Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23980
標題: 水溶性XCC菌纖維分解酶基因選殖及表達 與放線菌轉麩胺醯胺酶的純化與結晶
Molecular cloning and protein expression of soluble cellulases from XCC and preliminary crystallization study of transglutaminases from Streptomyces kentuckense
作者: 江厚德
CHIANG, HOU-TE
關鍵字: cellulases;纖維分解酶麩胺醯胺酶;transglutaminases
出版社: 生物化學研究所
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摘要: 
十字花科等農作植物"黑腐"症係由Xanthomonas campestris pv. campestris(簡稱XCC)病原菌所造成,而病原菌能附著及穿透宿主組織(colonization)為感染機制的第一步,目前已知XCC的纖維分解酶(engXca, engXcb)與致病性有關,由XCC菌全基因體的定序結果顯示尚有7個似為纖維分解酶基因,代號分別為XCC0026、XCC0027、XCC0028、XCC1752、XCC2387 (為engXcb)、XCC3380、XCC3381、XCC3535、及XCC3521 (為engXca)。本研究旨在以分生選殖技術篩選及表達高水溶性的纖維分解酶,做為後續分子結構及功能的研究。實驗進行係以XCC0028及XCC3381為目標,蛋白質全長分別為317 a.a.(35 KDa)及221 a.a.(24 KDa),轉殖入具有高水溶性的融合蛋白GST, Nus, Trx, MBP等載體,增加其於菌體表達的水溶性。含上述融合蛋白的纖維分解酶XCC0028具有水溶性,DNS活性分析測試酵素活性不明顯,而含有Trx融合蛋白的XCC3381需要加入Urea增加水溶性。
轉麩胺醯胺酶(Transglutaminase簡稱TGase)催化麩醯胺酸(glutamine)與離胺酸(lysine)形成鍵結,使組織凝聚形成穩定的屏障,例如皮膚、頭髮、及凝血因子VIII等,在工業界常用於食品加工,增加製品的稠度及口感。本研究計畫前期旨在純化及結晶條件測試Streptomyces kentuckense菌的TGase,apo形式的TGase全長為517個胺基酸(57.4 kDa)。實驗進行係在大腸桿菌共同表達Trx融合蛋白的apoTGase與蛋白酶(TVMV),而後N-端的apo片段經TVMV切除後,再以Ni-NTA及15S-sepharose陽離子樹酯純化,並濃縮至24.8 mg/ml。初步使用沉澱劑MME550-2000,PEG400-8000等及配合各種緩衝溶液測試結晶條件後,可以觀察到proTGase會形成結晶的表徵。

Xanthomonas campestris pv. campestris (XCC) is the causative agent of black rot disease in crucifers, including cabbage, broccoli, cauliflower etc., resulting in serious agricultural yield loss worldwide. Invasion and colonization of most cruciferous plants by Xcc is accomplished through natural openings and plant tissue lesion. The ONSA/FAPBSP/Brazil group's study on the genomic sequence of XCC strain 33913 annotates nine putative cellulose genes related to plant pathogenicity (XCC0026, XCC0027, XCC0028, XCC1752, XCC2387 (engXCB), XCC3380, XCC3381, XCC3535, XCC3521 (engXCA)). In this study, we attempt to express soluble proteins two cellulose genes, 0028 and 3381, for functional and structural characterizations. The XCC0028 fused with -GST, -Nus, -Trx, or -MBP tag protein on the N-terminus can be expressed in soluble form. The DNS assay of these proteins did not show detectable enzymatic activity toward carboxymethyl cellulose. The Trx fusion protein of XCC3381 was expressed in aggregation but became soluble in 1.5 M urea solution.
Transglutaminases (EC 2.3.2.13) catalyzes the cross-linking of proteins by formation of a covalent bond between glutamine and lysine residues. This enzyme has been extensively used in food processing industry to improve the texture of meat product. The apo-transglutaminases (apoTGase) from Streptomyces kentuckense was expressed in E.Coli and purified to homogeneity for structural and functional study . The apoTGase containing a Trx fusion tag and TVMV protease cutting site on the N-terminus and TVMV protease were co-expressed in E. Coli strain BL21*(DE3). Then, the enzyme was purified by using Ni-NTA affinity and 15S cation-exchange chromatography, dialyzed and concentrated to 24.8 mg/ml. Preliminary screen of crystallization conditions by using various precipitants and biological buffers indicated formation of crystalline precipitations in PEG8000 solution. Further fine-tune crystallization condition to grow single crystal for X-ray diffraction study is achievable.
URI: http://hdl.handle.net/11455/23980
其他識別: U0005-0707200812275200
Appears in Collections:生物化學研究所

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