Small Ubiquitin-Like Modifier Protein 3 Enhances the Solubilization of Human Bone Morphogenetic Protein 2 in E. coli
- 116 Downloads
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.
KeywordshBMP2 SUMO3 Rosetta gami B(DE3) BL21 codon+ Fast protein liquid chromatography Alkaline phosphatase
The authors greatly acknowledge Dr. Muhammad Waheed Akhtar, Director School of Biological Sciences and his team from University of the Punjab, Lahore for their enormous support in the expression and purification experiments.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
- 7.Hissnauer, T. N., Stiel, N., Babin, K., et al. (2017). Recombinant human bone morphogenetic protein-2 (rhBMP-2) for the treatment of nonunion of the femur in children and adolescents: a retrospective analysis. BioMed Research International, 2017, 5. https://doi.org/10.1155/2017/3046842.CrossRefGoogle Scholar
- 11.Baskin, D. S., Ryan, P., Sonntag, V., Westmark, R., & Widmayer, M. A. (2003). A prospective, randomized, controlled cervical fusion study using recombinant human bone morphogenetic protein-2 with the CORNERSTONE-SR™ allograft ring and the ATLANTIS™ anterior cervical plate. Spine, 28(12), 1219–1224.PubMedGoogle Scholar
- 12.Schedel, H., Schneller, A., Vogl, T., Müller, H. F., Mäurer, J., Südkamp, N., Eisenschenk, A., & Felix, R. (2000). Dynamic magnetic resonance tomography (MRI): A follow-up study after femur core decompression and instillation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in avascular femur head necrosis. Rontgenpraxis; Zeitschrift fur radiologische Technik, 53(1), 16–24.PubMedGoogle Scholar
- 27.Marblestone, J. G., Edavettal, S. C., Lim, Y., Lim, P., Zuo, X., & Butt, T. R. (2006). Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO. Protein science: a publication of the Protein Society, 15(1), 182–189.CrossRefGoogle Scholar
- 28.Costa, S. J., Almeida, A., Castro, A., Domingues, L., & Besir, H. (2013). The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: A comparison with the traditional gene fusion technology. Applied Microbiology and Biotechnology, 97(15), 6779–6791.CrossRefPubMedGoogle Scholar
- 33.Webb, B., & Sali, A. (2014). Protein structure modeling with MODELLER. In D. Kihara (Ed.), Protein structure prediction (pp. 1–15). New York: Humana Press.Google Scholar
- 48.Kosinski, J., Cymerman, I. A., Feder, M., Kurowski, M. A., Sasin, J. M., & Bujnicki, J. M. (2003). A “FRankenstein's monster” approach to comparative modeling: Merging the finest fragments of fold-recognition models and iterative model refinement aided by 3D structure evaluation. Proteins: Structure, Function, and Bioinformatics, 53(S6), 369–379.CrossRefGoogle Scholar