Optimized bacterial production of nonglycosylated human transferrin and its half-molecules

Smit, Maarten H. de; Hoefkens, Peter; Jong, Gerard de; Duin, Jan van; Knippenberg, P.H. Van; Eijk, H.G. Van

doi:10.1016/1357-2725(95)00040-v

Cited by 16 publications

(16 citation statements)

References 46 publications

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“…Recombinant hTf proteins have been produced in other host cells. Even though expression yield of recombinant hTf in E. coli is higher (∼60 µg/mL) than in S2 cells, E. coli-derived recombinant hTf was expressed as nonfunctional inclusion bodies (10). The S2 cell system showed a similar or higher level than in other eukaryotic host cell systems (∼40 µg/mL in baby hamster kidney (BHK) mammalian cells (18), 7-20 µg/ mL in baculovirus-mediated Trichoplusia ni insect cells (13,14)).…”

Section: Resultsmentioning

confidence: 99%

“…hTf is composed of two globular lobes, each containing an affinity binding site for iron. hTf binds Fe 3+ , enters cells by binding to the transferrin receptor on the plasma membrane, releases the Fe 3+ , then exits and repeats this activity (10). hTf has two N-linked glycosylation sites at Asn 413 and Asn 611 in the C lobe, and the main structure of the N-glycan is a biantennnary oligosaccharide form that is terminally sialylated (9,11).…”

Section: Introductionmentioning

confidence: 99%

“…hTf has 38 cysteine residues that are all engaged in disulfide linkages (there are 11 in the C-lobe and 8 in the N-lobe) (12). This extensive number of disulfide bonds made it difficult to produce functional recombinant hTf in an Escherichia coli system (10). Functional recombinant hTf has been expressed in several insect cells using the baculovirus system, and the glycan structures were analyzed (11,13,14).…”

Section: Introductionmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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et al. 2004

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“…The numerous advantages of using a bacterial expression system are well established. However, production of hTF in E. coli met with extremely limited success (de Smit et al, 1995; Hershberger et al, 1991; Hoefkens et al, 1996; Ikeda, Bowman, Yang, & Lokey, 1992; Steinlein & Ikeda, 1993). Due to an inability to correctly form 19 disulfide bonds (Fig.…”

Section: Recombinant Expressionmentioning

confidence: 99%

Transferrin-Mediated Cellular Iron Delivery

Luck

Mason

2012

Current Topics in Membranes

130

116

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Essential to iron homeostasis is the transport of iron by the bilobal protein human serum transferrin (hTF). Each lobe (N- and C-lobe) of hTF forms a deep cleft which binds a single Fe3+. Iron-bearing hTF in the blood binds tightly to the specific transferrin receptor (TFR), a homodimeric transmembrane protein. After undergoing endocytosis, acidification of the endosome initiates the release of Fe3+ from hTF in a TFR-mediated process. Iron-free hTF remains tightly bound to the TFR at acidic pH; following recycling back to the cell surface, it is released to sequester more iron. Efficient delivery of iron is critically dependent on hTF/TFR interactions. Therefore, identification of the pH-specific contacts between hTF and the TFR is crucial. Recombinant protein production has enabled deconvolution of this complex system. The studies reviewed herein support a model in which pH-induced interrelated events control receptor-stimulated iron release from each lobe of hTF.

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“…To our knowledge, Tf can be obtained either by recombination technology or by direct extraction from animal blood. Many attempts had been made to produce recombinant hTf in different expression systems including bacterials [4][5][6][7][8], mammalian cells [9][10][11], yeasts [12,13] and insect cells [14][15][16]. However, the results displaying protein inactivation or low yield were not satisfactory.…”

Section: Introductionmentioning

confidence: 98%

A simple method for obtaining transferrins from human plasma and porcine serum: Preparations and properties

Zhang

et al. 2008

Journal of Chromatography B

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Optimized bacterial production of nonglycosylated human transferrin and its half-molecules

Cited by 16 publications

References 46 publications

Untitled

Untitled

Transferrin-Mediated Cellular Iron Delivery

A simple method for obtaining transferrins from human plasma and porcine serum: Preparations and properties

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