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

Kanagarajan, Selvaraju and Carlsson, Magnus and Chakane, Sandeep and Kettisen, Karin and Smeds, Emanuel and Kumar, Ranjeet and Ortenlof, Niklas and Gram, Magnus and Akerstrom, Bo and Bulow, Leif and Zhu, Li-Hua (2021). Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers. International Journal of Biological Macromolecules. 184 , 955-966
[Research article]

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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.

Authors/Creators:Kanagarajan, Selvaraju and Carlsson, Magnus and Chakane, Sandeep and Kettisen, Karin and Smeds, Emanuel and Kumar, Ranjeet and Ortenlof, Niklas and Gram, Magnus and Akerstrom, Bo and Bulow, Leif and Zhu, Li-Hua
Title:Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
Series Name/Journal:International Journal of Biological Macromolecules
Year of publishing :2021
Volume:184
Page range:955-966
Number of Pages:12
Publisher:ELSEVIER
ISSN:0141-8130
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 2 Engineering and Technology > 209 Industrial Biotechnology > Medical Biotechnology
Keywords:HBOCs, Fetal hemoglobin, Heme-binding protein, Plant-made pharmaceuticals, Plant molecular farming, Oxygen delivery, Oxygen therapeutics
URN:NBN:urn:nbn:se:slu:epsilon-p-113252
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-113252
Additional ID:
Type of IDID
DOI10.1016/j.ijbiomac.2021.06.102
Web of Science (WoS)000682782900011
ID Code:25139
Faculty:LTV - Fakulteten för landskapsarkitektur, trädgårds- och växtproduktionsvetenskap
Department:(LTJ, LTV) > Department of Plant Breeding (from 130101)
Deposited By: SLUpub Connector
Deposited On:01 Sep 2021 07:26
Metadata Last Modified:01 Sep 2021 07:31

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