Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21588
標題: Expression of N-acrtyl D-glucosamine 2-epimerase gene in Escherichia coli
大腸桿菌中表現N-acetyl D-glucosamine 2-epimerase基因
作者: 陳淑芬
Chen, Shu Fen
關鍵字: 唾液酸
sialic acid
腎素結合蛋白
包涵體
renin binding protein
N-acetyl D-glucosamine 2-epimerase
inclusion body
出版社: 分子生物學研究所
摘要: Sialic acid唾液酸 ( Sialic acid ) 為含縮基(-COOH) 的九碳醣。存在於動物體內醣蛋白、醣脂質末端上;與細胞間 (cell-cell recognition ) 辨識有相當大的關聯。此特性被應用於新藥之開發,例如感冒病毒抑制劑,zanamivir及消炎藥Sialyl Lewis X之化合物。目前正積極研發以酵素方法生產sialic acid。N-acetyl D-glucosamine 2-epimerase (GlcNAc 2-epimerase) 酵素在sialic acid 合成上扮演一關鍵之角色,此基因大部分來自真核細胞,在E. coli 大量表現後形成包涵體 (inclusion body) 。 本實驗針對包涵體之形成與改善方法做一探討,將GlcNAc 2-epimerase 基因構築於pQE-30轉型入E. coli 菌株中,發現大部分蛋白質皆形成不可溶蛋白質,可溶性蛋白質約佔表現量之20%。嘗試各種方法增進GlcNAc 2-epimerase之可溶性蛋白質產量,利用不同IPTG濃度於28℃與17℃溫度誘導GlcNAc 2-epimerase基因表現,對於可溶性蛋白質並無提增效果。 E. coli菌株中以JM109,XLI-Blue,及Nove Blue對於GlcNAc 2-epimerase基因表現能力較好,但所表現的GlcNAc 2-epimerase可溶性蛋白質無太大差異。為了使GlcNAc 2-epimerase基因表現較多的可溶性蛋白質,在GlcNAc 2-epimerase基因構築於pET32之表現載體,形成thioredoxin-GlcNAc 2-epimerase融合蛋白質,結果顯示thioredoxin 對GlcNAc 2-epimerase 蛋白質可溶性提昇無影響,此融合作用反而使GlcNAc 2-epimerase 活性降低10倍。輔助蛋白質GroES及GroEL與GlcNAc 2-epimerase共同表現時,GlcNAc 2-epimerase之可溶性蛋白質,未隨著GroES及GroEL之表現量增加而有所提增。 由於包涵體形成原因複雜,且對於形成機制尚未明瞭,目前所用之改善方法其效果因蛋白質而異。本研究設計一套簡單、快速、且可回收蛋白質的篩選系統,用來篩選可溶性蛋白質之基因。以5.5mM MgCl2濃度之PCR條件進行error-pone PCR突變GlcNAc 2-epimerase 基因,並將PCR 產物直接選殖於此表現載體上,利用載體上所帶有的篩選標記間接判斷蛋白質之溶解性。利用此系統從中篩選在E. coli 菌體內水溶性較佳又具GlcNAc 2-epimerase 活性變異酵素。
N-acetyl D-glucosamine 2-epimerase (GlcNAc 2-epimerase) has been used for the synthesis of sialic acid. GlcNAc 2-epimerase gene was cloned into pQE-30 and expressed in E. coli in which only 20% of GlcNAc 2-epimerase produced was soluble form. The expression of GlcNAc 2-epimerase gene in E. coli strains JM109, XLI-Blue, and Nova Blue, was higher than that in strain DH5a. IPTG concentration and temperature (28℃and 17℃) had not significantly effect on the expression of GlcNAc 2-epimerase gene. To increase the amount of soluble GlcNAc 2-epimerase, the gene was fused to the thioredoxin gene and expressed in E. coli BL21 (DE3). However, the solubility of GlcNAc 2-epimerase was still not improved. The specific activity of the fused GlcNAc 2-epimerase decreased 10-fold in the crude cell extract. Soluble GlcNAc 2-epimerase was also not increased in the co-expression of GroES or GroEL with GlcNAc 2-epimerase or thioredoxin-GlcNAc 2-epimerase gene. When the sialic acid aldolase gene was fused to N-terminal of GlcNac 2-epimerase gene and expressed in E. col Nova Blue, no GlcNAc 2-epimerase activity was detected in the crude cell extract and all fusion proteins were insoluble. A T-vector was constructed for the rapid and selective screening of the gene which expresses soluble form protein. This vector contained 6x His tag, multiple cloning sites, and LacZ-a fragment under the control of T5 promoter of the pQE-30. Two XcmI sites in multiple cloning sites will facilitate the direct cloning of mutated genes from error-prone PCR.
URI: http://hdl.handle.net/11455/21588
Appears in Collections:分子生物學研究所

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