Possibilities of Using Fetal Hemoglobin as a Platform for Producing Hemoglobin-Based Oxygen Carriers (HBOCs)

Abstract

The expression levels of fetal hemoglobin (HbF) in bacterial recombinant systems are higher compared with normal adult hemoglobin (HbA). However, heme disorientation in globins are often observed in recombinant production processes, both for HbA and HbF, although the degree of heme oriental disorder is much lower for HbF. In addition, the heme disorientation can be converted to a normal conformation by an oxidation-reduction process. A chromatographic cleaning process involving a strong anion exchanger can be utilized to remove such unstable and nondesirable forms of Hb.

Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin

“…The gene expressing wildtype recombinant HbF (rHbF) was previously inserted into the pETDuet-1 plasmid from Novagen [21] . Site-directed mutagenesis was used to mutate the rHbF gene at specific sites of interest, and the method has been described previously [22] .…”

“…The E. coli containing rHbF, αA19C, γC93A or αA19C/γC93A were all cultivated in the same manner and the expression and purification procedures were based on previous protocols [21] , [22] , [23] , with some modifications. 500 ml Terrific Broth media in 2 l Erlenmeyer flasks were inoculated with overnight starter culture and immediately induced with 0.1 mM isopropyl β- D -1-thiogalactopyranoside (IPTG, Saveen & Werner) and supplemented with 0.24 mM δ-aminolevulinic acid (Sigma-Aldrich).…”

Artificial Oxygen Carriers—Past, Present, and Future—a Review of the Most Innovative and Clinically Relevant Concepts

“…In contrast, disposability of bovine blood is nearly unlimited. Instead of using Escherichia coli or Saccharomyces cerevisiae, there are now efforts to obtain recombinant Hgb (Varnado et al, 2013) from plants, e.g., from Nicotiana benthamiana (Eriksson and Bülow, 2017), and furthermore researchers are focusing more and more an fetal Hgb, which is more stable than adult Hgb (Ratanasopa et al, 2016;Simons et al, 2018). Free, unprocessed mammal Hgb that is not encased with any type of membrane is associated with typical problems (Table 3) (Cardenas et al, 2017).…”

Glutaraldehyde-polymerized hemoglobin and tempol (PolyHb-tempol) has superoxide dismutase activity that can attenuate oxidative stress on endothelial cells induced by superoxide anion

“…Hemoglobin (Hb) is both an oxygen carrier and pro-oxidant that participates in complex redox processes during conditions of oxidative stress. As a blood substitute, Hb-based oxygen carriers (HBOCs) can exert both inflammatory and toxic effects from oxidative processes involving the reactive group [1][2][3][4], such as the formation of ferric ions (Fe 3þ ), ferryl ions (Fe 4þ ), ferryl heme radicals ( H-Fe 4þ ), ferryl protein radicals ( P-Fe 4þ ), hemichromes, protein radical-induced globin chain cross-links, free heme/iron and non-heme radical species [5][6][7]. Adverse microvascular effects of cell-free Hb have also hindered the development of safe and efficacious HBOCs.…”