Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.

Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by th...

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Autores principales:	Johan Seijsing, Malin Lindborg, John Löfblom, Mathias Uhlén, Torbjörn Gräslund
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e4060617ee8a4ebb811b810ebd70a15b
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spelling	oai:doaj.org-article:e4060617ee8a4ebb811b810ebd70a15b2021-11-18T08:46:07ZRobust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.1932-620310.1371/journal.pone.0081350https://doaj.org/article/e4060617ee8a4ebb811b810ebd70a15b2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24260574/?tool=EBIhttps://doaj.org/toc/1932-6203Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.Johan SeijsingMalin LindborgJohn LöfblomMathias UhlénTorbjörn GräslundPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 11, p e81350 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Johan Seijsing Malin Lindborg John Löfblom Mathias Uhlén Torbjörn Gräslund Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.
description	Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.
format	article
author	Johan Seijsing Malin Lindborg John Löfblom Mathias Uhlén Torbjörn Gräslund
author_facet	Johan Seijsing Malin Lindborg John Löfblom Mathias Uhlén Torbjörn Gräslund
author_sort	Johan Seijsing
title	Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.
title_short	Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.
title_full	Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.
title_fullStr	Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.
title_full_unstemmed	Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.
title_sort	robust expression of the human neonatal fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with egfp.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/e4060617ee8a4ebb811b810ebd70a15b
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Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.

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