以獨立血基質傳送受器chuA質體提昇人類血紅蛋白於大腸桿菌中之表現

輸血是指將人類的血液成分輸入患者體內以達到治療的目的，而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，目前最具有潛力是利用大腸桿菌生產所需的血紅素。先前實驗室利用了定點突變的方式使血紅素與一氧化氮的反應速率降低；連結氧氣的胜肽調節因子，可有效的修飾血紅素對於氧氣的親和力，使血紅素在適當的環境下釋放氧氣，由於大腸桿菌血基質合成的速率遠低於血紅蛋白合成速率，容易導致蛋白質結構不穩定且易在細胞內形成包含體。因此我們嘗試將血基質輸送系統chuA轉殖進入大腸桿菌中，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複製並與pET29c進行接合反應，使chuA能利用pET29c上的T7啟動子啟動並與先前建構的質體(pET-Hb/MAP)做共同表現，希望可以增加可溶性血紅素蛋白的表現量。

關鍵字

血基質輸送系統；血基質；血紅素；大腸桿菌

並列摘要

Blood transfusion is transferring human blood component into the patient to achieve treatment . 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.However , the results showed that the expression of chuA using its chromosomal original promoter did not enhance soluble amount of Hb expression in E. coli BL21 (DE3). In this study, we attempted to replace the original chuA promoter with T7 promoter to enhance heme transport and soluble hemoglobin expression. chuA gene was amplified from pET-Hb/MAP/chuA using polymerase chain reaction and inserted into pET29c vector. pET29c-chuA and pET-Hb/MAP were then co-transformed for Hb expression in E. coli.

並列關鍵字

Heme ； Hemoglobin ； Escherichia coli ； Heme-transport

參考文獻

[1]Nagai, K., & Thogersen, H. C. (1984). Generation of Beta-globin by Sequence-specific Proteolysis of A Hybrid Protein Produced in Escherichia coli. Nature , 309(5971) , 810-812.

[2] Hoffman, S. J., Looker, D. L., Roehrich, J. M., Cozart, P. E., Durfee, S. L., Tedesco, J. L. and Stetler, G. L. (1990) Expression of Fully Functional Tetrameric Human Hemoglobin in Escherichia coli. 87, 8521-8525.

Recombinant Haemoglobin Designed for Use as A Blood Substitude, Nature.

[5]Weickert , M. J. and Curry , S. R. (1997) Turnover of Recombinant Human Hemoglobin in Escherichia coli Occurs Rapidly for Insoluble And Slowly for Soluble Globin , Arch. Biochem. Biophys . 348, 337-346.

[6]Michael J. Weickert, Maria Pagratis, Christopher B. Glascock , and Richard Blackmore.(1998) A Mutation That Improves Soluble Recombinant Hemoglobin Accumulation in Escherichia coli in Heme Excess,Applied and Environmental Microbiology 640-647 , 0099-2240

國際替代計量

以獨立血基質傳送受器chuA質體提昇人類血紅蛋白於大腸桿菌中之表現

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