於Escherichia coli.表達具有功能性Streptomyces mobaraensis之轉醯基酵素

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Title:	於Escherichia coli.表達具有功能性Streptomyces mobaraensis之轉醯基酵素 Functional expression of transglutaminase from Streptomyces mobaraensis in Escherichia coli
Authors:	羅翊心 Yi-Hsin Lo
Contributors:	江建民嘉南藥理科技大學：生物科技研究所
Keywords:	麩醯基轉移酵素蛋白純化大腸桿菌 Escherichia coli protein purification transglutaminase
Date:	2008
Issue Date:	2008-12-29 15:24:01 (UTC+8)
Abstract:	Transglutaminase (TGA) 可以將蛋白質上的麩醯胺酸 (Gln) 和離胺酸 (Lys) 殘基側鏈以共價鍵連結在一起，目前在食品、生醫、製藥等領域都廣泛的被應用。在此篇研究中，我們利用大腸桿菌以水溶性的方式表達放線菌中的 TGA 基因。本篇研究使用了兩種載體，一種是 pET21b，為胞內表達型式；另一種為pETYebF，為胞外表達型式。為了探討 pro-region 及其前方加上 T7-tag 對於表達的影響，而分別做出三種 insert genes (PTGA 、T7PTGA 、T7ΔPTGA)。在胞內表達型式方面，PTGA 可以被成左漲b胞內以水溶性表達，但純化後會很快聚集沉澱，而 T7PTGA 更可以較短時間成孕H水溶性在胞內表達，而且純化到的 T7PTGA 並不會沉澱，也可以在 4℃ 保存達 6 個月以上。而以 YebF-PTGA 型式表達時，並無法有效被分泌到胞外，多數都以胞涵體的形式存在，而胞外部份經活化後並無明顯活性。以 LB 培養基、在 25℃、0.1mM IPTG 誘導時，所獲得的 T7PTGA 經 dispase 活化後可達 62U/mL；以ZYP 培養基及自動誘導 (autoinduction) 方式所表達的 T7PTGA，經 dispase 活化後活性更可達 140U/ml。這是目前表達重組 TGA 蛋白相當高量的研究報導，可以提供一個不須要進行蛋白質再折疊的方式進行高效的突變研究。 Transglutaminases (TGA) are enzymes that catalyze the acyl-transfer reaction between the γ-carboxyamide group of peptide or protein-bound glutaminyl residues, and primary amines. Recently, the protein was wildly used in food, biomedicine and pharmaceuticals. In this study, we expressed the TGase from Streptomyces mobaraensis in soluble form in Escherichia coli. Two vectors were used, pET21b for intracellular expression and pETYebF for extracellular expression. In order to evaluate the effects of pro-region and T7-tag, three insert genes, PTGA, T7PTGA, and T7PTGA, will amplified by PCR and constructed into relative vectors. The PTGA was expressed in soluble form intracellularly in E. coli. However, it precipitated soon after Ni2+ column purification. T7PTGA was expressed more efficiently than that of PTGA, and it did not precipitate after Ni2+ purification. It can be stored 4℃ for at least 6 month. YebFPTGA was expressed not only in periplasm but also in inclusion bodied. The extracellular broth did not clearly detect the TGA activity. When culture in LB, 25℃, induced by 0.1mM IPTG, 62U/ml TGA activity can be achieved after dispase activation. When culture in ZYP and autoinduction condition were applied, T7PTGA expressed more efficiently and 140U/ml TGA activity can be achieved. This procedure provide an easy way to acquire a large quantity of TGA and it enable the high-throughput screening of mutant libraries without the restraint of refolding.
Relation:	校內校外均不公開
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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