Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
Kanagarajan, Selvaraju ; Carlsson, Magnus L.R. ; Chakane, Sandeep LU ; Kettisen, Karin LU ; Smeds, Emanuel LU ; Kumar, Ranjeet ; Ortenlöf, Niklas LU ; Gram, Magnus LU
; Åkerström, Bo
LU
and Bülow, Leif
LU
, et al.
(2021)
In International Journal of Biological Macromolecules
184.
p.955-966
- 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.... (More)
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.
(Less)
- author
- Kanagarajan, Selvaraju
; Carlsson, Magnus L.R.
; Chakane, Sandeep
LU
; Kettisen, Karin
LU
; Smeds, Emanuel
LU
; Kumar, Ranjeet
; Ortenlöf, Niklas
LU
; Gram, Magnus
LU
; Åkerström, Bo
LU
and Bülow, Leif
LU
, et al. (More)
- Kanagarajan, Selvaraju
; Carlsson, Magnus L.R.
; Chakane, Sandeep
LU
; Kettisen, Karin
LU
; Smeds, Emanuel
LU
; Kumar, Ranjeet
; Ortenlöf, Niklas
LU
; Gram, Magnus
LU
; Åkerström, Bo
LU
; Bülow, Leif
LU
and Zhu, Li Hua
(Less)
- organization
- publishing date
- 2021-08-01
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Fetal hemoglobin, HBOCs, Heme-binding protein, Oxygen delivery, Oxygen therapeutics, Plant molecular farming, Plant-made pharmaceuticals
- in
- International Journal of Biological Macromolecules
- volume
- 184
- pages
- 12 pages
- publisher
- Elsevier
- external identifiers
-
- scopus:85109199884
- pmid:34153360
- ISSN
- 0141-8130
- DOI
- 10.1016/j.ijbiomac.2021.06.102
- language
- English
- LU publication?
- yes
- id
- 061ba15a-115d-4118-93bd-fc1f3af00043
- date added to LUP
- 2021-08-18 12:59:59
- date last changed
- 2024-09-21 23:41:01
@article{061ba15a-115d-4118-93bd-fc1f3af00043,
abstract = {{<p>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.</p>}},
author = {{Kanagarajan, Selvaraju and Carlsson, Magnus L.R. and Chakane, Sandeep and Kettisen, Karin and Smeds, Emanuel and Kumar, Ranjeet and Ortenlöf, Niklas and Gram, Magnus and Åkerström, Bo and Bülow, Leif and Zhu, Li Hua}},
issn = {{0141-8130}},
keywords = {{Fetal hemoglobin; HBOCs; Heme-binding protein; Oxygen delivery; Oxygen therapeutics; Plant molecular farming; Plant-made pharmaceuticals}},
language = {{eng}},
month = {{08}},
pages = {{955--966}},
publisher = {{Elsevier}},
series = {{International Journal of Biological Macromolecules}},
title = {{Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers}},
url = {{http://dx.doi.org/10.1016/j.ijbiomac.2021.06.102}},
doi = {{10.1016/j.ijbiomac.2021.06.102}},
volume = {{184}},
year = {{2021}},
}