Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
Artikel i vetenskaplig tidskrift, 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.

Heme-binding protein

Plant-made pharmaceuticals

Fetal hemoglobin

Plant molecular farming

Oxygen therapeutics

Oxygen delivery

HBOCs

Författare

Selvaraju Kanagarajan

Sveriges lantbruksuniversitet (SLU)

Magnus L.R. Carlsson

Sveriges lantbruksuniversitet (SLU)

Sandeep Chakane

Kemiska Institutionen

Karin Kettisen

Kemiska Institutionen

Emanuel Smeds

Lunds universitet

Ranjeet Kumar

Chalmers, Biologi och bioteknik, Kemisk biologi

Niklas Ortenlöf

Lunds universitet

Magnus Gram

Lunds universitet

Bo Åkerström

Lunds universitet

Leif Bülow

Kemiska Institutionen

Li Hua Zhu

Sveriges lantbruksuniversitet (SLU)

International Journal of Biological Macromolecules

0141-8130 (ISSN)

Vol. 184 955-966

Ämneskategorier

Biokemi och molekylärbiologi

Bioprocessteknik

Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

DOI

10.1016/j.ijbiomac.2021.06.102

PubMed

34153360

Mer information

Senast uppdaterat

2021-07-14