Gary L. Stetler

Expression of recombinant human hemoglobin in Escherichia coli.

Publisher Summary This chapter introduces an expression system that, in combination with high cell density fermentation and efficient purification methods, is capable of producing large quantities of soluble rHb. Soluble rHb in crude or clarified cell lysate is readily quantitated using standard difference spectroscopy techniques, in either the Soret or visible region of the spectrum. The expression plasmid utilizes synthetic α-globin and β-globin genes with usefully placed restriction enzyme sites. This facilitates cassette insertion of specific mutations into the globin genes and allows analyses of structure-function relationships in hemoglobin. A comparison of the amount of total globins present in the E. coli lysate at harvest with the amount of hemoglobin present in the crude lysate indicates that most of the globin synthesized is assembled into soluble tetrameric hemoglobin. This system has been used to design and express an engineered form of human hemoglobin that has some of the properties desired in a hemoglobin-based blood substitute.