Production of human serum transferrin in Escherichia coli

Ikeda, Richard A.; Bowman, Barbara H.; Yang, Funmei; Lokey, Laurie K.

doi:10.1016/0378-1119(92)90737-a

Cited by 19 publications

(12 citation statements)

References 11 publications

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“…Although this suggests that more convenient yeast expression systems (31) can be considered, the functional attributes of the recombinant proteins, such as receptor binding, will have to be evaluated. Although it is even possible to produce recombinant transferrins in procaryotic systems such as E. coli (32), there is a lack of suitable post-translational processing (i.e. glycosylation proteolytic processing) (33).…”

Section: Discussionmentioning

confidence: 99%

Production and Characterization of Chimeric Transferrins for the Determination of the Binding Domains for Bacterial Transferrin Receptors

Retzer

Kabani

Button

et al. 1996

Journal of Biological Chemistry

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Pathogenic bacteria in the Neisseriaceae and Pasteurellaceae possess outer membrane proteins that specifically bind transferrin from the host as the first step in the iron acquisition process. As a logical progression from prior studies of the ligand-receptor interaction using biochemical approaches, we have initiated an approach involving the production of recombinant chimeric transferrins to further identify the regions of transferrin involved in receptor binding. In order to prepare bovine/human hybrids, the bovine transferrin gene was cloned, sequenced, and compared with the existing human transferrin gene sequence. After identification of potential splice sites, hybrid transferrin genes were constructed using the polymerase chain reaction-based approach of splicing by overlap extension. Five hybrid genes containing sequences from both bovine and human transferrin were constructed. Recombinant transferrins were produced in a baculovirus expression vector system and affinity-purified using concanavalin A-Sepharose. The recombinant proteins were analyzed for reactivity against polyclonal and monoclonal antibodies and assessed for binding to Neisseria meningitidis transferrin receptor proteins in solidphase binding assays and affinity isolation experiments. These experiments enabled us to localize the regions of human transferrin predominantly involved in binding to the N. meningitidis receptor to amino acid residues 346 -588. The construction of these chimeras provides unique tools for the investigation of transferrin binding to receptors from both human and bovine bacterial pathogens.

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Section: Discussionmentioning

confidence: 99%

Production and Characterization of Chimeric Transferrins for the Determination of the Binding Domains for Bacterial Transferrin Receptors

Retzer

Kabani

Button

et al. 1996

Journal of Biological Chemistry

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“…Site-specific and domain replacement mutants will allow further characterization of iron binding and release both in itro and in i o. Over the past few years, much attention has been focused upon expressing unglycosylated and single-lobed half-molecules for structure\folding analyses by Xray crystallography, NMR spectroscopy and CD [11][12][13][14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

“…Bacterial expression systems have been reported [14][15][16]. Their primary advantages over the BHK mammalian expression systems are that selection of expression clones is rapid and that large cultures can be rapidly and inexpensively produced.…”

Section: Introductionmentioning

confidence: 99%

High-yield production of functionally active human serum transferrin using a baculovirus expression system, and its structural characterization

Ali

João²,

Csonga³

et al. 1996

Biochemical Journal

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Recently, there has been much interest in expressing recombinant human serum transferrin (HST) and mutants thereof for structural and functional studies. We have developed a baculovirus expression system for the rapid and efficient production of large quantities of HST (> 20 mg/l). Like native HST, the recombinant protein can bind two ferric ions in the presence of bicarbonate, and is actively taken up by receptor-mediated endocytosis. Secondary structure calculations from CD measurements indicate a content of 42% alpha-helix and 28% beta-sheet. This is the first reported use of a non-mammalian expression system to produce functional HST, and will provide a practical tool to allow expression of a wide range of HST variants for mutagenesis studies.

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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

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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Production of human serum transferrin in Escherichia coli

Cited by 19 publications

References 11 publications

Production and Characterization of Chimeric Transferrins for the Determination of the Binding Domains for Bacterial Transferrin Receptors

Production and Characterization of Chimeric Transferrins for the Determination of the Binding Domains for Bacterial Transferrin Receptors

High-yield production of functionally active human serum transferrin using a baculovirus expression system, and its structural characterization

Transferrin-Mediated Cellular Iron Delivery

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