在大腸桿菌中表達具有功能性的果糖轉移酶

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Title:	在大腸桿菌中表達具有功能性的果糖轉移酶 Funtional expression of fructosyltransferase from Aspergillus in E.coli
Authors:	姚怡杏
Contributors:	嘉南藥理科技大學：生物科技系暨研究所江建民劉坤湘
Keywords:	水溶性表達重組蛋白果糖轉移酶 recominbinant protein soluble expression fructosyltransferase
Date:	2009
Issue Date:	2011-10-27 14:43:02 (UTC+8)
Abstract:	來自麴菌的果糖轉移酶 (FT，fructosyltransferase) 可將蔗糖反應生成蔗果三醣或蔗果四醣等果寡糖。本篇研究將麴菌的果糖轉移酶基因轉殖入大腸桿菌，藉由原核系統來表達果糖轉移酶。使用大腸桿菌生產重組蛋白時，常發生三級結構折疊錯誤而形成胞涵體，導致水溶性蛋白質的產率降低的現象。我們藉由降低誘導溫度，避免因蛋白質生產速度過快，及利用T7 tag 及蛋白麥芽糖鍵結蛋白質 (maltose binding protein，MBP)來做為融合伴，可表達出水溶性之果糖轉移酶。而且 MBP 更使得表達的重組果糖轉移酶較不易聚集沉澱。當利用共表達分子伴侶 (chaperone) 的方式來改善水溶性蛋白質的表達時，DnaK-DnaJ-GrpE (簡稱KJE)及GroEL-GroES (簡稱ELS) 系統都無法提高FT 的水溶性表達。補充較不常使用的tRNA 時，也無法提高水溶性FT 的表達。當將培養基改成 ZYP，且使用乳糖為誘導劑時，可以有效增加果糖轉移酶的水溶性表達量。表達出的果糖轉移酶可經由鎳管柱快速純化。本研究結果顯示能快速獲得大腸桿菌表達的果糖轉移酶，可供未來在結構及功能方面深入研究。 Abstract Fructosyltransferase (FT, sucrose fructosyltransferase) from Aspergellus joponicus catalyzes the reaction of the transfer of fructosyl group and causes the formation of fructooligosaccharide. The functional expression of frcutosyltransferase in E. coli was reported in this study. To overcome the misfolding and inclusion body formation that cause the lower yield of soluble protein, the T7 tag and MBP fusion were fused with the ORF FT gene and reduced expression temperature were performed. The soluble form recombinant FT could be obtained. The MBP fusion further prevented the FT from aggregation and precipitation. To investigate the effect of chaperones, E. coli KJE and ELS systems was coexpressed. The results show that both systems have little effect on the soluble expression of FT. The supply of rare tRNA show little effect in increasing soluble expression of FT either. The soluble expression of FT were significantly increased when ZYP-505 medium and lactose as inducer were used. The expressed FT can be quickly purified by Ni+2 column. This method can facilitate the fruther study in structure-function relationship. Keywords: fructosyltransferase, recominbinant protein, soluble expression
Relation:	校內校外均不公開，學年度：97， 90 頁
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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