HUMAN HEMOGLOBIN EXPRESSION IN ESCHERICHIA-COLI - IMPORTANCE OF OPTIMAL CODON USAGE

The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time. Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter. In this system, beta-globin accumulated to approximately 10% of total E. coli proteins. Alpha-globin was not expressed in the T7 system using the native cDNA. For the expression of alpha-globin, synthetic genes containing optimal E. coli codons were constructed. Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter. Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter. The two proteins combined intracellularly with endogenous heme, which was concomitantly over produced to yield tetrameric hemoglobin as roughly 5-10% of total E. coli protein. Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage. The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha-chains. N-Terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta-1 methionine residue. Two additional mutants, beta-1 Val --> Met and beta-1 Val --> Ala were produced using the T7 system. Functional and structural properties of the purified hemoglobins will be discussed in the following papers.