Expression of functional soluble human α-globin chains of hemoglobin in bacteria

Abstract

Individual, soluble human α-globin chains were expressed in bacteria with exogenous heine and methionine aminopeptidase. The yields of soluble α chains in bacteria were comparable to those of recombinant non-α chains expressed under the same conditions. Molecular mass and gel-filtration properties of purifled recombinant α chains were the same as those of authentic human α chains. Biochemical and biophysical properties of isolated α chains were identical to those of native human α chains as assessed by UV/vis, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy which contrasts with previous results of refolded precipitated α chains made in the presence of heine in vitro (M. T. Sanna et al., J. Biol. Chem. 272, 3478-3486, 1997). Mixtures of purified, soluble recombinant α-globin and native β-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 α chains are stable in bacteria and fold properly in vivo and that they then can assemble with free β chains to form hemoglobin heterotetramers in vivo as well as in vitro. (C) 2000 Academic Press.