Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.

The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial d...

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Autores principales:	Wilhelm Bohr, Michael Kupper, Kurt Hoffmann, Ralf Weiskirchen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/fb6015fabc9143cd99a3748038213ff6
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spelling	oai:doaj.org-article:fb6015fabc9143cd99a3748038213ff62021-11-18T07:00:58ZRecombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.1932-620310.1371/journal.pone.0016000https://doaj.org/article/fb6015fabc9143cd99a3748038213ff62010-12-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21209863/?tool=EBIhttps://doaj.org/toc/1932-6203The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated.Wilhelm BohrMichael KupperKurt HoffmannRalf WeiskirchenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 12, p e16000 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Wilhelm Bohr Michael Kupper Kurt Hoffmann Ralf Weiskirchen Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.
description	The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated.
format	article
author	Wilhelm Bohr Michael Kupper Kurt Hoffmann Ralf Weiskirchen
author_facet	Wilhelm Bohr Michael Kupper Kurt Hoffmann Ralf Weiskirchen
author_sort	Wilhelm Bohr
title	Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.
title_short	Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.
title_full	Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.
title_fullStr	Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.
title_full_unstemmed	Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.
title_sort	recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/fb6015fabc9143cd99a3748038213ff6
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Recombinant expression, purification, and functional characterisation of connective tissue growth factor and nephroblastoma-overexpressed protein.

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