Recombinant human hemoglobin: Expression and refolding of β-globin fromEscherichia coli

Fronticelli, Clara; O’Donnell, J; Brinigar, William S.

doi:10.1007/bf01025477

Cited by 33 publications

(37 citation statements)

References 20 publications

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“…The denatured globin could be refolded and reconstituted with hemin in the presence of native, reduced a-chains to produce functional, tetrameric Hb (47,(90)(91)(92). Subsequently, Nagai developed a similar system for obtaining unfolded a-globin and reconstituting it with native bchains (92,125,146).…”

Section: Historical Overview Of Recombinant Hbmentioning

confidence: 99%

Development of Recombinant Hemoglobin-Based Oxygen Carriers

Varnado

Mollan

Birukou

et al. 2013

Antioxidants & Redox Signaling

102

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Significance: The worldwide blood shortage has generated a significant demand for alternatives to whole blood and packed red blood cells for use in transfusion therapy. One such alternative involves the use of acellular recombinant hemoglobin (Hb) as an oxygen carrier. Recent Advances: Large amounts of recombinant human Hb can be expressed and purified from transgenic Escherichia coli. The physiological suitability of this material can be enhanced using protein-engineering strategies to address specific efficacy and toxicity issues. Mutagenesis of Hb can (i) adjust dioxygen affinity over a 100-fold range, (ii) reduce nitric oxide (NO) scavenging over 30-fold without compromising dioxygen binding, (iii) slow the rate of autooxidation, (iv) slow the rate of hemin loss, (v) impede subunit dissociation, and (vi) diminish irreversible subunit denaturation. Recombinant Hb production is potentially unlimited and readily subjected to current good manufacturing practices, but may be restricted by cost. Acellular Hb-based O 2 carriers have superior shelf-life compared to red blood cells, are universally compatible, and provide an alternative for patients for whom no other alternative blood products are available or acceptable. Critical Issues: Remaining objectives include increasing Hb stability, mitigating iron-catalyzed and iron-centered oxidative reactivity, lowering the rate of hemin loss, and lowering the costs of expression and purification. Although many mutations and chemical modifications have been proposed to address these issues, the precise ensemble of mutations has not yet been identified. Future Directions: Future studies are aimed at selecting various combinations of mutations that can reduce NO scavenging, autooxidation, oxidative degradation, and denaturation without compromising O 2 delivery, and then investigating their suitability and safety in vivo. Antioxid. Redox Signal. 18, 2314-2328.

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Section: Historical Overview Of Recombinant Hbmentioning

confidence: 99%

Development of Recombinant Hemoglobin-Based Oxygen Carriers

Varnado

Mollan

Birukou

et al. 2013

Antioxidants & Redox Signaling

102

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“…Alternatively, tetrameric hemoglobins in which only the ␣-or ␤-subunits are recombinant (␣rHbA and ␤rHbA) can be obtained by in vitro refolding of the expressed globin. ␤-Globin has thus been expressed (5,6) and refolded in the presence of native ␣-chains to yield recombinant hemoglobin whose conformational and functional properties resemble those of natural HbA (5,6). In contrast, development of an analogous ␣-globin expression system was not as successful (7), although it is highly desirable as a tool for elucidating the tetrameric assembly and cooperativity of HbA.…”

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confidence: 99%

Assembly of Human Hemoglobin

Sanna

Razynska

Karavitis

et al. 1997

Journal of Biological Chemistry

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The ␣-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native ␤-chains. The functional and structural properties of the expressed ␣-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer. The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl ؊ and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the ␣ 1 ␤ 1 and ␣ 1 ␤ 2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed ␣-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA. These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics. In addition to incorporation into a HbA tetramer, the ␣-globin refolds and incorporates heme in the absence of the partner ␤-chain. Although the CO binding kinetics of recombinant ␣-chains were the same as that of native ␣-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component. These results show that recombinant ␣-chain assumes alternating conformations in the absence of ␤-chain and indicate that the isolated ␣-chain exhibits a higher degree of conformational flexibility than the ␣-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed ␣-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.

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“…These last two systems, however, result in globin chains containing N-terminal methionine, which may affect functional properties of hemoglobin. More recently, Shen et al (6) expressed soluble hemoglobin tetramers lacking N-terminal methionine in bacteria by coexpression of ␣-and ␤-globin cDNAs with methionine aminopeptidase cDNA.In order to further the understanding of hemoglobin assembly and folding, production of soluble single-chain hemoglobin variants is critical; however, expression of recombinant, soluble, individual globin chains has not been realized to date (4,5,7,8). Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization.…”

mentioning

confidence: 99%

“…In order to further the understanding of hemoglobin assembly and folding, production of soluble single-chain hemoglobin variants is critical; however, expression of recombinant, soluble, individual globin chains has not been realized to date (4,5,7,8). Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization.…”

mentioning

confidence: 99%

“…Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization. After solubilization, chains must then be renatured in vitro and form correctly folded native globin chains, which then must properly assemble to form authentic hemoglobin tetramers (1,(7)(8)(9). This process is labor intensive, and efficiency of tetramer reconstitution from denatured globin chains is very low.…”

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confidence: 99%

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Expression of Soluble Human β-Globin Chains in Bacteria and Assembly in Vitro with α-Globin Chains

Yamaguchi

Pang

Reddy

et al. 1996

Journal of Biological Chemistry

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Authentic soluble human ␤-globin chains were produced in Escherichia coli using an expression plasmid (pHE2␤) containing full-length cDNAs coding for human ␤-globin chain and methionine aminopeptidase. Spectral properties of the purified ␤-globin were identical to those of authentic ␤-globin. Soluble ␤-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric ␤-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric ␤-globin. Monomeric forms of ␤-globin, like authentic native ␤-globin, formed tetrameric hemoglobin (Hb) A (␣ 2 ␤ 2 ) in vitro upon incubation with ␣-globin, while dimeric forms did not. When ␤-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the ␤-globin chain monomers assembled with ␣-globin and formed hemoglobin tetramers. ␣-Globin was more thermally unstable than ␤-globin, while assembled tetramers promoted higher stability. Disulfide-bonded ␤-globin dimers showed a slight increase in thermal stability compared with ␤-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of ␣ chains favors assembly with ␤-globin, ␤-␤ dimers cannot bind ␣ chains, and that Hb A tetramer formation results in the most thermally stable species.The development of molecular biological techniques to replace selectively individual amino acids has helped further our understanding of the relationship between structure and function of hemoglobin. Initial reports described production of normal and modified human ␤-globin in bacteria employing a fusion protein expression vector (1). An expression system was later developed in which ␣-and ␤-globin chains were coexpressed, resulting in formation of soluble tetrameric hemoglobin in yeast (2, 3). Recent studies described coexpression of human ␣-and ␤-globin in Escherichia coli, which resulted in formation of soluble tetrameric hemoglobins (4), as well as a system for high expression of insoluble ␤-globin chains in E. coli. (5). These last two systems, however, result in globin chains containing N-terminal methionine, which may affect functional properties of hemoglobin. More recently, Shen et al. (6) expressed soluble hemoglobin tetramers lacking N-terminal methionine in bacteria by coexpression of ␣-and ␤-globin cDNAs with methionine aminopeptidase cDNA.In order to further the understanding of hemoglobin assembly and folding, production of soluble single-chain hemoglobin variants is critical; however, expression of recombinant, soluble, individual globin chains has not been realized to date (4,5,7,8). Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization. After solubilization, chains must then be renatured in vitro and form correctly folded native globin chains, which...

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Recombinant human hemoglobin: Expression and refolding of β-globin fromEscherichia coli

Cited by 33 publications

References 20 publications

Development of Recombinant Hemoglobin-Based Oxygen Carriers

Development of Recombinant Hemoglobin-Based Oxygen Carriers

Assembly of Human Hemoglobin

Expression of Soluble Human β-Globin Chains in Bacteria and Assembly in Vitro with α-Globin Chains

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