Enhancement of Soluble Recombinant Human Hemoglobin Production in Escherichia coli BL21 byTransformation Heme Transport Receptor Gene chuA
| Autor: | Chun-Hao Lin, 林俊豪 |
|---|---|
| Rok vydání: | 2013 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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