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“In cell” biotinylation and immobilization of hBMP-2 (human Bone Morphogenetic Protein 2) on polymeric surfaces

Author:
Tsitouroudi, F., Karatza, A., Karoulias, S., Pantazaki, A., Andriotis, E.G., Achilias, D.S., Choli-Papadopoulou, T.
Source:
Biochemical engineering journal 2017 v.123 pp. 1-12
ISSN:
1369-703X
Subject:
alizarin, alkaline phosphatase, biotin, biotinylation, bone formation, bone morphogenetic proteins, bones, cement, genes, humans, lysine, orthopedics, osteoblasts, staining
Abstract:
Bone Morphogenetic Protein 2 (BMP-2) is a very well-known factor that induces bone formation in vitro (Yamaguchi et al., 1991) [1] and in vivo (Wang et al., 1990) [2] and is widely used in orthopedics for the reconstitution of bone tissue. A full description of a purification and refolding (Long et al., 2006; Von Einem et al., 2010) [3,4] protocol for the “in cell” biotinylation of hBMP-2 is given in this paper: cloning of the mature hBMP-2 gene in vector pAN5 for the integration of the biotinylation sequence into the N-terminus and subsequently in vector pET29c for simultaneous overproduction and biotinylation of the protein. The biotinylation sequence codes for the specific 14mer – peptide with the unique recognizable lysine for biotin covalent interaction, ensuring the “in cell” biotinylation of hBMP-2 without functional distortion. In addition, the targeted biotinylation process does not affect the osteogenic properties of the protein when free in solution or after its functional immobilization onto “bone cement” (Vaishya et al., 2013) [5] materials. This is supported by the observation of the enhancement of differentiation of mesenchymal cells into osteoblasts on the “bio-functionalized” polymeric surfaces verified by the alizarin red staining and the alkaline phosphatase assay.
Agid:
5656132