Site-Specific Introduction of Negative Charges on the Protein Surface for Improving Global Functions of Recombinant Fetal Hemoglobin

Abstract

Due to its compatible oxygen-transporting abilities, hemoglobin (Hb) is a protein of interest in the development of artificial oxygen therapeutics. Despite continuous formulation attempts, extracellular Hb solution often exhibits undesirable reactions when applied in vivo. Therefore, protein engineering is frequently used to examine alternative ways of controlling the unwanted reactions linked to cell-free Hb solutions. In this study, three mutants of human fetal hemoglobin (HbF) are evaluated; single mutants αA12D and αA19D, and a double mutant αA12D/A19D. These variants were obtained by site-directed mutagenesis and recombinant production in E. coli, and carry negative charges on the surface of the α-subunit at the designated mutation sites. Through characterization of the mutant proteins, we found that the substitutions affected the protein in several ways. As expected, the isoelectric points (pIs) were lowered, from 7.1 (wild-type) down to 6.6 (double mutant), which influenced the anion exchange chromatographic procedures by shifting conditions toward higher conductivity for protein elution. The biological and physiological properties of HbF could be improved by these small modifications on the protein surface. The DNA cleavage rate associated with native HbF could be reduced by 55%. In addition, the negatively charged HbF mutant had an extended circulation time when examined in a mouse model using top load Hb additions. At the same time, the mutations did not affect the overall structural integrity of the HbF molecule, as determined by small-angle X-ray scattering. In combination with circular dichroism and thermal stability, modest structural shifts imposed by the mutations could possibly be related to changes in secondary structure or reorganization. Such local deformations were too minor to be determined within the resolution of the structural data; and overall, unchanged oxidation and heme loss kinetics support the conclusion that the mutations did not adversely affect the basic structural properties of Hb. We confirm the value of adding negatively charged residues onto the surface of the protein to improve the global functions of recombinant Hb.

Introducing Negatively Charged Residues on the Surface of Fetal Hemoglobin Improves Yields in Escherichia coli

“…Using the conventional purification protocol with cation exchange and anion exchange resins, we achieved yields of purified HbF (>95% purity) in the range ∼40–50 mg Hb [heme] /L flask culture (n > 3), in comparison to ∼15 mg/L reported previously ( Ratanasopa et al, 2016 ). The rHbF double mutant αA12D/A19D with 50 ± 4 mg/L showed significantly improved yields compared to wt rHbF, which was not detected in the previous study ( Kettisen et al, 2021 ). Nevertheless, these values are still low in terms of general recombinant protein yields, but for laboratory-scale Hb production, we demonstrate that even in flask cultures, unoptimized cultivation parameters lead to substantially more time and larger culture volumes needed to acquire sufficient amounts of Hb for characterization studies.…”

“…The yields of the rHbFγ2 and rHbFα4/γ2 mutants were lower, 16 ± 2 and 29 ± 12 Hb [heme] /L flask culture, respectively, due to the formation of non-functional HbF resulting in considerable losses. To confirm the benefits of the adapted flask culture protocol, we tried expressing three other HbF mutants reported elsewhere ( Kettisen et al, 2021 ). Using the conventional purification protocol with cation exchange and anion exchange resins, we achieved yields of purified HbF (>95% purity) in the range ∼40–50 mg Hb [heme] /L flask culture (n > 3), in comparison to ∼15 mg/L reported previously ( Ratanasopa et al, 2016 ).…”

“…The functionality of the mutant HbF was assessed several-fold by spectrophotometric analysis of the anticipated ligand states, autoxidation, and isoelectric point determination, respectively. The different spectra were produced according to previously described methods ( Kettisen et al, 2021 ). Briefly, the HbF mutants were examined on a Cary 60 UV-Vis spectrophotometer (Agilent Technologies) in different ligand-bound and oxidation states of hemoglobin: Hb-CO, Hb-O 2 , deoxy Hb (Fe 2+ ), and ferric Hb (Fe 3+ ).…”

“…The α -subunit mutations are as follows: α11 (A9) Lys→Glu, α56 (E5) Lys→Glu (Hb Shaare Zedek ( Abramov et al, 1980 )), α78 (EF7) Asn→Asp (Hb J-Singa ( Wong et al, 1984 )), and α90 (FG2) Lys→Glu (Hb Sudbury). The αK11E mutation was selected from a design perspective in part for its proximity to α12, which is a potential site for adding surface-located negative charge as demonstrated previously ( Kettisen et al, 2021 ). The other mutations have been reported as naturally occurring mutations in the Hb variant database ( Giardine et al, 2014 ).…”

“…The other mutations have been reported as naturally occurring mutations in the Hb variant database ( Giardine et al, 2014 ). Compared to a previously reported HbF mutant (αA12D/A19D) where two negatively charged residues were placed relatively closely on the A helix ( Kettisen et al, 2021 ), the mutation sites presented here were selected also to provide a more dispersed spread of negative charges on the surface of the α -subunit. In addition to introducing negative charges on the α -subunit, the γ-subunit was modified similarly.…”