| Summary: | 碩士 === 國立中正大學 === 化學工程研究所 === 99 === 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 including hepatitis C and human immunodeficiency virus. One approach is to produce recombinant human hemoglobin (Hb) in Escherichia coli (E. coli). Particularly, the rapid reaction rate of Hb with nitric oxide (NO) causing blood vessel constriction should be avoided for the design of a recombinant Hb mutant. To reduce the NO reaction rate with Hb, we constructed a mutant α29 Leu->Phe and β67 Val->Trp using the method of site-directed mutagenesis to reduce the space for the reaction oxygen and NO in the distal heme pocket. In addition, the expression of Hb suffers from low protein production due to the high stability of globin RNA secondary structure near the start codon and the lack of heme transport system in laboratory E. coli stains. To increase the Hb expression in E. coli, we constructed a synonymous mutant of globin to reduce its RNA secondary structure near the start codon and introduced the heme transporter, ChuA, from E. coli O157:H7.
|
|---|
