Publisher Summary This chapter discusses the production of human hemoglobin in Escherichia coli ( E. coli ) using cleavable fusion protein expression vector. The engineering of Hb is a powerful method of investigating various functional and structural aspects of Hb. This is the only method of making human hemoglobin in a microorganism until soluble and functional Hb is produced in E. coli. Hb made by this method has, at the N termini, an extra Met residue, which arises from the initiation AUG codon. The oxygen binding of this Hb is only slightly different from that of human hemoglobin A. The soluble and functional form of human hemoglobin is produced by coexpressing α- and β-globins in the yeast Saccharomyces cerevisiae. The N termini of both subunits were processed correctly and hence this is the first authentic functional Hb tetramer to be produced in a microorganism. Despite these developments, the method is valuable because it permits the preparation from E. coli of large amounts of Hb that is chemically identical to human Hb.