鳳梨莖部凝集素融合蛋白功能性表現與活性分析

凝集素廣泛存在各種生物體內，具有高度醣類結合專一性之蛋白質，其稱為外源凝集素、植物凝集素、血球凝集素、親醣蛋白質或醣結合蛋白等。醣類研究是繼基因體時代及蛋白質體學研究後重要的領域之一。凝集素是醣類或醣蛋白質研究中，重要的分析工具，其後續的應用發展持續研究中。而過去研究已由鳳梨莖部選出具葡萄糖結合專一性之凝集素。本研究利用穀胱甘肽S-轉移酶（Glutathione S-transferase, GST) 序列融合鳳梨莖部凝集素基因，將全長基因藉由EcoI及XhoI限制酶切位構築至pGEX-4T-1表現載體，即命名為pGEX-lec。其穀胱甘肽S-轉移酶標記(GST-tag)序列融合鳳梨凝集素，轉型至BL21(DE3)，定序後進行表現蛋白質，進而測試表現蛋白質條件，在溫度、時間及不同濃度IPTG等因素測試，經由實驗證明利用低溫誘導表現有較多水溶性蛋白質，較不易形成內涵體，以利於其後續實驗，利用GST親和力管柱進行純化水溶性蛋白質，並進行紅血球凝集活性等試驗，確認融合蛋白功能性與鳳梨莖部純化之凝集素活性相似。本研究成功建立將重組凝集素蛋白質表達系統；此系統利於後續量化生產及進行更深入的蛋白質功能分析。

關鍵字

凝集素；鳳梨莖部凝集素；穀胱甘肽S轉移酶醣類結合蛋白質

並列摘要

Lectins are carbohydrate-binding proteins that can agglutinate cells and precipitate glycoconjugates. Plants contain the lectins that bind preferentially to mannose or glucose occurred in many monocot or dicot species. The lectins are important tool for the research of carbohydrates and to identify their function in glycobiology. One of the mainly protein in pineapple stem has been identified as a lectin. The lectin denoted as ACL (Ananas Comosus Lectin) has been isolated, purified and cloned from the pineapple stem. The coding region of ACL clone was introduction into expression vector, pGEX-4T-1 with GST-tag fusion, named pGEX-lec and transformed to Escherichia coli BL21 (DE3). Overexpression of the recombinant ACL (rACL) in the lower culture temperature the water-soluble protein of rACL was found. The proteins subjected to a glutathione affinity column to purify the GST-fusion rACL. Western analysis of rACL confirmed that rACL is proper expressed. In this study, rACL exhibited a similar hemagglutation activity to purified native ACL (nACL) that could agglutinate erythrocytes. Functional expression of rACL and establishing the produce system is an important step forward for future study of the ACL.

並列關鍵字

Lectin ； Pineapple stem lectin ； Glutathione S-transferase, GST ； Carbohydrate-binding protein

參考文獻

林志偉, 2009. 鳳梨莖部凝集素純化與特性分析, 光電與材料科技研究所碩士論文. 國立虎尾科技大學

鄒啟煇, 2010. 以石英晶體微天平探討重組鳳梨莖部凝集素醣類結合活性, 光電與材料科技研究所碩士論文. 國立虎尾科技大學

Barre, A., Bourne, Y., Van Damme, E.J., Peumans, W.J., Rouge, P., 2001. Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process. Biochimie 83, 645-651.

Barre, A., Van Damme, E.J., Peumans, W.J., Rouge, P., 1996. Structure-function relationship of monocot mannose-binding lectins. Plant Physiology 112, 1531-1540.

Bourne, Y., Roig-Zamboni, V., Barre, A., Peumans, W.J., Astoul, C.H., Van Damme, E.J., Rouge, P., 2004. The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold. The Journal of Biological Chemistry 279, 527-533.

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鳳梨莖部凝集素融合蛋白功能性表現與活性分析

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