Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli

Hart, Roger A.; Kallio, Pauli T.; Bailey, James E.

doi:10.1128/aem.60.7.2431-2437.1994

Cited by 15 publications

(5 citation statements)

References 25 publications

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“…Heme is a prosthetic group of hemoglobin. It has been observed that the absence of adequate heme production during recombinant human hemoglobin biosynthesis in E. coli results in an insoluble aggregate of hemoglobin (27), the removal of heme from human hemoglobin in vitro results in partial unfolding and severely reduced solubility (28), and the insoluble form of VHb overexpressed in E. coli lacks the heme group (29). Therefore, the high percentage of insoluble VHb fraction obtained at the end of 22 h of cultivation as observed in Figure 5 may result from the inadequate heme production in the recombinant E. coli cells.…”

Section: Resultsmentioning

confidence: 99%

Coexpression of Vitreoscilla Hemoglobin Reduces the Toxic Effect of Expression of D-Amino Acid Oxidase in E. coli

Chien

Kuan

et al. 2004

Biotechnol. Prog.

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Expression of the gene (daao) encoding D-amino acid oxidase (DAAO) in Escherichia coli typically results in a marked decrease of cell viability, and it has generally been assumed that the consumption of intracellular D-alanine by DAAO is responsible for this effect. Vitreoscilla hemoglobin (VHb) gene (vgb) was coexpressed with Rhodosporidium toruloides D-amino acid oxidase in E. coli BL21(DE3) and BL21(DE3)pLysS, expression hosts differing in the stringency of suppressing basal transcription. Not only was the toxic effect of DAAO on cell growth relieved but also the pronounced cell lysis of BL21(DE3)pLysS caused by the expression of DAAO was prevented by coexpressing VHb with DAAO. As a result of the higher cell density achieved, DAAO activity about 1.5-fold higher than that of DAAO-expressing control strains could be obtained by DAAO/VHb-coexpressing strains. The relieving effect of VHb on DAAO toxicity resulted from its oxygen-binding ability. The low availability of free intracellular oxygen reduced DAAO activity and consequently its toxicity.

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Section: Resultsmentioning

confidence: 99%

Coexpression of Vitreoscilla Hemoglobin Reduces the Toxic Effect of Expression of D-Amino Acid Oxidase in E. coli

Chien

Kuan

et al. 2004

Biotechnol. Prog.

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“…It has been described that inhibition of haem biosynthesis in human myeloid leukaemic cells resulted in the accumulation of inactive haem-deficient myeloperoxidase in the endoplasmic reticulum of the cells [45]. Similarly, supplementing the growth medium of E. coli with precursors of haem biosynthesis was found to be essential for solubilization of recombinant Vitreoscilla haemoglobin [46], and the haem-deficient apoform of horseradish peroxidase C was shown to accumulate as an inactive protein in inclusion bodies of E. coli, with haemin being essential for in itro folding and activation of the enzyme [47].…”

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confidence: 99%

Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding

List

Klatt

Werner

et al. 1996

Biochemical Journal

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Nitric oxide synthase (NOS) catalyses the conversion of L-arginine into L-citrulline and nitric oxide. Recently we have developed a method for expression of recombinant rat brain NOS in baculovirus-infected Sf9 cells and purification of the enzymically active enzyme [Harteneck, Klatt, Schmidt and Mayer (1994) Biochem J. 304, 683-686]. To study how biosynthetic manipulation of the NOS cofactors haem, FAD/FMN, and tetrahydrobiopterin (H4biopterin) affects the properties of the isolated enzyme, Sf9 cells were infected in the absence and presence of haemin chloride (4 microg/ml), riboflavin (0.1.mM), and the inhibitor of H4biopterin biosynthesis 2,4-diamino-6-hydroxypyrimidine (10 mM). In the absence of haemin, NOS was expressed to a very high level but remained predominantly insoluble. Purification of the soluble fraction of the expressed protein showed that it had poor activity (0.35 micromol of citrulline x mg(-1) x min(-1)) and was haem-deficient (0.37 equiv. per monomer). Supplementing the culture medium with haemin resulted in pronounced solubilization of the expressed enzyme, which had a specific activity of approximately 1 micromol of citrulline x mg(-1) x min(-1) and contained 0.95 equiv. of haem per monomer under these conditions. Unexpectedly, the amount of H(4) biopterin endogenously present in the different NOS preparations positively correlated with the amount of enzyme-bound haem (y = 0.066+0.430x; r = 0.998). Radioligand binding experiments demonstrated that haem-deficient enzyme preparations containing 30-40% of the holoenzyme bound only approximately 40% of H4biopterin as compared with haem-saturated controls. These results suggest that the prosthetic haem group is essentially involved in the correct folding of NOS that is a requisite for solubilization of the protein and tight binding of H4biopterin.

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“…In E. coli, accumulation of soluble rHb (11,26), human cystathionine ␤-synthase (12), rat cytochrome b 5 (32), and Vitroscilla hemoglobin (VHb) (9) is limited by heme availability, as indicated by the fact that ␦-aminolevulinic acid, a heme precursor, is required to increase heme and protein accumulation (12,26,32). Insoluble VHb aggregates do not contain heme, suggesting that heme may stabilize VHb and thus prevent aggregation (9).…”

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A Mutation That Improves Soluble Recombinant Hemoglobin Accumulation in Escherichia coli in Heme Excess

Weickert

Pagratis

Glascock

et al. 1999

Appl Environ Microbiol

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High-level expression of soluble recombinant human hemoglobin (rHb) in Escherichia coli was obtained with several hemoglobin variants. Under identical conditions, two rHbs containing the Presbyterian mutation (Asn-108→Lys) in β-globin accumulated to approximately twofold less soluble globin than rHbs containing the corresponding wild-type β-globin subunit accumulated. The β-globin Providence(asp) mutation (Lys-82→Asp) significantly improved soluble rHb accumulation compared to the wild-type β-globin subunit and restored soluble accumulation of rHbs containing the Presbyterian mutation to wild-type levels. The Providenceasp substitution introduced a negatively charged residue into the normally cationic 2,3-bisphosphoglycerate binding pocket, potentially reducing the electrostatic repulsion in the absence of the polyanion. The average soluble globin accumulation when there was coexpression of di-α-globin and β-Lys-82→Asp-globin (rHb9.1) and heme was present in at least a threefold molar excess was 36% ± 3% of the soluble cell protein in E. coli. The average total accumulation (soluble globin plus insoluble globin) was 56% ± 7% of the soluble cell protein. Fermentations yielded 6.0 ± 0.3 g of soluble rHb9.1 per liter 16 h after induction and 6.4 ± 0.2 g/liter 24 h after induction. The average total globin yield was 9.4 g/liter 16 h after induction. High-level accumulation of soluble rHb in E. coli depends on culture conditions, the protein sequence, and the molar ratio of the heme cofactor added.

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Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli

Cited by 15 publications

References 25 publications

Coexpression of Vitreoscilla Hemoglobin Reduces the Toxic Effect of Expression of D-Amino Acid Oxidase in E. coli

Coexpression of Vitreoscilla Hemoglobin Reduces the Toxic Effect of Expression of D-Amino Acid Oxidase in E. coli

Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding

A Mutation That Improves Soluble Recombinant Hemoglobin Accumulation in Escherichia coli in Heme Excess

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