High expression level of recombinant human erythropoietin in the milk of non-transgenic goats

Toledo, Jorge R.; Sánchez, Oliberto; Seguí, Raquel Montesino; García, Gerardo; Montañez, Margela; Alfonso, P; Rodríguez, María Pilar; Cremata, José A.

doi:10.1016/j.jbiotec.2005.10.019

Cited by 54 publications

(42 citation statements)

References 30 publications

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“…rhEPO was expressed in goat milk by adenoviral transduction and purified from the skimmed milk pool from three nuliparous 1-year-old Toggenburg goats milked on days 32-38 after the induction of lactation [20]. The purity was greater than 99% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining.…”

Section: Production Of Epo In Goat Milkmentioning

confidence: 99%

“…N-Glycans releasing, fluorophore labeling and normal-phase (NP) HPLC separation rhEPO from goat milk was enzymatically deglycosylated with peptide-N 4 -(N-acetyl-b-D-glucosaminyl) asparagine amidase (PNGase F) and the N-glycans recovered as described elsewhere [20,21]. Lyophilized oligosaccharides were labeled with 4ABA and resolved on a TSK Amide-80 column 4.6 · 250 mm; TosoHaas, Japan) [23].…”

Section: Oligosaccharide Identification Using Lectin Binding Assaysmentioning

confidence: 99%

“…An adenoviral vector, carrying the hEPO cDNA, allowed the expression of the recombinant glycoprotein at levels over 2 g/L in goat milk. However, the milk-derived recombinant hormone (rhEPO-milk), showed a lower molecular weight, more basic isoforms and a very low in vivo hematopoietic activity compared to the described homologous rhEPO produced in CHO cell cultures, presumably due to differences in the glycosylation of rhEPO produced in milk compared to that produced in CHO cell line [20]. Moreover, we have also established a continuous, non-transformed epithelial cell line (GMGE) from primary culture of goat mammary gland and expressed rhEPO in this system (EPO-GMGE).…”

mentioning

confidence: 99%

“…Recently, our group has expressed hEPO in the milk of mice and goats (Capra hircus) by adenoviral transduction of the mammary secretory epithelial cells [19,20]. An adenoviral vector, carrying the hEPO cDNA, allowed the expression of the recombinant glycoprotein at levels over 2 g/L in goat milk.…”

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confidence: 99%

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Monosialylated biantennary N-glycoforms containing GalNAc–GlcNAc antennae predominate when human EPO is expressed in goat milk

Montesino

Toledo

Sánchez

et al. 2008

Archives of Biochemistry and Biophysics

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Recently, our group reported the expression of recombinant human erythropoietin in goat milk (rhEPO-milk) as well as in the mammary epithelial cell line GMGE (EPO-GMGE) by cell culture using the adenoviral transduction system. N-Glycosylation characterization of rhEPO-milk by Normal-Phase HPLC profiling of the fluorophore, 4-aminobenzoic acid-labeled enzymatically released N-glycan pool from rhEPO-goat milk, combined with MALDI, ESI-MS and LC/MS, revealed that low branched, core-fucosylated, N-glycans predominate. The labeled N-glycans were separated into neutral and charged fractions by anion exchange chromatography and the charged N-glycans were found to be mostly a2,6-monosialylated with Neu5Ac or Neu5Gc in a ratio of 1:1. Unlike the N-glycans from rhEPO produced in CHO cells, where the glycans are multiantennary highly sialylated, core-fucosylated oligosaccahrides, or even in the goat mammary gland epithelial cell line cultured in vitro in which multiantennary, core-and outer-arm fucosylated, monosialylated Nglycans are the most abundant species, a large proportion of the N-glycans from rhEPO-milk were monosialylated, biantennary, antennae mostly terminating with the more unusual GalNAc-GlcNAc motive and without outer-arm fucosylation. These findings, emphasizing the difference in the N-glycan repertoire between the rhEPO-milk and EPO-GMGE, are consistent with the principle that glycosylation is cell-type dependent and that the cell environment is crucial as well.

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Section: Production Of Epo In Goat Milkmentioning

confidence: 99%

Section: Oligosaccharide Identification Using Lectin Binding Assaysmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Monosialylated biantennary N-glycoforms containing GalNAc–GlcNAc antennae predominate when human EPO is expressed in goat milk

Montesino

Toledo

Sánchez

et al. 2008

Archives of Biochemistry and Biophysics

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“…Ele relatou a detecção de EPOrh no leite de cabras utilizando o protocolo descrito em Toledo et al (2005).…”

Section: Processamento Das Amostras De Leiteunclassified

Modelos para a produção de eritropoietina recombinante humana in vivo e in vitro com vetores plasmideais em ovinos

Giassetti¹

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Biosafety evaluation of recombinant protein production in goat mammary gland using adenoviral vectors: Preliminary study

Jiménez

Pose

Moltó

et al. 2012

Biotechnology Journal

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The production of recombinant proteins in the milk of non-transgenic goats can be achieved by transducing the mammary gland with recombinant adenoviral vectors. However, this process involves several regulatory issues. The current study evaluates the biosafety of this production system. We present a preliminary biosafety profile based on detection of adenoviral particles in different body fluids and the antibody response after adenoviral transduction of the goat mammary gland. In addition, two methods of adenoviral inactivation in milk were tested. Although adenoviral particles were detected in the milk until day 4 after transduction, they were absent in serum, saliva, urine and feces. Anti-adenovirus antibodies were detected in serum and milk. The virus inactivation methods neutralized adenoviral particles and preserved the immunological identity of the recombinant protein. These results support the idea of a safe production of recombinant proteins using adenoviral vectors.

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High expression level of recombinant human erythropoietin in the milk of non-transgenic goats

Cited by 54 publications

References 30 publications

Monosialylated biantennary N-glycoforms containing GalNAc–GlcNAc antennae predominate when human EPO is expressed in goat milk

Monosialylated biantennary N-glycoforms containing GalNAc–GlcNAc antennae predominate when human EPO is expressed in goat milk

Modelos para a produção de eritropoietina recombinante humana in vivo e in vitro com vetores plasmideais em ovinos

Biosafety evaluation of recombinant protein production in goat mammary gland using adenoviral vectors: Preliminary study

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