The overexpression of a nonfusion product of human β-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time. Expression of β-globin from its native cDNA required the use of the strong bacteriophage T7 promoter. In this system, β-globin accumulated to approximately 10% of total E. coli proteins. α-Globin was not expressed in the T7 system using the native cDNA. For the expression of α-globin, synthetic genes containing optimal E. coli codons were constructed. Neither synthetic α-nor β-globin gene alone was expressed from the lac or tac promoter. Globin expression was achieved when the two synthetic α-and β-globin genes were combined as an operon downstream of the lac promoter. The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E. coli protein. Cloning the α-and β-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage. The recombinant β-globin from the T7 expression system was purified and reconstituted in vitro with heme and native a chains. N-Terminal analyses showed that the β-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional β1 methionine residue. Two additional mutants, β1 Val → Met and β1 Val Ala were produced using the T7 system. Functional and structural properties of the purified hemoglobins will be discussed in the following papers.
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