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Research article2021Peer reviewedOpen access

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

Kanagarajan, Selvaraju; Carlsson, Magnus L. R.; Chakane, Sandeep; Kettisen, Karin; Smeds, Emanuel; Kumar, Ranjeet; Ortenlof, Niklas; Gram, Magnus; Akerstrom, Bo; Bulow, Leif; Zhu, Li-Hua

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 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.

Keywords

HBOCs; Fetal hemoglobin; Heme-binding protein; Plant-made pharmaceuticals; Plant molecular farming; Oxygen delivery; Oxygen therapeutics

Published in

International Journal of Biological Macromolecules
2021, Volume: 184, pages: 955-966 Publisher: ELSEVIER