| 1. |
- Chakane, Sandeep, et al.
(author)
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Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein
- 2017
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In: Redox Biology. - : Elsevier BV. - 2213-2317. ; 12, s. 114-120
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Journal article (peer-reviewed)abstract
- Hemoglobin (Hb) is well protected inside the red blood cells (RBCs). Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA). Hb induced cleavage of supercoiled pDNA (sc pDNA) which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2) and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA) could easily induce complete pDNA cleavage while fetal hemoglobin (HbF) was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals.
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| 2. |
- Hajizadeh, Solmaz, et al.
(author)
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Composite imprinted macroporous hydrogels for haemoglobin purification from cell homogenate
- 2018
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In: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1534, s. 22-31
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Journal article (peer-reviewed)abstract
- Purification of haemoglobin (Hb) has been studied for many years due to its ability to act as an oxygen carrier and its possible use in urgent clinical treatment. In this study, different types of chromatography columns were developed for Hb purification. Two of them showed satisfactory results as affinity chromatography columns and were thus studied more extensively. The affinity adsorbents were prepared by molecular imprinting techniques. In the first case, Pickering emulsion polymerization was used to prepare affinity adsorbents based on molecular imprinting technology. The imprinted particles were immobilized via covalent bonds on the surface of cryogel, a macroporous hydrogel produced by free radical polymerization under sub-zero temperature. In the second case, the affinity sites for Hb were formed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The dynamic binding capacity of the composite cryogel with the immobilized particles and the directly imprinted acrylamide cryogel was found to be 5.2 mg/g and 3.6 mg/g, respectively. The affinity columns showed high selectivity towards Hb in spite of the presence of serum albumin as well as other interfering substances in non-clarified cell homogenates. The maximum capacity in batch mode, the fluid flow and other physical and chemical properties of these columns were investigated.
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| 3. |
- Hajizadeh, Solmaz, et al.
(author)
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Rapid Separation of Human Hemoglobin on a Large Scale From Non-clarified Bacterial Cell Homogenates Using Molecularly Imprinted Composite Cryogels
- 2021
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In: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 9
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Journal article (peer-reviewed)abstract
- The production of a macroporous hydrogel column, known as cryogel, has been scaled up (up to 150 mL) in this work for the purification of human hemoglobin from non-clarified bacterial homogenates. Composite cryogels were synthesized in the presence of adult hemoglobin (HbA) to form a molecularly imprinted polymer (MIP)network where the affinity sites for the targeted molecule were placed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The MIP composite cryogel column was first evaluated in a well-defined protein mixture. It showed high selectivity toward HbA in spite of the presence of serum albumin. Also, when examined in complex non-clarified E. coli cell homogenates, the column showed excellent chromatographic behavior. The binding capacity of a 50 mL column was thus found to be 0.88 and 1.2 mg/g, from a protein mixture and non-clarified cell homogenate suspension, respectively. The recovery and purification of the 50 mL column for separation of HbA from cell suspension were evaluated to be 79 and 58%, respectively. The MIP affinity cryogel also displayed binding and selectivity toward fetal Hb (HbF) under the same operational conditions.
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| 4. |
- Kanagarajan, Selvaraju, et al.
(author)
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Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
- 2021
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In: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 184, s. 955-966
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Journal article (peer-reviewed)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. 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.
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| 5. |
- Kettisen, Karin
(author)
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Engineering of Human Fetal Hemoglobin
- 2021
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Doctoral thesis (other academic/artistic)abstract
- In blood, the oxygen-transporting protein hemoglobin (Hb) governs the oxygenation of cells and tissues. Naturally, this protein has claimed a place in the center of the research field of artificial oxygen therapeutics. Such Hb-based products have used cell-free Hb purified from human, bovine, invertebrate, or recombinant sources to create hemoglobin-based oxygen carriers (HBOCs). However, administration of these cell-free Hb products into the bloodstream initiates several unwanted adverse events. The inherent toxic reactivity of Hb related to heme-mediated oxidation, nitric oxide scavenging, and heme release give rise to serious side effects. These issues have persisted despite attempts at finding a formulation strategy to tame native Hb outside the red blood cell. This dissertation describes work regarding the engineering of human fetal Hb (HbF) for screening of beneficial protein design strategies on the protein itself, both in terms of retaining oxidative stability and from a production perspective. Oxidative side effects are central to the extracellular toxicity exhibited by Hb. We examined the effect of modifying redox active cysteine residues in HbF by removing and/or adding cysteine at the conserved hotspot γCys93 and a surface-located site on the α-subunit. The conserved cysteine was important for the oxidative stability of the protein and removal produced a more unstable Hb molecule. In contrast, the addition of cysteine on the surface of the α-subunit alleviated damaging reactions during oxidative conditions by providing an alternative oxidation hotspot. As the surface of the α-subunit appeared to be promising as a target area for mutagenesis, we explored a set of mutants where alanine residues were substituted into negatively charged aspartic acid. This lowered the pI of HbF and reduced the DNA cleavage rate without affecting the overall structural integrity. We also observed an extended half-life in vivo as well as unchanged oxidation and heme loss rates, indicating that improved functions could be attained with modification of the net surface charge without adversely affecting key functions and stability.We continued to focus on the surface of HbF and created mutants with more dramatically changed net surface charge by replacing positive surface residues with negatively charged residues. We improved Hb yields in the crude extracts during recombinant expression in E. coli with this strategy, but non-target Hb fractions were present in significant quantities in two of the three mutant samples. This indicated an unbalanced assembly of subunits in the HbF variants carrying the γ-subunit mutations, leading to the formation of homotetramers. In addition, the chosen γ-subunit mutations contributed to a more oxidatively unstable Hb molecule, as seen by increased autoxidation rates. The best-performing negatively charged mutant was subjected to a more in-depth characterization study. The crystal structure of this mutant was solved and thus confirmed the surface-exposed locations of the mutations. The mutant showed no significant differences in oxidation rate reactions but differed in reduction and heme loss rates from wild-type HbF in reactions governed by the α-subunit. In contrast to a previously studied HbF mutant, this mutant did not show any increased effect on retention time in vivo. However, a dramatic decrease in DNA cleavage rate was seen, indicating a much less damaging behavior towards important cellular components.We conclude that there are strategies for modifying HbF itself for the improvement of protein properties towards a better, and more easily produced Hb for HBOC development. The suggested modifications to the HbF protein presented in this work could be used in combination with other protein engineering strategies for Hb development.
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| 6. |
- Kettisen, Karin, et al.
(author)
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Introducing Negatively Charged Residues on the Surface of Fetal Hemoglobin Improves Yields in Escherichia coli
- 2021
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In: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 9
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Journal article (peer-reviewed)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.
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| 7. |
- Kettisen, Karin, et al.
(author)
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Potential Electron Mediators to Extract Electron Energies of RBC Glycolysis for Prolonged in Vivo Functional Lifetime of Hemoglobin Vesicles
- 2015
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In: Bioconjugate Chemistry. - : American Chemical Society (ACS). - 1520-4812 .- 1043-1802. ; 26:4, s. 746-754
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Journal article (peer-reviewed)abstract
- Developing a functional blood substitute as an alternative to donated blood for clinical use is believed to relieve present and future blood shortages, and to reduce the risks of infection and blood type mismatching. Hemoglobin vesicle (HbV) encapsulates a purified and concentrated human-derived Hb solution in a phospholipid vesicle (liposome). The in vivo safety and efficacy of HbV as a transfusion alternative have been clarified. Auto-oxidation of ferrous Hb in HbV gradually increases the level of ferric methemoglobin (metHb) and impairs the oxygen transport capabilities. The extension of the functional half-life of HbV has recently been proposed using an electron mediator, methylene blue (MB), which acts as a shuttle between red blood cells (RBC) and HbV. MB transfers electron energies of NAD(P)H, produced by RBC glycolysis, to metHb in HbV. Work presented here focuses on screening of 15 potential electron mediators, with appropriate redox potential and water solubility, for electron transfer from RBC to HbV. The results are assessed with regard to the chemical properties of the candidates. The compounds examined in this study were dimethyl methylene blue (DMB), methylene green, azure A, azure B, azure C, toluidine blue (TDB), thionin acetate, phenazine methosulfate, brilliant cresyl blue, cresyl violet, gallocyanine, toluylene blue, indigo carmine, indigotetrasulfonate, and MB. Six candidates were found to be unsuitable because of their insufficient diffusion across membranes, or overly high or nonexistent reactivity with relevant biomolecules. However, 9 displayed favorable metHb reduction. Among the suitable candidates, phenothiazines DMB and TDB exhibited effectiveness like MB did. In comparison to MB, they showed faster reduction by electron-donating NAD(P)H, coupled with showing a lower rate of reoxidation in the presence of molecular oxygen. Ascertaining the best electron mediator can provide a pathway for extending the lifetime and efficiency of potential blood substitutes.
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| 8. |
- Kettisen, Karin, et al.
(author)
-
Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin
- 2018
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In: Redox Biology. - : Elsevier BV. - 2213-2317. ; 19, s. 218-225
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Journal article (peer-reviewed)abstract
- Redox active cysteine residues including βCys93 are part of hemoglobin's “oxidation hotspot”. Irreversible oxidation of βCys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active γCys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of γCys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at α19 on the exposed surface of the α-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.
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| 9. |
- Kettisen, Karin, et al.
(author)
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Site-Specific Introduction of Negative Charges on the Protein Surface for Improving Global Functions of Recombinant Fetal Hemoglobin
- 2021
-
In: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Journal article (peer-reviewed)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.
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| 10. |
- Kettisen, Karin, et al.
(author)
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Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant
- 2023
-
In: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 10
-
Journal article (peer-reviewed)abstract
- Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli. In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6 Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli, we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H2O2-mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain.
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