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Small Ubiquitin-Like Modifier Protein 3 Enhances the Solubilization of Human Bone Morphogenetic Protein 2 in E. coli.
- Source :
-
Applied biochemistry and biotechnology [Appl Biochem Biotechnol] 2018 Sep; Vol. 186 (1), pp. 256-270. Date of Electronic Publication: 2018 Mar 24. - Publication Year :
- 2018
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Abstract
- Small ubiquitin-like modifier (SUMO) fusion technology is widely used in the production of heterologous proteins from prokaryotic system to aid in protein solubilization and refolding. Due to an extensive clinical application of human bone morphogenetic protein 2 (hBMP2) in bone augmentation, total RNA was isolated from human gingival tissue and mature gene was amplified through RT-PCR, cloned (pET21a), sequence analyzed, and submitted to GenBank (Accession no. KF250425). To obtain soluble expression, SUMO3 was tagged at the N-terminus of hBMP2 gene (pET21a/SUMO3-hBMP2), transferred in BL21 codon+, and ~ 40% soluble expression was obtained on induction with IPTG. The dimerized hBMP2 was confirmed with Western blot, native PAGE analysis, and purified by fast protein liquid chromatography with 0.5 M NaCl elution. The cleavage of SUMO3 tag from hBMP2 converted it to an insoluble form. Computational 3D structural analysis of the SUMO3-hBMP2 was performed and optimized by molecular dynamic simulation. Protein-protein interaction of SUMO3-hBMP2 with BMP2 receptor was carried out using HADDOCK and inferred stable interaction. The alkaline phosphatase assay of SUMO3-hBMP2 on C2C12 cells showed maximum 200-ng/ml dose-dependent activity. We conclude that SUMO3-tagged hBMP2 is more suited for generation of soluble form of the protein and addition of SUMO3 tag does not affect the functional activity of hBMP2.
- Subjects :
- Blotting, Western
Bone Morphogenetic Protein 2 genetics
Bone Morphogenetic Protein 2 metabolism
Chromatography, Liquid methods
Cloning, Molecular
Dimerization
Gingiva metabolism
Humans
Molecular Dynamics Simulation
Native Polyacrylamide Gel Electrophoresis
Protein Binding
RNA genetics
RNA isolation & purification
Recombinant Fusion Proteins genetics
Reverse Transcriptase Polymerase Chain Reaction
Solubility
Ubiquitins genetics
Ubiquitins metabolism
Bone Morphogenetic Protein 2 physiology
Escherichia coli genetics
Ubiquitins physiology
Subjects
Details
- Language :
- English
- ISSN :
- 1559-0291
- Volume :
- 186
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Applied biochemistry and biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 29574511
- Full Text :
- https://doi.org/10.1007/s12010-018-2736-0