title: Expression of functional soluble human alpha-globin chains of hemoglobin in bacteria.
abstract: Individual, soluble human alpha-globin chains were expressed in bacteria with exogenous heme and methionine aminopeptidase. The yields of soluble alpha chains in bacteria were comparable to those of recombinant non-alpha chains expressed under the same conditions. Molecular mass and gel-filtration properties of purified recombinant alpha chains were the same as those of authentic human alpha chains. Biochemical and biophysical properties of isolated alpha chains were identical to those of native human alpha chains as assessed by UV/vis, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy which contrasts with previous results of refolded precipitated alpha chains made in the presence of heme in vitro (M. T. Sanna et al., J. Biol. Chem. 272, 3478-3486, 1997). Mixtures of purified, soluble recombinant alpha-globin and native beta-globin chains formed heterotetramers in vitro, and oxygen- and CO-binding properties as well as the heme environment of the assembled tetramers were experimentally indistinguishable from those of native human Hb A. UV/vis, CD, and NMR spectra of assembled Hb A were also the same as those of human Hb A. These results indicate that individual expressed alpha chains are stable in bacteria and fold properly in vivo and that they then can assemble with free beta chains to form hemoglobin heterotetramers in vivo as well as in vitro.
authors.affiliation: Division of Hematology, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. adachi@email.chop.edu
authors.forename: K
authors.initials: K
authors.lastname: Adachi
authors.forename: T
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Y
authors.initials: Y
authors.lastname: Yang
authors.forename: P T
authors.initials: PT
authors.lastname: Konitzer
authors.forename: J
authors.initials: J
authors.lastname: Pang
authors.forename: K S
authors.initials: KS
authors.lastname: Reddy
authors.forename: M
authors.initials: M
authors.lastname: Ivanova
authors.forename: F
authors.initials: F
authors.lastname: Ferrone
authors.forename: S
authors.initials: S
authors.lastname: Surrey
mesh_terms: D005914:Globins / Q000737:chemistry / Q000235:genetics*; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D011487:Protein Conformation; D013057:Spectrum Analysis
chemical_list: D006454:Hemoglobins; D005914:Globins