Details
| Original language | English |
|---|---|
| Article number | e00249 |
| Journal | Biotechnology Reports |
| Volume | 18 |
| Early online date | 4 Apr 2018 |
| Publication status | Published - Jun 2018 |
Abstract
Biologically active human bone morphogenetic protein-4 (hBMP-4) was successfully produced in a prokaryotic host. For this aim, hBMP-4 cDNA was cloned in Escherichia coli (E. coli) and the protein was produced in a non-active aggregated form. After washing and solubilization, in vitro refolding of the rhBMP-4 monomer was performed using rapid dilution. In this study, different refolding conditions were tested for the dimerization of rhBMP-4 by one-factor-at-a-time variation. The dimerization process was found to be sensitive to pH, protein concentration and the presence of aggregation suppressors. In contrast, redox conditions and ionic strength did not impact refolding as expected. The dimer was separated from the remaining monomer, aggregates and host cell contaminants in a single step using cation-exchange membrane chromatography. The rhBMP-4 dimer produced in E. coli was biologically active as demonstrated by its capability to induce trophoblast differentiation and primitive streak induction of human pluripotent stem cells (hPSCs).
Keywords
- Cation-exchange membrane chromatography, Inclusion bodies, Recombinant human bone morphogenetic protein-4 (rhBMP-4), Refolding
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Biotechnology Reports, Vol. 18, e00249, 06.2018.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Solubilization and renaturation of biologically active human bone morphogenetic protein-4 from inclusion bodies
AU - Gieseler, Gesa Maria
AU - Ekramzadeh, Kimia
AU - Nölle, Volker
AU - Malysheva, Svitlana
AU - Kempf, Henning
AU - Beutel, Sascha
AU - Zweigerdt, Robert
AU - Martin, Ulrich
AU - Rinas, Ursula
AU - Scheper, Thomas
AU - Pepelanova, Iliyana
N1 - Funding information: This research project was partly supported by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) as part of the research program cluster Biokatalyse2021, P43. Another part of this work was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (from Regenerative Biology to Reconstructive Therapy) at the Hannover Medical School.
PY - 2018/6
Y1 - 2018/6
N2 - Biologically active human bone morphogenetic protein-4 (hBMP-4) was successfully produced in a prokaryotic host. For this aim, hBMP-4 cDNA was cloned in Escherichia coli (E. coli) and the protein was produced in a non-active aggregated form. After washing and solubilization, in vitro refolding of the rhBMP-4 monomer was performed using rapid dilution. In this study, different refolding conditions were tested for the dimerization of rhBMP-4 by one-factor-at-a-time variation. The dimerization process was found to be sensitive to pH, protein concentration and the presence of aggregation suppressors. In contrast, redox conditions and ionic strength did not impact refolding as expected. The dimer was separated from the remaining monomer, aggregates and host cell contaminants in a single step using cation-exchange membrane chromatography. The rhBMP-4 dimer produced in E. coli was biologically active as demonstrated by its capability to induce trophoblast differentiation and primitive streak induction of human pluripotent stem cells (hPSCs).
AB - Biologically active human bone morphogenetic protein-4 (hBMP-4) was successfully produced in a prokaryotic host. For this aim, hBMP-4 cDNA was cloned in Escherichia coli (E. coli) and the protein was produced in a non-active aggregated form. After washing and solubilization, in vitro refolding of the rhBMP-4 monomer was performed using rapid dilution. In this study, different refolding conditions were tested for the dimerization of rhBMP-4 by one-factor-at-a-time variation. The dimerization process was found to be sensitive to pH, protein concentration and the presence of aggregation suppressors. In contrast, redox conditions and ionic strength did not impact refolding as expected. The dimer was separated from the remaining monomer, aggregates and host cell contaminants in a single step using cation-exchange membrane chromatography. The rhBMP-4 dimer produced in E. coli was biologically active as demonstrated by its capability to induce trophoblast differentiation and primitive streak induction of human pluripotent stem cells (hPSCs).
KW - Cation-exchange membrane chromatography
KW - Inclusion bodies
KW - Recombinant human bone morphogenetic protein-4 (rhBMP-4)
KW - Refolding
UR - http://www.scopus.com/inward/record.url?scp=85046145900&partnerID=8YFLogxK
U2 - 10.1016/j.btre.2018.e00249
DO - 10.1016/j.btre.2018.e00249
M3 - Article
AN - SCOPUS:85046145900
VL - 18
JO - Biotechnology Reports
JF - Biotechnology Reports
M1 - e00249
ER -