High-level production of animal-free recombinant transferrin from saccharomyces cerevisiae

Finnis, C.; Payne, Tom; Hay, Joanna; Dodsworth, Neil; Wilkinson, D. T.; Morton, P. H.; Saxton, Malcolm J.; Tooth, David; Evans, Robert W.; Goldenberg, Hans; Scheiber-Mojdehkar, Barbara; Ternes, Nina; Sleep, Darrell

doi:10.1186/1475-2859-9-87

Cited by 38 publications

(23 citation statements)

References 35 publications

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“…41) to create pDB3827. The HSA DIII cassette was obtained as a Not I fragment from pDB3827 and then ligated into pSAC35 to produce pDB3855, which was used to transform S. cerevisiae .…”

Section: Methodsmentioning

confidence: 99%

Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor

et al. 2012

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Albumin is the most abundant protein in blood where it has a pivotal role as a transporter of fatty acids and drugs. Like IgG, albumin has long serum half-life, protected from degradation by pH-dependent recycling mediated by interaction with the neonatal Fc receptor, FcRn. Although the FcRn interaction with IgG is well characterized at the atomic level, its interaction with albumin is not. Here we present structure-based modelling of the FcRn–albumin complex, supported by binding analysis of site-specific mutants, providing mechanistic evidence for the presence of pH-sensitive ionic networks at the interaction interface. These networks involve conserved histidines in both FcRn and albumin domain III. Histidines also contribute to intramolecular interactions that stabilize the otherwise flexible loops at both the interacting surfaces. Molecular details of the FcRn–albumin complex may guide the development of novel albumin variants with altered serum half-life as carriers of drugs.

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“…41) to create pDB3827. The HSA DIII cassette was obtained as a Not I fragment from pDB3827 and then ligated into pSAC35 to produce pDB3855, which was used to transform S. cerevisiae .…”

Section: Methodsmentioning

confidence: 99%

Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor

et al. 2012

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“…While offering several advantages (high yields and low cost), for unknown reasons very little full-length hTF was successfully produced in this system until quite recently [17]. In contrast, the use of common baker’s yeast, Saccharomyces cerevisiae , has produced homogenous non-glycosylated hTF in higher yields (~1.5 g/L) than previously reported in any yeast system [18]. …”

Section: Introductionmentioning

confidence: 99%

Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.)

Steere

Bobst²,

Zhang

et al. 2012

Journal of Inorganic Biochemistry

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The Fe3+ binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin™ (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin™) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin™ is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin™ is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin™ bind Fe3+ tightly yet reversibly. Although previously shown to be capable of delivering Fe3+ to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin™ might be suitable for consideration in biopharmaceutical applications.

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“…While the P. pastoris expression system offers several advantages (high yields and low cost), until recently no significant amount of full-length hTF has been produced in this system (Mizutani et al, 2010). Recent efforts using common baker’s yeast, Saccharomyces cerevisiae , have produced homogenous non-glycosylated hTF at significantly higher yields (~1.5 g/l) than previously observed in any yeast system (Finnis et al, 2010). …”

Section: Recombinant Expressionmentioning

confidence: 95%

Transferrin-Mediated Cellular Iron Delivery

Luck

Mason

2012

Current Topics in Membranes

132

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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High-level production of animal-free recombinant transferrin from saccharomyces cerevisiae

Cited by 38 publications

References 35 publications

Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor

Structure-based mutagenesis reveals the albumin-binding site of the neonatal Fc receptor

Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.)

Transferrin-Mediated Cellular Iron Delivery

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