TY - JOUR
T1 - Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
AU - Kanagarajan, Selvaraju
AU - Carlsson, Magnus L. R.
AU - Chakane, Sandeep
AU - Kettisen, Karin
AU - Smeds, Emanuel
AU - Kumar, Ranjeet
AU - Ortenlof, Niklas
AU - Gram, Magnus
AU - Akerstrom, Bo
AU - Bulow, Leif
AU - Zhu, Li-Hua
PY - 2021
Y1 - 2021
N2 - 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 alpha- and two identical gamma-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the alpha- and gamma-subunits of HbF were fused in order to stabilize the alpha-subunits and facilitate balanced expression of alpha- and gamma-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.
AB - 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 alpha- and two identical gamma-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the alpha- and gamma-subunits of HbF were fused in order to stabilize the alpha-subunits and facilitate balanced expression of alpha- and gamma-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.
KW - HBOCs
KW - Fetal hemoglobin
KW - Heme-binding protein
KW - Plant-made pharmaceuticals
KW - Plant molecular farming
KW - Oxygen delivery
KW - Oxygen therapeutics
KW - HBOCs
KW - Fetal hemoglobin
KW - Heme-binding protein
KW - Plant-made pharmaceuticals
KW - Plant molecular farming
KW - Oxygen delivery
KW - Oxygen therapeutics
UR - https://res.slu.se/id/publ/113252
U2 - 10.1016/j.ijbiomac.2021.06.102
DO - 10.1016/j.ijbiomac.2021.06.102
M3 - Journal article
SN - 0141-8130
VL - 184
SP - 955
EP - 966
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
ER -