Application of an SPR-based receptor assay for the determination of biologically active recombinant bone morphogenetic protein-2.
Human bone morphogenetic protein-2 (hBMP-2) is a member of the human transforming growth factor-beta superfamily. Biologically active bone morphogenetic protein-2 (BMP-2) is a dimeric protein that binds in the first step of the signal transduction cascade to specific receptors on the cell-surface. This specific interaction of the dimeric protein with the extracellular ligand-binding domain (ECD) of the receptor was used to develop a receptor-based assay based on an optical biosensor system (Biacore 2000, Biacore AB, Uppsala, Sweden). The ECD of the BMP-receptor type IA, tagged with the Fc part of IgG (BMPR-IA-Fc), was immobilised on the surface of a dextran-protein A-coated sensor chip. Calibration curves were obtained with purified and biologically active recombinant hBMP-2 (rhBMP-2) that showed a linear range from approximately 5 to 250 nM rhBMP-2. Moreover, this assay was used to quantitatively follow the generation of biologically active protein during the renaturation from unfolded and reduced monomers to biologically active dimers. A refolding mixture containing renatured dimeric rhBMP-2 and not correctly folded monomers, was used as the sample solution without any further pre-treatment. It was proven that only the biologically active dimers were recognised by the immobilised receptor, so the generation of biologically active rhBMP-2 during the renaturation process could be monitored directly and rapidly. Furthermore, the results from the optical sensor obtained during the renaturation process showed a good correlation with the data obtained by non-reducing SDS-PAGE analysis carried out at the end of the renaturation process. These data show that the disulphide-bonded dimer corresponds to the biologically active protein capable of binding the BMP-receptor type IA.
Wendler
Janine
J
Division of Natural Products, National Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124, Braunschweig, Germany. Janine.Wendler@gbf.de
Vallejo
Luis Felipe
LF
Rinas
Ursula
U
Bilitewski
Ursula
U
D055396:Bone Morphogenetic Protein 2; D052005:Bone Morphogenetic Protein Receptors, Type I; D019485:Bone Morphogenetic Proteins / Q000032:analysis* / Q000235:genetics; D019281:Dimerization; D006801:Humans; D007118:Immunoassay; D011489:Protein Denaturation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis; D020349:Surface Plasmon Resonance / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000032:analysis* / Q000235:genetics
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D017346:Protein Serine-Threonine Kinases; C498211:BMPR1A protein, human; D052005:Bone Morphogenetic Protein Receptors, Type I