Expression and Purification of Recombinant Hemoglobin in Escherichia coli

Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis

Analysis of human α globin gene mutations that impair binding to the α hemoglobin stabilizing protein

Alpha hemoglobin stabilizing protein (AHSP) reversibly binds nascent α globin to maintain its native structure and facilitate its incorporation into hemoglobin A. Previous studies indicate that some naturally occurring human α globin mutations may destabilize the protein by inhibiting its interactions with AHSP. However, these mutations could also affect hemoglobin A production through AHSP-independent effects, including reduced binding to β globin. We analyzed 6 human α globin variants with altered AHSP contact surfaces. Alpha globin amino acid substitutions H103Y, H103R, F117S, and P119S impaired interactions with both AHSP and β globin. These mutations are destabilizing in biochemical assays and are associated with microcytosis and anemia in humans. By contrast, K99E and K99N α globins bind β globin normally but exhibit attenuated binding to AHSP. These mutations impair protein folding and expression in vitro and appear to be mildly destabilizing in vivo. In Escherichia coli and erythroid cells, α globin K99E stability is rescued on coexpression with AHSP mutants in which binding to the abnormal globin chain is restored. Our results better define the biochemical properties of some α globin variants and support the hypothesis that AHSP promotes α globin chain stability during human erythropoiesis