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

Individual, soluble human. alpha-globin chains were expressed in bacteria w ith exogenous heme and methionine aminopeptidase. The yields of soluble Lu chains in bacteria were comparable to those of recombinant non-alpha chains expressed under the same conditions. Molecular mass and gel-filtration pro perties of purified recombinant alpha chains were the same as those of auth entic human alpha chains. Biochemical and biophysical properties of isolate d alpha chains were identical to those of native human. alpha chains as ass essed by UV/vis, circular dichroism (CD), and nuclear magnetic resonance (N MR) spectroscopy which contrasts with previous results of refolded precipit ated alpha chains made in the presence of heme in vitro (M. T. Sanna ct at, J, Biol Chem, 272, 3478-3486, 1997), Mixtures of purified, soluble recombi nant alpha-globin and native beta-globin chains formed heterotetramers in v itro, and oxygen- and GO-binding properties as well as the heme environment of the assembled tetramers were experimentally indistinguishable from thos e 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 individua l expressed alpha chains are stable in bacteria and fold properly in vivo a nd that they then can assemble with free beta chains to form hemoglobin het erotetramers in vivo as well as in vitro, (C) 2000 Academic Press.