title: Production of unmodified human adult hemoglobin in Escherichia coli.
abstract: We have constructed a plasmid (pHE2) in which the synthetic human alpha- and beta-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed alpha and beta chains of component a, respectively, have the expected masses. The remaining 10% of the beta chain in component a corresponds in mass to the beta chain plus methionine. In component b, both alpha and beta chains have the correct masses without detectable N-terminal methionine (< 2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the alpha and beta chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the beta chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.
authors.forename: T J
authors.initials: TJ
authors.lastname: Shen
authors.forename: N T
authors.initials: NT
authors.lastname: Ho
authors.forename: V
authors.initials: V
authors.lastname: Simplaceanu
authors.forename: M
authors.initials: M
authors.lastname: Zou
authors.forename: B N
authors.initials: BN
authors.lastname: Green
authors.forename: M F
authors.initials: MF
authors.lastname: Tam
authors.forename: C
authors.initials: C
authors.lastname: Ho
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D006441:Hemoglobin A / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015183:Restriction Mapping
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005914:Globins; D006441:Hemoglobin A