Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
Journal article, 2021

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.

Oxygen delivery

HBOCs

Plant-made pharmaceuticals

Oxygen therapeutics

Plant molecular farming

Fetal hemoglobin

Heme-binding protein

Author

Selvaraju Kanagarajan

Swedish University of Agricultural Sciences (SLU)

Magnus L.R. Carlsson

Swedish University of Agricultural Sciences (SLU)

Sandeep Chakane

Lund University

Karin Kettisen

Lund University

Emanuel Smeds

Lund University

Ranjeet Kumar

Chalmers, Biology and Biological Engineering, Chemical Biology

Niklas Ortenlöf

Lund University

Magnus Gram

Lund University

Bo Åkerström

Lund University

Leif Bülow

Lund University

Li Hua Zhu

Swedish University of Agricultural Sciences (SLU)

International Journal of Biological Macromolecules

0141-8130 (ISSN) 18790003 (eISSN)

Vol. 184 955-966

Subject Categories

Biochemistry and Molecular Biology

Bioprocess Technology

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

DOI

10.1016/j.ijbiomac.2021.06.102

PubMed

34153360

More information

Latest update

2/25/2022