表現血基質傳送受器 chuA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 Introducing Heme Transport Receptor Gene chuA

張雅芬(Chang, Yafen)

指導教授 ： 黃光策

國立中正大學/工學院/化學工程學系/碩士(2012年)

摘要

為了避免經由輸血的血液可能受到HIV及C型肝炎病毒的感染，研發安全且可有效攜氧的紅血球替代物，被世人所重視。本實驗是利用大腸桿菌表現生產人類血紅素。而此種方法有以下優點：不需經由人體提供血液，進而避免疾病感染；利用定點突變血紅蛋白，以降低其副作用。藉由純化得到可溶的α、β球蛋白鏈，由於缺乏血基質，容易導致蛋白結構不穩定且易在膜內形成包涵體 (inclusion body) 。非致病性的大腸桿菌在外膜上缺乏血基質接受器，在外在環境中提供的血基質亦不足提供其大量生產血紅素。在多種大腸桿菌的菌株中，外膜上的ChuA可幫助細胞利用胞外的血基質。為了增加可溶性血紅素的含量，因此在先前建構的質體 (pET-Hb/MAP) 嵌入來自大腸桿菌O157:H7的ChuA基因利用大腸桿菌BL21 (DE3 △clpP::km)，在37°C條件下表現蛋白。實驗結果發現使用大腸桿菌O157:H7的ChuA啟動子來表現ChuA時，不會影響血紅素的總產量，但亦未能增加可溶性血紅素的含量。

關鍵字

大腸桿菌 ； 血紅素

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). The advantage of the E. coli expression system include easy mass production without the need of human blood supply, optimizing the Hb function using the site-directed mutagenesis, genetically-engineered tetramer formation without the use of chemical crosslinker, and the prevention of disease transmission. Although human α- and β- globin chains can be harvested from soluble fraction of cells, the globins chain are unstable or sent to inclusion body unless heme incorporated into globins. The nonpathogenic strains of E. coli lack heme receptor in the outer membrane and the endogenous synthesis of heme is not sufficient for the Hb production. In several pathogenic Escherichia strains, the outer membrane protein ChuA has been recently responsible for hemin utilization. To increase the amount of globins in the soluble fraction, we cloned ChuA from E. coli O157:H7 chromosome and ligased it into our previous constructed plasmid pET-Hb/MAP. The protein expression was carried our at 37°C using the strain BL21 (DE3 △clpP::km). The results show that the expression of ChuA controlled by its orginal promoter in E. coli O157:H7 does not affect the total and soluble amount of Hb expression in E. coli BL21.

並列關鍵字

Escherichia coli ； Hemoglobin

參考文獻

Bellelli, A. and Brunori, M. "Hemoglobin allostery: variations on the theme," Biochim Biophys Acta, 10 (72), 3 (2011).

Bunn, H. F. "The use of hemoglobin as a blood substitute," American Journal of Hematology, 42 (1), 112 (1993).

Chang, T. M. "Red blood cell substitutes," Baillieres Best Pract Res Clin Haematol, 13 (4), 651 (2000).

D'Agnillo, F. and Chang, T. M. "Absence of hemoprotein-associated free radical events following oxidant challenge of crosslinked hemoglobin-superoxide dismutase catalase," Free Radic Biol Med, 24 (6), 906 (1998).

Doherty, D. H., Doyle, M. P., Curry, S. R., Vali, R. J., Fattor, T. J., Olson, J. S. and Lemon, D. D. "Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin," Nat Biotechnol, 16 (7), 672 (1998).