- 學位論文
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
Binding affinity of carbohydrates to the recombinant pineapple stem lectin by quartz crystal microbalance
摘要
凝集素廣泛地存在各種生物體內，為具有高度醣類結合專一性之蛋白質，其被稱為植物凝集素、血球凝集素、親醣蛋白質、醣結合蛋白等。醣類研究是繼基因體時代與蛋白質體學後相當重要的領域之一；凝集素可以做為醣類或醣蛋白研究中重要的分析工具。近年來有相關研究，利用石英晶體微天平(Quartz Crystal Microbalance, QCM)生物感測器分析凝集素與醣類結合活性。鳳梨莖部凝集素(native Ananas Comosus Lectin, nACL)為Jacalin-related lectin，有紅血球凝集活性及Glucose/Mannose結合專一性。本研究利用大腸桿菌大量表現具有His-tag融合的重組鳳梨莖部凝集素(recombinant Ananas Comosus Lectin, rACL)。利用親和力管柱內復性及純化得到具有血球凝集活性之rACL。rACL具有對D-Glucose、D-Mannose、D-Glucosamine、Trehalose、Fructose、Sucrose等醣類結合專一性；其比活性為1.22x105 HAU/mg。每300ml之菌液可回收約18μg之重組蛋白質。以QCM分析rACL與nACL之醣類結合活性及結合動力學。利用Scatchard plot方法得到ovalbumin與rACL的結合Kd值為4.7mM及與nACL的Kd值為1.8mM。本研究建立凝集素分析之QCM醣晶片，經0.1M Formic acid對醣晶片進行再生，可進行二十次以上之分析工作。表現系統的建立也可以幫助我們瞭解鳳梨莖部凝集素結構與功能之關係。而未來利用蛋白質工程，將可進行其醣類結合位置之相關特性探討，進而了解鳳梨莖部凝集素之活性調節與生理功能。而此表現系統的建立也可以用來生產大量之重組蛋白質，並可評估及比較鳳梨莖部純化與重組蛋白質之生產效益。
並列摘要
Lectin is carbohydrate-binding protein 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. One of the predominant proteins in pineapple stem has been identified as a lectin. The lectin designated ACL (Ananas Comosus Lectin) has been isolated and purified from the pineapple stem. A cDNA clone encoding the ACL from pineapple stem has been cloned and sequenced. Overexpression of recombinant ACL (rACL) in the Escherichia coli BL21 (DE3) has been established but the rACL accumulated as inactive inclusion bodies. In this study, on-column refolded His-tagged rACL exhibited a similar hemagglutination activity to purified native ACL (nACL). D-glucose, D-mannose, D-glucosamine, fructose, sucrose and trehalose inhibited rACL-induced agglutination of human erythrocytes. The specific activity of rACL was 1.22x105 HAU/mg. Immobilization of the lectins or carbohydrate ligan on quartz crystal chips was established for analysis of binding affinity of carbohydrates to the rACL or the nACL by Quartz Crystal Microbalance (QCM) biosensor. The QCM analysis confirmed the rACL has identical carbohydrate binding specificity with nACL. The Scatchard anaysis of a glycoprotein, ovalbumin binding with rACL and nACL were determined the dissociation constant Kd to be 4.7mM and 1.8mM by QCM, repactively. Furthermore, the direct functional expression of the recombinant lectin into soluble form and to improve the production efficiency is for future study.