Abstract
Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.
Highlights
Human hemoglobin (Hb) in the red blood cells (RBCs) of vertebrates has an essential role in oxygen transport
We have expressed recombinantly fused endotoxin-free HbF (rfHbF) in N. benthamiana leaves through transient expression with the viral pJL-TRBO-rfHbF vector delivered by Agrobacterium using the scalable agrospray method
Anal ysis of protein extracts with SDS-PAGE (Fig. 1a) showed the expression of the rfHbF protein in the leaves sprayed with Agrobacterium harboring the target gene, while as expected, no rfHbF bands were detected in the extract of leaves without agrospray or those sprayed with the empty vector
Summary
Human hemoglobin (Hb) in the red blood cells (RBCs) of vertebrates has an essential role in oxygen transport. Patient refusal of donated blood for religious, ethical or medical reasons further affects the use [2]. These factors have driven efforts to develop “Hemoglobin-based oxygen carriers” (HBOCs) as a potential resource for emergency clinical treat ments of acute anemia, resulting from severe blood loss. A number of different Hb forms have been described, two major types are adult Hb (HbA) and fetal Hb (HbF). Transfusion of adult donor blood (containing HbA) to neonates has demonstrated an increased risk of a devastating intestinal disease, necrotizing enterocolitis and immune sensitization [14]. Alto gether, this makes HbF may be an interesting future alternative to cur rent RBC transfusions for anemic preterm infants and possibly in other clinical applications
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