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

Abstract

Fetal hemoglobin (HbF) has been developed into an important alternative protein for oxygen therapeutics. Such applications require extensive amounts of proteins, which only can be achieved via recombinant means. However, the expression of vertebrate hemoglobins in heterologous hosts is far from trivial. There are several issues that need to be dealt with. These include, among others, the solubility of the globin chains, equimolar expression of the globin chains, and access to high levels of free heme. In this study, we examined the impact of introducing negative charges on the surface of HbF. Three different HbF mutants were examined, carrying four additional negative charges on the α-subunit (rHbFα4), two additional negative charges on the γ-subunit (rHbFγ2) or a combination of these (rHbFα4/γ2). The increase in negative surface charge in these HbF mutants required the development of an alternate initial capture step in the downstream purification procedures. For the rHbFα4 mutant, we achieved a significantly enhanced yield of purified HbF with no apparent adverse effects on Hb functionality. However, the presence of non-functional Hb portions in the rHbFγ2 and rHbFα4/γ2 samples reduced the yields significantly for those mutants and indicated an imbalanced expression/association of globin chains. Furthermore, the autoxidation studies indicated that the rHbFγ2 and rHbFα4/γ2 mutants also were less oxidatively stable than rHbFα4 and wt rHbF. The study further verified the need for an improved flask culture protocol by optimizing cultivation parameters to enable yield-improving qualities of surface-located mutations.

Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant

“…Four mutations at surface-exposed locations on the α-subunit were previously selected for introducing negative charges on HbF ( Kettisen and Bulow, 2021 ). The four mutations were as follows, α11 (A9) Lys→Glu, α56 Lys→Glu, α78 (EF7) Asn→Asp, and α90 (FG2) Lys→Glu, and this HbF variant will henceforth be called rHbFα4.…”

“…At the end of cultivation, the cultures were bubbled with carbon monoxide (CO) gas to stabilize the protein, and CO bubbling was repeated after each subsequent downstream processing step. The cultivation and purification procedures have been described in more detail elsewhere ( Kettisen and Bulow, 2021 ). The purity of the Hb fractions was estimated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using densitometric analysis.…”

“…Optimization of culture conditions can markedly improve HbF production and a lesser fraction of non-desirable Hb (consisting of e.g., the soluble β-type subunit homotetramers) were seen with HbF compared to HbA ( Ratanasopa et al, 2016 ). We recently revisited a laboratory-scale protocol for shake flask HbF production and found that wt HbF yield in Escherichia coli flask cultures could be increased to ∼30 mg purified Hb/L ( Kettisen and Bulow, 2021 ).…”

“…As the subunits still must be able to readily combine with each other, as well as retain Hb functionality as an oxygen carrier, the choice of mutation sites for this strategy should focus on surface-exposed sites located far away from the subunit contact interfaces and key structures of the heme pocket. We have previously identified one HbF mutant that exhibited a significantly higher yield than recombinant wt HbF (wt rHbF) in E. coli cultures ( Kettisen and Bulow, 2021 ). The present study aims to examine this particular HbF mutant in a more in-depth characterization study.…”

“…The present study aims to examine this particular HbF mutant in a more in-depth characterization study. The HbF mutant harbors substitutions of three positive lysine residues on the surface of the α-subunit into negatively charged glutamic acid, combined with an asparagine residue substituted for aspartate ( Kettisen and Bulow, 2021 ). The mutation sites are spread out across the surface of the alpha chain at 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).…”