利用再折疊法製備Pseudomonas syringae VasH重組蛋白並探討其結構與活性分析

Recombinant protein preparation by refolding to study the structure and activity of Pseudomonas syringae VasH

許之騰(Chih-Teng Sheu)

指導教授 ： 林乃君

共同指導教授 ： 徐駿森

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10876

摘要

重組蛋白 (recombinant protein) 表現在現代的生技產業是不可或缺的技術。其中，利用大腸桿菌作為宿主的優勢是可以低成本地獲得大量的重組蛋白。但是在過程當中，許多重組蛋白因為折疊錯誤而形成不具有活性的不可溶沉澱，稱之為包涵體 (inclusion body)。再折疊法 (refolding) 則是利用離散劑 (chaotropic agent) 將折疊錯誤的蛋白質沉澱變性而溶解，在去除離散劑之後，使蛋白質折疊成正確的構型。VasH 在細胞中扮演依賴 sigma factor 54的轉錄調控因子，並且發現其參與 Pseudomonas syringae 第六型分泌系統 (Type VI secretion system) 分泌蛋白 Hcp2 之轉錄，但是以大腸桿菌做為宿主進行重組蛋白 His-VasH 表現時卻都是位於不可溶的包涵體。本篇選擇以快速稀釋法 (rapid dilution) 先將變性蛋白質滴入含有精胺酸 (arginine) 的再折疊緩衝溶液 (refolding buffer)，再將大體積的再折疊緩衝溶液通過鎳親和性管柱 (Ni2+-NTA column) 以捉住其中的 His-VasH 重組蛋白，最後以含有咪唑 (imidazole) 的溶液流洗出以達到濃縮的效果。首先，我們以生物資訊學的方式了解 VasH 為一種 AAA protein (ATPase associated with diverse cellular activities)，具有典型的AAA功能區塊並且歸類於第三群細菌增強子結合蛋白 (bacterial enhancer binding protein)。由於過去研究發現再折疊緩衝溶液中的精胺酸將影響蛋白質與管柱的結合，所以我們首先以綠色螢光蛋白 (eGFP) 在含有不同濃度精胺酸緩衝液中與管柱結合，以肉眼觀察綠色螢光蛋白與鎳親和性管柱的結合情形和計算蛋白質回收率，得知鎳親和性管柱可容許的精胺酸濃度為100 mM。接著，將不同量的蛋白質加入等體積的再折疊緩衝溶液中，以肉眼觀察沉澱與光度計測定OD600決定稀釋比例，發現每100 mL 的再折疊緩衝溶液可以容納2 mg 的蛋白質進行再折疊。最後，我們對以此方法製備出的 His-VasHAAA 與可溶的 MBP-VasHAAA 進行活性分析，證明此方法可以製備出具有活性的 VasHAAA，而 MBP 也確定能夠在細胞中幫助 VasHAAA 蛋白質正確折疊成有活性的構型。透過本論文的研究，對於未來製備 VasH 重組蛋白進行結構分析有所助益。

關鍵字

再折疊法 ； 重組蛋白

並列摘要

Heterologous expression of recombinant proteins has become a powerful tool in the field of medicine and biotechnology. Escherichia coli is one of the most favorable host for recombinant protein expression due to its rapid, robust and economic advantages. However, many exogenous proteins may not be folded correctly in E.coli. Those mis-folded proteins become insoluble and precipitate as inclusion bodies. Refolding is a method developed to directly recover proteins from inclusion bodies. The process begins with solubilization of inclusion body with solution containing high concentration of chaotropic compounds, such as urea or guanidinium hydrochloride. The denature proteins undergo refolding while those chaotropic compounds are gradually removed. In this study, we used the AAA domain of VasH (VasHAAA), a sigma 54-dependent transcriptional regulator, of Pseudomonas syringae to demonstrate a simple and convenient refolding method for His-tagged recombinant protein by drip dilution following Ni2+-NTA column purification. Bioinformatics research of VasH in this study provides essential understanding of this AAA protein. Sequence alignment with other bacterial enhancer binding proteins reveals that VasH possesses common features of most AAA proteins and is classified in group III. From structural modeling, interaction between ATP and AAA domain of VasH suggests that VasH is a functional ATPase. We had determined the arginine concentration compatible for Ni2+-NTA column is 100 mM, and maximum protein accommodation for 100 mL refolding buffer is 2 mg. Finally, activity of refolded His-VasHAAA is verified and MBP is proven to enhance protein folding to improve recombinant protein solubility. This study provides reasearchers a feasible method to prepare recombinant VasH for further investigation.

並列關鍵字

refolding ； recombinant protein

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