Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae

Abstract

Recombinant human HbA, produced by co-expressing a-globin and P-globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N-terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen-binding studies demonstrated that the yeast-derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb-based oxygen carrier.For many years there has been interest in the potential of cell-free haemoglobin as a substitute for red cells in oxygen delivery (for reviews see Winslow, 1989;Vandegriff and Winslow, 1991). Research into the development of red-cell substitutes has intensified because of increasing concern over viral contamination of donated blood. To be clinically useful, cell-free Hb requires modification, both to stabilise the tetramer against dissociation into dimers and to reduce the oxygen affinity, thereby allowing efficient tissue oxygenation. Such properties have been achieved by cross-linking Hb chemically using a variety of intramolecular and intermolecular cross-linking agents (for reviews see Chang, 1988;Vandegriff and Winslow, 1991).Currently, Hb for modification is purified from outdated stocks of donated blood. Increasingly, however, it appears that this source of Hb will become limiting (Surgenor et al., 1990). Recombinant-DNA techniques now make it possible to produce recombinant Hb (rHb) in genetically engineered microorganisms. This ensures a source of protein which is free of human-blood-derived infectious agents such as hepatitis B and human immunodeficiency virus.Human rHb was first synthesised using Escherichia coli as a host (Nagai and Therrgersen, 1984;Hoffman and Nagai, 1991 as insoluble, intracellular fusion proteins which required solubilisation followed by specific proteolytic cleavage to produce authentic N-termini. Functional tetrameric Hb was then reconstituted in vitro by mixing the globin subunits and exogenous haem. While this approach has proved valuable for production of small quantities of wild-type HbA and modified Hb for structural and functional studies, it would be extremely costly to synthesise rHb on a large scale using these methods.More recently, rHb has been synthesised in E. coli (Hoffman et al., 1990) and Saccharomyces...

Development of Recombinant Hemoglobin-Based Oxygen Carriers

“…To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars in the erythrocytes (135). Expression of rHb in E. coli and S. cerevisiae was also accomplished with impressive yields (i.e., 2%-10% of the total cellular protein content consists of Hb) (2,28,52,62,74,79,96,97,136,156). Although each system has its own set of advantages and drawbacks, most recent efforts have focused on E. coli production systems.…”

Tetrameric Hemoglobin Expressed in Escherichia coli

“…Our previous report has shown that the ␤ chain can also be expressed from cDNA clones as insoluble aggregates using a T7 promoter without the use of a fusion protein, thus circumventing some of the laborious problems associated with a fusion protein expression system (9). Unlike yeast (16,17) and the fusion protein expression systems, this construction leaves the initiator methionine on the N terminus. Characterization of three N-terminal ␤ chain mutants produced in E. coli using the T7 expression system (␤1 ϩ Met,…”

Properties of a recombinant human hemoglobin double mutant: Sickle hemoglobin with Leu‐88 (β) at the primary aggregation site substituted by Ala

“…There are several systems currently available for producing normal or sickle recombinant Hb (Nagai et al, 1985;Baudin-Chich et al, 1990;Wagenbach et al, 1991;Adachi et al, 1992Bihoreau et al, 1992Coghlan et al, 1992;Looker et al, 1992;Martin de Llano et al, 1993a, 1993bShen et al, 1993;Vallone et al, 1993). Previous reports have shown that the recombinant sickle Hb produced in yeast closely resembles natural Hb, i.e., , 1993a, 1993b).…”