Expression, purification, and refolding of a recombinant human bone morphogenetic protein 2 in vitro
| Autor: | Liangjun Zhu, Yanhong Zhang, Yinsun Ma, Juming Yao, Mingying Yang, Sijia Min |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Bone Regeneration
Protein Renaturation Bone Morphogenetic Protein 2 medicine.disease_cause Bone morphogenetic protein 2 Bone and Bones Chromatography Affinity Protein Refolding law.invention chemistry.chemical_compound Transforming Growth Factor beta law Cell Line Tumor Escherichia coli medicine Humans Cloning Molecular Bone regeneration Inclusion Bodies Chemistry Biological activity Hydrogen-Ion Concentration Recombinant Proteins Biochemistry Cell culture Bone Morphogenetic Proteins Recombinant DNA Urea Biotechnology |
| Zdroj: | Protein Expression and Purification. 75:155-160 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2010.07.014 |
| Popis: | In this work, the recombinant human bone morphogenetic protein 2 (rhBMP-2) gene was cloned from MG-63 cells by RT-PCR, and the protein was expressed in Escherichia coli expression system, purified by Ni–NTA column under denaturing conditions and refolded at 4 °C by urea gradient dialysis. We found that the protein refolding yield was increased with the increase of pH value from pH 6.0 to pH 9.0. The yield was 42% and 96% at pH 7.4 and pH 9.0, respectively, while that at pH 6.0 was only 3.4%. The cell culture results showed that the rhBMP-2 refolded at pH 7.4 urea gradient dialysis had higher biological activity for MG-63 cell proliferation and differentiation than that refolded at pH 9.0 since pH 7.4 is closer to the conditions in vivo leading to the formation of dimers through the interchain disulfide bond. Moreover, the biological activity for MG-63 was promoted with the increase of rhBMP-2 concentration in the cell culture medium. This work may be important for the in vitro production and biomedical application of rhBMP-2 protein. |
| Databáze: | OpenAIRE |
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