次可逆相變循環(inverse transition cycling, ITC)之後，就可以從蛋白質粗萃取混合液中選擇性地分離出ELP融合蛋白(fusion protein)，再經特異性酶切或者改變環境條件而引發内含肽(Intein)發生自我剪切去除ELP標籤，從而得到單一的目的蛋白，從而實現簡單快速分離纯化重组蛋白。目前，利用類彈性蛋白結合內蛋白子之技術，已成功應用於原核大腸桿菌和植物表達系统中。而這種利用類彈性蛋白標籤達到分離纯化重组蛋白的方法，具有技術簡單、操作快速、成本低、易於擴大等優點。
本研究主要是藉由基因轉殖技術來改善重組蛋白分離與純化之問題，利用ELP標籤來達到回收與純化之目的，而本研究分為兩部分，第一部分為利用雙質體系統同時表面展示(Surface display)ELP及OPH，利用ELP標籤來回收外膜囊泡，並利用OPH活性來判定是否回收成功;第二部分為分別建構並表達三個酵素系統，使其彼此間專一性的結合成一巨大複合體後，利用ELP來純化此複合體，最終達到回收三個酵素之效果。

The separation and purification of recombinant proteins has been a difficult issue in the field of biochemistry for a long time, The use of conventional protein purification techniques such as affinity chromatography, is not only high cost and time-consuming, the purification efficiency is also not high. Hence, the development of a simple protein separation and purification technique is the primary goal.

Elastin-like polypeptide (ELP) tag is an extremely promising recombinant protein purification tag. The artificial polypeptide consists of repeats of the penta-peptide (VPGXG) with a reversible phase transition at a specific temperature. ELP fusion protein, a target protein fused to the ELP at the gene level, equally retains this behavior. Hence, after isolating a recombinant ELP fusion protein from the protein solution by several runs of inverse transition cycling (ITC), the ELP tag can be proteolytically removed, or be removed through intein-mediated self cleavage which responds to condition shift in the solution, and then the pure target protein can be recovered. At present, the method is used successfully in expression systems such as E.coli and plants. This non-chromatographic protein purification method is simple, rapid, cost- effective, and easy to scale up.

In this study, genetic engineering techniques are used to improve the purification of recombinant proteins, specifically, the utilization of ELP tag to achieve the aim of recovering and purifying the proteins. This study consists of two parts. The first part is to surface display both ELP and organophosphorus hydrolase(OPH) by using a two-vector system, then recover the outer membrane vesicles through the ELP tag, and make use of the reactivity of OPH to determine the success of the recovery in the end. The second part is to construct and express three enzymes respectively, then by making use of their specificity to each other, combine them into a huge enzyme complex. This enzyme complex is purified through the ELP tag and the three enzymes are recovered eventually.

摘要 iii
Abstract iv
致謝 v
總目錄 vi
表目錄 x
圖目錄 xi
縮寫表 xiv
第一章 緒論 1
1.1 研究背景 1
1.2 研究動機與目的 2
1.3 研究內容 2
第二章 文獻回顧 5
2.1 蛋白質的純化方法 5
2.1.1 根據蛋白質溶解度不同而分離 5
2.1.2 根據蛋白質分子大小的差別而分離 5
2.1.3 根據配體特異性親和力而分離 6
2.2 類彈性蛋白(Elastin-like-polypeptide, ELP) 7
2.2.1 ELP之相變原理 8
2.2.2 ELP之純化方法 9
2.2.3 ELP標籤之選擇 10
2.2.4 ELP標籤的切除方法 11
2.3 外膜囊泡(Outer membrane vesicles, OMVs) 11
2.3.1 OMVs的結構 12
2.3.2 OMVs的應用 13
2.4 有機磷水解酶(Organophosphorus hydrolase, OPH) 13
2.5 纖維素水解酵素(Cellulase) 14
2.6 蛋白質交互作用 15

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