Expression, Aufreinigung und Charakterisierung von rekombinantem hCTGF (CCN2) und rNOV (CCN3) in einem eukaryontischen Zellsystem

The family of the CCN proteins is currently represented by 6 individual members. The eponymous members are CYR61 (Cysteine rich protein 61, CCN1), CTGF (Connective Tissue Growth Factor, CCN2) and NOV (Nephroblastoma-Overexpressed protein, CCN3). The most prominent member of this family of proteins, CTGF, has been identified as a possible biomarker for the diagnosis of fibrotic diseases. CTGF is known as a target gene and as a ‘downstream mediator’ of the TGF-beta1 signaling cascade. CTGF is involved into scarring process of organ tissues by stimulating the over expression and deposition of ECM proteins (i.e. collagens). It is reported that several cell types (e.g. endothelial cells) respond with proliferation after stimulating with CTGF. Another member of this family of proteins, NOV, has been shown as a first member of CCN protein family cell growth inhibiting properties and aberrantly reduced expression during tumourigenesis. The first reports suggest that these two CCN proteins are acting opposite to each other in regulating of ECM protein expression. Moreover, they also reciprocally influence their own expression in vitro when one of them is temporally over expressed. For the investigation of biological functions and structural details of CCN2 we have established stable transfected HEK and Flp-In™ 293 clones as productive sources for recombinant human CTGF. The overexpression of rat NOV was induced in COS-7 cells infected adenoviral vector Ad-CMV-rNOV. Both secreted and recombinant proteins were isolated from culture medium of the cells by using of heparin affinity chromatography. This method allows producing of sufficient amounts of both CCNs for structural and functional studies. The identity of purified rhCTGF and rrNOV was demonstrated by ‘Trypsin In-Gel-digesting’ followed by ESI-MS/MS mass spectrometry measurement of proteolytic peptides. The total mass of purified recombinant CCN proteins was further determined by MALDI-TOF/TOF mass spectroscopy. Biological activity of both proteins was demonstrated by using of a Smad3-sensitive reporter gene (i.e. (CAGA)12-MLP-Luc) and BrdU proliferation assay in EA hy 926 cells. Treatment with specific glycosidases further revealed that both CCN proteins are N-glycosylated confirming to bioinformatical analysis of the amino acid sequence of both CCNs. Because of the N-glycosylation both CCN-proteins differ to commercially available recombinant CNN-proteins expressed in E. coli.