Human Hemoglobin Expression in Escherichia Colt Importance of Optimal Codon Usaget

Ronald A. Hernan, Stephen G. Sligar, Hilda L. Hui, Robert W. Noble, Mark E. Andracki, Joseph A. Walder, Roxanne Y. Walder

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)8619-8628
Number of pages10
JournalBiochemistry
Volume31
Issue number36
DOIs
StatePublished - Feb 1 1992

ASJC Scopus subject areas

  • Biochemistry

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    Hernan, R. A., Sligar, S. G., Hui, H. L., Noble, R. W., Andracki, M. E., Walder, J. A., & Walder, R. Y. (1992). Human Hemoglobin Expression in Escherichia Colt Importance of Optimal Codon Usaget. Biochemistry, 31(36), 8619-8628. https://doi.org/10.1021/bi00151a032