Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens

Kyogoku, Kenji; Yoshida, Kazuhiro; Watanabe, Hiroyuki; Yamashita, Takashi; Kawabe, Yoshinori; Motono, Makoto; Nishijima, Ken ichi; Kamihira, Masamichi; Iijima, Shinji

doi:10.1263/jbb.105.454

Cited by 23 publications

(16 citation statements)

References 31 publications

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“…As shown in Fig. 2a (right panel), the anti-Fc antibody detected several molecular species of TNFR/ Fc in the yolk, as was previously reported (Kyogoku et al 2008). The major band above 61 kD was broad (61-73 kD, and there appeared to be several protein bands), possibly by differential glycosylation (see below).…”

Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedsupporting

confidence: 84%

“…The major band above 61 kD was broad (61-73 kD, and there appeared to be several protein bands), possibly by differential glycosylation (see below). There were several bands of smaller sizes (about 55 kD and a fainter band), which were yolkspecific because they were not observed in a serum sample (Kyogoku et al 2008). The smaller size protein species may have been possibly generated by partial proteolysis.…”

Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedmentioning

confidence: 96%

“…The basic characteristics of the genetically manipulated chickens that expressed TNFR/Fc were described previously (Kyogoku et al 2008). TNFR/ Fc was purified from the yolk of G 0 #711, containing 30-60 lg/mL TNFR/Fc, in a similar manner to scFv/ Fc, except that the sample was dialyzed against PBS.…”

Section: Detection Of Sialic Acid In Tnfr/fc By Lectin Blotmentioning

confidence: 99%

“…We previously reported that TNFR/Fc accumulated in the yolk of genetically manipulated chickens (Kyogoku et al 2008). Three and 10 putative sites have been identified in TNFR/Fc for the addition of N-and Oglycan, respectively.…”

Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedmentioning

confidence: 99%

“…In addition to endogenous chicken IgY, human and mouse IgGs, as well as Fc-fused proteins, injected into the blood stream of hens can be recovered from the yolk of laid eggs (Kawabe et al 2006;Morrison et al 2002). Due to this transport system, several recombinant proteins with the human Fc domain accumulated in the yolk of genetically manipulated chickens (Kamihira et al 2005(Kamihira et al , 2009Kyogoku et al 2008;Penno et al 2010): the single-chained Fv fragment fused to Fc (scFv/Fc), the extracellular domain of the TNF receptor fused to Fc (TNFR/Fc), and erythropoietin fused to Fc (EPO/Fc), as well as humanized antibody, were deposited in the yolk when they were expressed under the control of a ubiquitous promoter. Structural analysis of N-glycan on scFv/Fc purified from the serum revealed the considerable proportions of sialylated glycans (Kamihira et al 2009), which implies the potential to obtain sialylated Fc-fused proteins from the egg yolk of genetically manipulated chickens.…”

Section: Introductionmentioning

confidence: 99%

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Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan

et al. 2013

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The transgenic chicken is a candidate for the production of biopharmaceutical proteins with several economic superiorities. In general, the addition of sialic acid at the terminal of N-glycan is important for the bioactivity of biopharmaceuticals including plasma half-life; however, sialic acid has not been detected in the N-glycan of proteins produced in the egg white of genetically manipulated chickens. In this study, the extracellular domain of the TNF receptor and single chain Fv fused to Fc (referred to as TNFR/Fc and scFv/Fc, respectively) were purified from the egg yolk of genetically manipulated chickens and their sialylation in N-glycan was examined. In contrast to the glycan in egg white, yolk-derived proteins were partly sialylated. Lectin blot showed the existence of a2,6-sialic acid on TNFR/Fc, which disappeared with the removal of N-glycan by PNGase. In scFv/Fc, up to 7 % of N-glycan contained sialic acid. Disialyl glycans, which were detected in serumderived scFv/Fc in a previous study, were not found in the yolk sample. Ovarian follicular tissue, which surrounds growing yolk, expressed several neuraminidases, suggesting the partial truncation of glycan during the yolk transfer process from the blood.

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Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedsupporting

confidence: 84%

Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedmentioning

confidence: 96%

Section: Detection Of Sialic Acid In Tnfr/fc By Lectin Blotmentioning

confidence: 99%

Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedmentioning

confidence: 99%