轉形血基質傳送受器chuA啟動子以提升大腸桿菌可溶性重組人類血紅蛋白產量

Enhancement of Soluble Recombinant Human Hemoglobin Production in Escherichia coli BL21 by Transformation Heme Transport Receptor Gene chuA

林俊豪(Chun-Hao Lin)

指導教授 ： 黃光策

國立中正大學/工學院/化學工程學系/碩士(2013年)

摘要

使用安全且有效攜帶氧氣的血液替代物，可避免經由輸血時受到HIV和C型肝炎病毒的影響。目前最具生產力、便宜的方法是利用大腸桿菌生產所需要的血紅素。先前實驗室利用定點突變使血紅素與一氧化氮的反應速率降低；連結氧氣調節胜肽調節血紅素修飾對氧氣的親和力，並加入血基質輸送系統chuA嘗試增加可溶性血紅素的表現量。結果顯示利用大腸桿菌BL21(DE3) chuA的啟動子並沒有增加可溶性血紅素蛋白的表現量。
此外，用另一種宿主不含RNase E之大腸桿菌BL21 StarTM(DE3)也無法增加蛋白質的表現量。因此我們嘗試置換血基質輸送系統chuA所夾帶的啟動子，使用強啟動子T7 promoter來增加血基質蛋白質表現量，將實驗室先前所建構的質體(pET-Hb/MAP;chuA)利用PCR將chuA取出並與pET29c進行ligation，使chuA能利用pET29c上的T7啟動子啟動，幫助ChuA蛋白質可以表現將血基質傳送至大腸桿菌內，增加可溶性血紅素的含量。

關鍵字

血紅素 ； 大腸桿菌

並列摘要

Various attempts have been made to find a safe and efficient substitute for human blood due to the increased prevalence of the blood-borne infections such as hepatitis C and human immunodeficiency virus (HIV). Recent advances in molecular biology have led to the development of a plasmid expression system to synthesize and produce recombinant human hemoglobin (rHb) in Escherichia coli (E. coli). Previously, our laboratory has constructed Hb mutants to decrease the nitric oxide consumption by Hb and to create a site for conjugation with an erythrocytic Band 3 derived peptide for modulation of oxygen affinity using site-directed mutagenesis. To increase the soluble protein production, the heme-transport system chuA from E. coli O157:H7 was introduced to the protein expression system. The results show that the expression of chuA using its original promoter did not affect soluble amount of Hb expression in E. coli BL21 (DE3). In addition, the protein expression was not improved when another E.coli host, BL21 StarTM (DE3) that carries the RNase E-defected gene, was used. To ensure the protein expression of ChuA for heme transport, we replaced the original promoter of chuA with T7 promoter. We amplified chuA gene from pET-Hb/MAP;chuA using polymerase chain reaction and inserted into pET29c vector with T7 promoter and T7 terminator.

並列關鍵字

Hemoglobin ； Escherichia

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