表現血基質運輸蛋白 chuA 以提高大腸桿菌之血紅素重組蛋白產量

Enhancement of Recombinant Human Hemoglobin Production in Escherichia coli containing Heme Transport Gene chuA

陳怡文(YI-WEN CHEN)

指導教授 ： 黃光策

國立中正大學/工學院/化學工程學系/碩士(2016年)

摘要

由於全血輸血受交叉配對、疾病傳播和血液儲存的限制，要開發一個安全和有效的紅血球替代物的是一個巨大的挑戰。利用大腸桿菌作為宿主生產重組人類血紅素的 α 和 β 鏈是目前最具潛力的方式，但血基質不足會使血紅素的結構變得不穩定，並且容易被降解。然而，非致病性大腸桿菌的內源性血基質生產不足以提供重組血紅素的需要，也缺乏將血基質由細胞外輸送至胞內利用的血基質運輸蛋白，為了提高血基質的產量以穩定血紅素，複製大腸桿菌 O157：H7 染色體中之 chuA 基因引入質體 pET-29c 中，之後在質體 pET-Hb（β67W-β95C-synoα-α29F）-MAP 中插入 chuA 基因與 T7 啟動子和終止子建構pET-Hb（β67W-β95C-synoα-α29F）-MAP-chuA，並將之轉型至大腸桿菌 BL21（DE3）和 JM109（DE3）中，能表現 chuA 並增加細菌生長速率。然而引入 chuA 基因後並沒有提高血紅素的表現量。此外，兩種宿主相比，JM109（DE3）重組蛋白表現量比BL21（DE3）佳。

關鍵字

血基質運輸 ； 血基質 ； 血紅素 ； 大腸桿菌

並列摘要

Whole blood transfusions are limited by cross matching, disease transmission and blood storage. To develop a safe and effective red blood cell substitute is being a great challenge. Using Escherichia coli (E. coli)as a host to produce soluble recombinant human hemoglobin alpha and beta chains is attractive. Insufficient heme would let the structure of hemoglobin become unstable and easy to be degraded. However, the endogenous heme production from the non-pathogenic stain of E. coli is insufficient to supply the need for recombinant hemoglobin production. Additionally, the non-pathogenic stain of E. coli lacks of heme transporter that can transport extracellular heme inti cells for utilization. In order to increase the supply of heme and stabilize hemoglobin, we cloned chuA gene from the chromosome of E. coli O157: H7 into plasmid pET-29c and then subcloned the chuA gnen with the T7 promoter and terminator to plasmid pET-Hb(β67W-β95C-synoα-α29F)-MAP becoming pET-Hb(β67W-β95C -synoα-α29F)-MAP-chuA. When transformed into E. coli BL21(DE3) and JM109(DE3), plasmid pET-Hb(β67W-β95C-synoα-α29F)-MAP-chuA was able to express chuA and increased the cell growth. However, the introduction of chuA did not improve the hemoglobin expression. Moreover, the hemoglobin expression of E. coli JM109(DE3) is better than that in E. coli BL21(DE3).

並列關鍵字

Heme ； Hemoglobin ； Escherichia coli ； Heme-transport

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