Recombinant Human Hemoglobin Research Articles

WITHDRAWN: Recent Advances in the Microbial Synthesis of Human Hemoglobin

Hemoglobin is a major binding protein in human red blood cells and plays an important role in the storage and transportation of oxygen. In recent years, the use of recombinant hemoglobin as a new type of oxygen carrier has attracted widespread attention. So far, the research on recombinant hemoglobin technology is in its early stages and there are still many problems, such as low yield and easy degradation. In this review, we summarize the molecular strategies for recombinant synthesis of human hemoglobin in microorganisms through metabolic engineering and synthetic biology techniques in recent years, and explore the possibility of preparing hemoglobin-based oxygen carriers based on fetal hemoglobin, in order to improve the yield of recombinant human hemoglobin and the sustained stability during protein production through protein engineering strategies, and to possibly reduce the toxic side effects of recombinant hemoglobin products.

A Convenient Self-Removing Affinity Tag Method for the Simple Purification of Tagless Recombinant Proteins.

In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTagTM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform.

Rational Mutagenesis to Engineer Heme Stability in Recombinant Human Hemoglobin to Design Potential Hemoglobin Based Oxygen Carrier

Certain life-saving medical conditions like surgery, war wound and trauma necessitate blood transfusions. The transfusion process is associated with several risks and side effects. Heterologous expression of human hemoglobin for developing HBOCs seems to be an alternative to blood transfusion. However, such hemoglobins suffer quite a few disadvantages as described in Varnado et al. A major problem that prevents wider use of HBOCs is the dissociation of “heme” from its protein matrix. The released heme causes serious renal cytotoxicity which needs to be minimized. We successfully engineered heme stability into myoglobin by covalent linkage with a side chain from the apoprotein mimicking a cyanobacterial globin.Efforts are on in translating the heme stability in human hemoglobin. Based on rational mutagenesis, we were indeed able to engineer heme stability to an extent into recombinant human hemoglobin. Specific mutations in the α- and β- chains individually caused significant increase (∼3-4 fold) in the hemin retention. These mutations did not show any major influence on the structural stability and heme coordination of the protein. Moreover, engineered rHb mutants exhibited autooxidation rates similar to the wild type protein. Such preliminary success in engineering heme stability holds promise for the production of stable recombinant HBOCs.

Potential role of the compound Eucommia bone tonic granules in patients with osteoarthritis and osteonecrosis: A retrospective study.

Osteoarthritis is a major source of pain, disability, and socioeconomic cost worldwide. Osteonecrosis is a disabling disorder that frequently occurs in the younger population aged from 20-50 years. The compound Eucommia bone tonic granules, a traditional Chinese medicine, can alleviate the damage of osteoarthritis and osteonecrosis. To investigate the potential role of the compound Eucommia bone tonic granules (Eucommia) in the treatment of patients with osteoarthritis and osteonecrosis. One-hundred forty osteoarthritis and osteonecrosis cases admitted to our hospital from January 2013 to December 2017 were selected. Patients were divided into two groups: Eucommia-meloxicam group and meloxicam group. Clinical efficacy and the Western Ontario and McMaster Universities Arthritis Index (WOMAC) score were evaluated according to the evaluation criteria of orthopedic diseases. The levels of bone-GLA protein, interleukin-17, recombinant human S100 calcium binding protein A12, sphingosine 1-phosphate, cystatin C, creatinine, and hemoglobin in peripheral blood were determined. The total effective rate in the two osteoarthritis groups was not different, but the total effective rate in the two osteonecrosis groups was significantly different. The overall efficacy of Eucommia-meloxicam group was superior to that of the meloxicam group. WOMAC showed that pain, stiffness, and dysfunction in the two groups of osteoarthritis and osteonecrosis before and after treatment were significantly different. The concentration of recombinant human S100 calcium binding protein A12, sphingosine 1-phosphate, cystatin C, creatinine, and hemoglobin before and after treatment in the Eucommia-meloxicam group and meloxicam group of osteoarthritis and osteonecrosis were significantly different, and the two treatment groups were significantly different from each other for osteoarthritis. Our findings indicate that Eucommia can effectively enhance the curative effect of meloxicam, and the combination of Eucommia and meloxicam is superior to meloxicam alone.

Genetically engineered haemoglobin wrapped covalently with human serum albumins as an artificial O2 carrier.

We describe the synthesis and O2 affinity of genetically engineered human adult haemoglobin (rHbA) wrapped covalently with recombinant human serum albumins (rHSAs) as an artificial O2 carrier used for a completely synthetic red blood cell (RBC) substitute. Wild-type rHbA [rHbA(wt)] expressed in yeast species Pichia pastoris shows an identical amino acid sequence and three-dimensional structure to those of native HbA. It is particularly interesting that two orientations of the prosthetic haem group in rHbA(wt) were aligned by gentle heating in the natural form. Covalent wrapping of rHbA(wt) with three rHSAs conferred a core-shell structured haemoglobin-albumin cluster: rHbA(wt)-rHSA3. Three variant clusters containing an rHbA mutant core were also created: Leu-β28 → Phe, Leu-β28 → Trp, and Leu-β28 → Tyr/His-β63 → Gln. Replacement of Leu-β28 with Trp decreased the distal space in the haem pocket, thereby yielding a cluster with moderately low O2 affinity which is nearly the same as that of human RBC.

Hemoglobin(βC93A)-Albumin Cluster: Mutation of Cysteine-β93 to Alanine Allows Moderate Reduction of O2 Affinity by Inositol Hexaphosphate.

Covalent wrapping of recombinant human hemoglobin (Cys-β93→Ala) variant rHb(βC93A) by human serum albumin (HSA) yielded the rHb(βC93A)-HSA3 cluster as an artificial O2 carrier as a red blood cell substitute. Complexation of inositol hexaphosphate to the central rHb(βC93A) core reduced the O2 affinity moderately, in much the same way as that of naked hemoglobin. This reduction might be attributable to the inert, small Ala-β93 residue, which cannot be reacted with the bulky maleimide crosslinker.

Mechanism of Human Apohemoglobin Unfolding.

Removal of heme from human hemoglobin (Hb) results in formation of an apoglobin heterodimer. Titration of this apodimer with guanidine hydrochloride (GdnHCl) leads to biphasic unfolding curves indicating two distinct steps. Initially, the heme pocket unfolds and generates a dimeric intermediate in which ∼50% of the original helicity is lost, but the α1β1 interface is still intact. At higher GdnHCl concentrations, this intermediate dissociates into unfolded monomers. This structural interpretation was verified by comparing GdnHCl titrations for adult human hemoglobin A (HbA), recombinant fetal human hemoglobin (HbF), recombinant Hb cross-linked with a single glycine linker between the α chains, and recombinant Hbs with apolar heme pocket mutations that markedly stabilize native conformations in both subunits. The first phase of apoHb unfolding is independent of protein concentration, little affected by genetic cross-linking, but significantly shifted toward higher GdnHCl concentrations by the stabilizing distal pocket mutations. The second phase depends on protein concentration and is shifted to higher GdnHCl concentrations by genetic cross-linking. This model for apoHb unfolding allowed us to quantitate subtle differences in stability between apoHbA and apoHbF, which suggest that the β and γ heme pockets have similar stabilities, whereas the α1γ1 interface is more resistant to dissociation than the α1β1 interface.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

Engineering the oxygen sensing regulation results in an enhanced recombinant human hemoglobin production by Saccharomyces cerevisiae.

Efficient production of appropriate oxygen carriers for transfusions (blood substitutes or artificial blood) has been pursued for many decades, and to date several strategies have been used, from synthetic polymers to cell-free hemoglobin carriers. The recent advances in the field of metabolic engineering also allowed the generation of different genetically modified organisms for the production of recombinant human hemoglobin. Several studies have showed very promising results using the bacterium Escherichia coli as a production platform, reporting hemoglobin titers above 5% of the total cell protein content. However, there are still certain limitations regarding the protein stability and functionality of the recombinant hemoglobin produced in bacterial systems. In order to overcome these limitations, yeast systems have been proposed as the eukaryal alternative. We recently reported the generation of a set of plasmids to produce functional human hemoglobin in Saccharomyces cerevisiae, with final titers of active hemoglobin exceeding 4% of the total cell protein. In this study, we propose a strategy for further engineering S. cerevisiae by altering the oxygen sensing pathway by deleting the transcription factor HAP1, which resulted in an increase of the final recombinant active hemoglobin titer exceeding 7% of the total cellular protein.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. (1)H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207-7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.

Development of a Method To Produce Hemoglobin in a Bioreactor Culture of Escherichia coli BL21(DE3) Transformed with a Plasmid Containing Plesiomonas shigelloides Heme Transport Genes and Modified Human Hemoglobin Genes

We describe a method for production of recombinant human hemoglobin by Escherichia coli grown in a bioreactor. E. coli BL21(DE3) transformed with a plasmid containing hemoglobin genes and Plesiomonas shigelloides heme transport genes reached a cell dry weight of 83.64 g/liter and produced 11.92 g/liter of hemoglobin in clarified lysates.

Protein Dynamics of Isolated Chains of Recombinant Human Hemoglobin Elucidated by Time-Resolved Resonance Raman Spectroscopy

Protein dynamics of isolated chains of recombinant human adult hemoglobin (rHb) following ligand photolysis were studied by time-resolved resonance Raman spectroscopy. In the time-resolved spectra, we observed frequency shifts of the iron-histidine stretching [ν(Fe-His)] and γ(7) bands and an intensity change of the ν(8) band for both the isolated α- and β-chains, showing that a primary metastable form was present in the initial tens of nanoseconds following the photolysis. Similar spectral changes were reported for human adult hemoglobin and rHb. Common spectral changes between isolated chains and hemoglobin indicated that structural changes reflected by the spectral changes were characteristic of the hemoglobin subunits. The heme modes suggested that the primary metastable form had a more disordered orientation of propionates and a less strained environment than the deoxy form. The spectral changes of the isolated α-chain were faster than those of the β-chain. In spite of the fact that the isolated β-chain formed a tetramer in a similar fashion to rHb, the spectral changes were much slower than those of rHb. The present study shows that intersubunit interactions affected the rates of the structural changes of the heme pocket. Characteristics of the tertiary structure dynamics of hemoglobin are discussed.

Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers.

A recombinant 130 kDa dihemoglobin which is made up of a single-chain tetra-α globin and four β globins has been expressed as a soluble protein in E. coli. The sequence of the single chain tetra-α is: αI-Gly-αII-(SerGlyGly)5Ser-αIII-Gly-αIV. This dihemoglobin has been purified and characterized in vitro by size exclusion chromatography, electrospray mass spectroscopy, equilibrium oxygen binding, and analytical ultracentrifugation. The observed values of P50 and nmax for the dihemoglobin are slightly lower than those observed for the recombinant hemoglobin rHb1.1 (a “monohemoglobin” comprised of two β globins and an αI-Gly-αII diα-globin chain). Titration of the deoxy form of dihemoglobin with CO shows that all eight heme centers bind ligand. In vivo, dihemoglobin showed increased circulating halflife and a reduced pressor response in conscious rats when compared to rHb1.1. These observations suggest that dihemoglobin is an oxygen carrying molecule with desirable in vivo properties and provides a platform for an isooncotic hemoglobin solution derived solely from a recombinant source. A 260 kDa tetrahemoglobin has also been produced by chemical crosslinking of a dihemoglobin that contains a Lys16Cys mutation in the C-terminal α-globin subunit. Tetrahemoglobin also shows reduced vasoactivity in conscious rats that is comparable to that observed for dihemoglobin.

Development of a Method To Produce Hemoglobin in a Bioreactor Culture of Escherichia coli BL21(DE3) Transformed with a Plasmid Containing Plesiomonas shigelloides Heme Transport Genes and Modified Human Hemoglobin Genes

We describe a method for production of recombinant human hemoglobin by Escherichia coli grown in a bioreactor. E. coli BL21(DE3) transformed with a plasmid containing hemoglobin genes and Plesiomonas shigelloides heme transport genes reached a cell dry weight of 83.64 g/liter and produced 11.92 g/liter of hemoglobin in clarified lysates.

Efficacy of low‐dose i.v. iron therapy in haemodialysis patients

i.v. iron therapy is more effective in maintaining adequate iron status in haemodialysis (HD) patients than oral iron therapy (OIT). However, data on lower doses of i.v. iron therapy are insufficient. A non-randomized, open-label study was performed to compare the efficacy of low-dose (<or=50 mg/week of iron sucrose) i.v. iron therapy (LD-IVIT) with OIT in HD patients with 100-800 microg/L serum ferritin levels over 4 months. Eighty-nine patients in the LD-IVIT group (40 men, 49 women; aged 61 +/- 13 years) and 30 patients in the oral iron therapy group (17 men, 13 women; aged 59 +/- 7 years) were evaluated. After 4 months of each treatment, serum ferritin levels increased from 398 +/- 137 to 529 +/- 234 microg/L in the LD-IVIT group (P < 0.01) but decreased from 351 +/- 190 to 294 +/- 175 microg/L in the OIT group (P < 0.01). In the LD-IVIT group, transferrin saturation (from 28% +/- 11% to 30% +/- 14%, P = 0.49), weekly doses of recombinant human erythropoietin (from 5822 +/- 2354 to 5636 +/- 2306 IU/week, P = 0.48) and haemoglobin (from 101 +/- 9 to 103 +/- 9 g/L, P = 0.15) levels remained stable. LD-IVIT may be one of the regimens that may be considered for maintaining iron status in HD patients. However, efficacy of LD-IVIT should be verified by further randomized study.

Effect of Reversed Heme Orientation on Circular Dichroism and Cooperative Oxygen Binding of Human Adult Hemoglobin

We found that recombinant human adult hemoglobin (rHb A) expressed in Escherichia coli showed heterogeneity of components with the intensity of a positive CD band at 260 nm and that it could be resolved into three components (SP-1, SP-2, and SP-3) by SP-Sepharose column chromatography. 1H NMR revealed that SP-1 is identical with native Hb A, while SP-2 and SP-3 largely contain the reversed heme isomer in both the alpha and beta subunits, with contents of approximately 50 and >80% in SP-2 and SP-3, respectively. Rotation of the heme 180 degrees about the 5,15-meso axis (reversed heme) causes an interexchange of the methyl groups at positions 2 and 7 with the vinyl groups at positions 8 and 3, respectively. To examine the effect of the modification of the heme-protein contact on the structure and function of Hb A, we compared the 1H NMR, CD, and oxygen binding properties of the three components with those of native Hb A. Native Hb A exhibits a distinct positive CD band in both the near-UV and Soret regions, but rHb A with reversed heme exhibits a very weak positive CD band at 260 nm and a prominent negative CD band in the Soret region. Cooperativity, as measured by Hill's n value, decreased from 3.18 (SP-1) to 2.94 (SP-2) to 2.63 (SP-3) with an increase in the reversed heme orientation. The effect of an allosteric effector, inositol hexaphosphate (IHP), on the oxygen binding properties was also reduced in rHb A with reversed heme. These results indicate that changes in the heme-globin contact exert a discernible influence on CD spectra and cooperative oxygen binding.

3P086 組換えヒト・ヘモグロビンのヘムの配向 : 逆配向ヘムを含むヘモグロビンの酸素結合特性(ヘム蛋白質))

3P086 組換えヒト・ヘモグロビンのヘムの配向 : 逆配向ヘムを含むヘモグロビンの酸素結合特性(ヘム蛋白質))

A Recombinant Human Hemoglobin with Anti-sickling Properties Greater than Fetal Hemoglobin

A new recombinant, human anti-sickling beta-globin polypeptide designated beta(AS3) (betaGly(16) --> Asp/betaGlu(22) --> Ala/betaThr(87) --> Gln) was designed to increase affinity for alpha-globin. The amino acid substitutions at beta22 and beta87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The beta16 substitution confers the recombinant beta-globin subunit (beta(AS3)) with a competitive advantage over beta(S) for interaction with the alpha-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (alpha(2)beta(AS3)(2)) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that beta(AS3) has a distinct advantage over beta(S) for dimerization with alpha-globin. When equal amounts of beta(S)- and beta(AS3)-globin monomers compete for limiting alpha-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the beta(AS3) polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of beta(AS3)-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit alpha-thalassemia. Therefore, constructs expressing the beta(AS3)-globin gene may be suitable for future clinical trials for sickle cell disease.

Future generations of red blood cell substitutes.

Polyhaemoglobins (PolyHb) and perfluorochemicals are in advanced phase III clinical trials and conjugated haemoglobins in phase II clinical trial. New recombinant human haemoglobin with no vasoactivity is being developed. A soluble macromolecule of PolyHb-catalase-superoxide dismutase is being studied as an oxygen carrier with antioxidant properties. New artificial red blood cells that are more like RBC are being developed. One is based on haemoglobin lipid vesicles. A more recent one is based on nano-dimension artificial red blood cells containing haemoglobin and RBC enzymes with membrane formed from composite copolymer of polyethylene glycol-polylactic acid. Their circulation time is double that of PolyHb.

Oxygen therapeutics (blood substitutes) in cardiac surgery

Discuss and summarize recent published literature related to the use of 'blood substitutes' in cardiac surgery patients. Concerns about safety, inventory and the cost of allogenic blood transfusion have led to the continued interest to find an ideal oxygen carrier 'blood substitute' for cardiac surgery patients. Two classes of oxygen therapeutics 'blood substitutes' are being investigated: (1) perfluorocarbons are synthetic fluorinated hydrocarbons which increase dissolved oxygen in the fluid phase and (2) hemoglobin-based oxygen carriers, modified to improve oxygen dissociation properties and side effects profiles of free hemoglobin. Hemoglobin-based oxygen carriers from bovine and human sources are being tested in phase III trials in cardiac surgery patients as well as a variety of other surgical and nonsurgical settings. Recombinant human hemoglobin and encapsulated hemoglobins of any source are emerging. Safety and cost effectiveness are being evaluated for their use as an alternative to blood transfusion or along with other strategies of blood conservation in cardiac surgery. One hemoglobin-based oxygen carrier (Hemopure) has been approved for use in humans in South Africa as well as another hemoglobin-based oxygen carrier (Gelenpol) and a perfluorocarbon (Perfluoron) in Russia. Phase III trials in Europe and North America will be concluded very soon. We anticipate seeing one or two products approved in North America and Europe by the year 2003/2004.