Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, recombinantly produced Hb, but the development has impeded safety and toxicity issues. Herewith we report successful production of fetal (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed two identical α- γ-subunits, held together hydrophobic interactions, hydrogen bonds, salt bridges. In our study, γ-subunits were fused order stabilize α-subunits facilitate balanced expression N. benthamiana. Efficient extraction purification methods enabled endotoxin-free (rfHbF) high quantity quality. The transiently expressed rfHbF was identified SDS-PAGE, Western blot liquid chromatography-tandem mass spectrometry analyses. purified possessed structural functional properties similar HbF, which confirmed biophysical, biochemical, vivo studies. results demonstrate potential plant systems producing Hb products for use as blood substitutes.