Characterisation of recombinant unglycosylated human serum transferrin purified from Saccharomyces cerevisiae

Sargent, Peter J.; Farnaud, Sébastien; Cammack, Richard; Zoller, Heinz; Evans, Robert W.

doi:10.1007/s10534-005-5532-6

Cited by 10 publications

(7 citation statements)

References 21 publications

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“…7). The native hTF contains two N-glycosylation sites (N413 and N611) [42], whereas the yeast-derived aglycosylated rhTF has two mutations of its N-glycosylation sites (N413Q and N611Q), rendering the protein without N-glycosylation [24]. As expected, the N-glycosylated nhTF showed a clear downward shift in electrophoretic mobility after PNGase F treatment, and the yeast-derived aglycosylated rhTF showed no change before and after the PNGase F treatment.…”

Section: Resultsmentioning

confidence: 99%

“…10B). The slower and faster migrating forms of rhTF reflected the conformational change of rhTF without or with bound iron [24, 30]. The pis-rhTF showed three bands in the urea-PAGE gel; the slowest and the fastest bands corresponded to the apo- and holo- forms of rhTF, respectively, whereas the middle band represented the monoferric form of rhTF.…”

Section: Resultsmentioning

confidence: 99%

“…Although both the insect cell (baculovirus) [23] and mammalian cell [21] expression systems have been shown to be able to express the bioactive rhTF, neither of them can provide enough quantity to be commercially available because of the low expression level and the high cost of production. Recently, bioactive rhTF has been expressed in Saccharomyces Cerevisiae [24] and become commercially available. This yeast-derived rhTF, however, still remains very expensive (Millipore, Billerica, MA).…”

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confidence: 99%

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Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.)

Zhang

Nandi

Bryan

et al. 2010

Protein Expression and Purification

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Transferrin is an essential ingredient used in cell culture media due to its crucial role in regulating cellular iron uptake, transport, and utilization. It is also a promising drug carrier used to increase a drug’s therapeutic index via the unique transferrin receptor-mediated endocytosis pathway. Due to the high risk of contamination with blood-borne pathogens from the use of human- or animal plasma-derived transferrin, recombinant transferrin is preferred for use as a replacement for native transferrin. We expressed recombinant human transferrin in rice (Oryza sativa L.) at a high level of 1% seed dry weight (10 g/kg). The recombinant human transferrin was able to be extracted with saline buffers and then purified by a one step anion exchange chromatographic process to greater than 95% purity. The rice-derived recombinant human transferrin was shown to be not only structurally similar to the native human transferrin, but also functionally the same as native transferrin in terms of reversible iron binding and promoting cell growth and productivity. These results indicate that rice-derived recombinant human transferrin should be a safe and low cost alternative to human or animal plasma-derived transferrin for use in cell culture-based biopharmaceutical production of protein therapeutics and vaccines.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.)

Zhang

Nandi

Bryan

et al. 2010

Protein Expression and Purification

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“…To date, the most optimized, well documented and widely available system utilizes baby hamster kidney (BHK) cells transfected with a plasmid in which the presence of the native signal peptide for human Tf allows secretion of the recombinant Tf into the tissue culture medium [305,345,350–352]. Since 2002, a strain of yeast ( S. cerevisiae ) has been developed and optimized for commercial production of large amounts of recombinant, nonglycosylated Tf, termed deltaferrin [353,354]. More recently production of Tf in rice ( Oryza sativa ) has also been reported [355].…”

Section: A Brief History Of Human Serum Transferrinmentioning

confidence: 99%

The long history of iron in the Universe and in health and disease

Sheftel

Mason

Ponka

2012

Biochimica et Biophysica Acta (BBA) - General Subjects

177

155

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Background Not long after the Big Bang, iron began to play a central role in the Universe and soon became mired in the tangle of biochemistry that is the prima essentia of life. Since life’s addiction to iron transcends the oxygenation of the Earth’s atmosphere, living things must be protected from the potentially dangerous mix of iron and oxygen. The human being possesses grams of this potentially toxic transition metal, which is shuttling through his oxygen-rich humor. Since long before the birth of modern medicine, the blood—vibrant red from a massive abundance of hemoglobin iron—has been a focus for health experts. Scope of Review We describe the current understanding of iron metabolism, highlight the many important discoveries that accreted this knowledge, and describe the perils of dysfunctional iron handling. General Significance Isaac Newton famously penned, “If I have seen further than others, it is by standing upon the shoulders of giants”. We hope that this review will inspire future scientists to develop intellectual pursuits by understanding the research and ideas from many remarkable thinkers of the past. Major Conclusions The history of iron research is a long, rich story with early beginnings, and is far from being finished.

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“…The rTf product was also shown to be functionally equivalent to plasma-derived transferrin as a cell culture supplement [26]. Electron paramagnetic resonance (EPR) was also used to confirm correct co-ordination of the ferric ions in the rTf expressed from S. cerevisiae [27]. …”

Section: Resultsmentioning

confidence: 99%

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

et al. 2010

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BackgroundAnimal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin.ResultsThe first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 μm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin.ConclusionsA Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics.

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Characterisation of recombinant unglycosylated human serum transferrin purified from Saccharomyces cerevisiae

Cited by 10 publications

References 21 publications

Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.)

Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.)

The long history of iron in the Universe and in health and disease

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

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