Isolation, renaturation and partial characterization of recombinant human transferrin and its half molecules from escherichia coli

Hoefkens, Peter; Smit, Maarten H. de; Jeu-Jaspars, Nel M.H. De; Huijskes-Heins, M.I.E.; Jong, Gerard de; Eijk, H.G. Van

doi:10.1016/1357-2725(96)00057-x

Cited by 22 publications

(14 citation statements)

References 30 publications

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“…Furthermore, it has been reported that N-glycan plays no role in binding of transferrin to its receptor (25). Similar findings were reported using a bacterial expression system that does not have a glycosylation process (26). In that study, recombinant N-and C-terminal half-hTf proteins were produced in E. coli, and there was no significant difference in iron-binding activities between each protein even though the original N-glycan structures exist only in the C-lobe.…”

Section: Resultssupporting

confidence: 76%

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et al. 2004

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Human transferrin (hTf) is a serum glycoprotein involved in Fe3+ transport. Here, a plasmid encoding the hTf gene fused with a hexahistidine (His6) epitope tag under Drosophila metallothionein promoter (pMT) was stably transfected into Drosophila melanogaster S2 cells as a nonlytic plasmid-based system. Following 3 days of copper sulfate induction, transfected S2 cells were found to secrete hTf into serum-free culture medium at a competitively high expression level of 40.8 microg/mL, producing 6.8 microg/mL/day in a 150-mL spinner flask culture. Purification of secreted recombinant hTf using immobilized metal affinity chromatography (IMAC) yielded 95.5% pure recombinant hTf with a recovery of 32%. According to MALDI-TOF mass spectrometry analysis, purified S2 cell-derived His6-tagged recombinant hTf had a molecular weight (76.4 kDa) smaller than that of native apo-hTf (78.0 kDa). 2-Dimensional gel electrophoresis patterns showed recombinant hTf had a simpler and less acidic profile compared to that of native hTf. These data suggest recombinant hTf was incompletely (noncomplex) glycosylated and lacked sialic acids on N-glycans. However, this difference in N-glycan structure compared to native hTf had no effect on the iron-binding activity of recombinant hTf. The present data show that a plasmid-based stable transfection S2 cell system can be successfully employed as an alternative for producing secreted functional recombinant hTf.

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Section: Resultssupporting

confidence: 76%

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et al. 2004

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“…1A. Tf alone has a dipeak EPR signal, with a g value of 4.3 (150-mT field strength), which is universally accepted as the signature spectrum of iron-replete holo-Tf and corresponds to the presence of bound high-spin Fe(III) iron (3,14). Addition of the catecholamine NE to Tf resulted in a rapid and distinct transformation of the Tf EPR signal (Fig.…”

Section: Resultsmentioning

confidence: 97%

Elucidation of the Mechanism by Which Catecholamine Stress Hormones Liberate Iron from the Innate Immune Defense Proteins Transferrin and Lactoferrin

Sandrini¹,

Shergill²,

Woodward³

et al. 2010

J Bacteriol

118

149

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The ability of catecholamine stress hormones and inotropes to stimulate the growth of infectious bacteria is now well established. A major element of the growth induction process has been shown to involve the catecholamines binding to the high-affinity ferric-iron-binding proteins transferrin (Tf) and lactoferrin, which then enables bacterial acquisition of normally inaccessible sequestered host iron. The nature of the mechanism(s) by which the stress hormones perturb iron binding of these key innate immune defense proteins has not been fully elucidated. The present study employed electron paramagnetic resonance spectroscopy and chemical iron-binding analyses to demonstrate that catecholamine stress hormones form direct complexes with the ferric iron within transferrin and lactoferrin. Moreover, these complexes were shown to result in the reduction of Fe(III) to Fe(II) and the loss of protein-complexed iron. The use of bacterial ferric iron uptake mutants further showed that both the Fe(II) and Fe(III) released from the Tf could be directly used as bacterial nutrient sources. We also analyzed the transferrin-catecholamine interactions in human serum and found that therapeutically relevant concentrations of stress hormones and inotropes could directly affect the iron binding of serum-transferrin so that the normally highly bacteriostatic tissue fluid became significantly more supportive of the growth of bacteria. The relevance of these catecholamine-transferrin/lactoferrin interactions to the infectious disease process is considered.

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“…The procedure for the re-naturation of recombinant proteins was adapted from Hoefkens et al [19]. Renaturation was performed by diluting the proteins in re-naturation buffer (0.1 mM Na-EDTA, 0.1 mM TriseCl, 1.0 mM GSH (reduced glutathione); pH 8.2) to a concentration of 20 mg/ml at 4 (C for 30 min.…”

Section: Scale-up Expression and Purification Of Recombinant Goldfishmentioning

confidence: 99%

Recombinant transferrin induces nitric oxide response in goldfish and murine macrophages

Stafford

Wilson

Belosevic

2004

Fish & Shellfish Immunology

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Isolation, renaturation and partial characterization of recombinant human transferrin and its half molecules from escherichia coli

Cited by 22 publications

References 30 publications

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Elucidation of the Mechanism by Which Catecholamine Stress Hormones Liberate Iron from the Innate Immune Defense Proteins Transferrin and Lactoferrin

Recombinant transferrin induces nitric oxide response in goldfish and murine macrophages

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