Small Ubiquitin-Like Modifier Protein 3 Enhances the Solubilization of Human Bone Morphogenetic Protein 2 in E. coli
| Autor: | Roquyya Gul, Muhammad Umair Hanif, Adnan Yaseen, Syed Shoaib Ahmed, Ayyaz Ali Khan, Muhammad Hassan Nawaz, Saima Chaudhary, Muhammad Shoaib, Salman Aziz, Muhammad Usman Mirza |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Recombinant Fusion Proteins Blotting Western Gingiva Bone Morphogenetic Protein 2 Heterologous lac operon Bioengineering Molecular Dynamics Simulation Bone morphogenetic protein Applied Microbiology and Biotechnology Biochemistry Bone morphogenetic protein 2 03 medical and health sciences Western blot Escherichia coli medicine Humans Cloning Molecular Ubiquitins Molecular Biology Gene 030102 biochemistry & molecular biology medicine.diagnostic_test Reverse Transcriptase Polymerase Chain Reaction Chemistry Fast protein liquid chromatography General Medicine Native Polyacrylamide Gel Electrophoresis 030104 developmental biology Solubility RNA Alkaline phosphatase Dimerization Chromatography Liquid Protein Binding Biotechnology |
| Zdroj: | Applied Biochemistry and Biotechnology. 186:256-270 |
| ISSN: | 1559-0291 0273-2289 |
| DOI: | 10.1007/s12010-018-2736-0 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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