[24] Production of Recombinant Human Hemoglobin A in Saccharomyces cerevisiae

The ability to produce human hemoglobin (Hb) in a genetically engineered host microorganism offers a number of valuable opportunities. First, a recombinant microorganism provides an attractive alternative to outdated stocks of red blood cells as a source of Hb for formulation into an Hb-based red cell substitute. Second, specific mutant Hbs can be synthesized and produced in the host for subsequent detailed structural and functional studies. Both Saccharomyces cerevisiae and Escherichia coli have been employed for the production of recombinant human Hb. This chapter presents a straightforward method for producing recombinant Hb (rHb) in S. cerevisiae and E. coli that avoids the refolding and reconstitution steps. It also describes methods for expression of recombinant human Hb A (rHb A) in S. cerevisiae, the purification of the protein, and techniques used for subsequent structural and functional characterization. Coexpression of α- and β-globins to produce rHb A can be achieved either by using a single plasmid carrying both α- and β-globin expression cassettes, or by cotransformation with two plasmids, each carrying α- and β-globin expression cassettes and complementary auxotrophic markers. Examples of both of these approaches are also discussed in the chapter.