Details
| Original language | English |
|---|---|
| Pages (from-to) | 80-87 |
| Number of pages | 8 |
| Journal | Process Biochemistry |
| Volume | 67 |
| Early online date | 5 Feb 2018 |
| Publication status | Published - Apr 2018 |
Abstract
Recombinant human bone morphogenetic protein-2 (rhBMP-2), a cystine-knot containing disulfide-linked homodimer, was produced in form of inclusion bodies (IBs) using E. coli BL21 (DE3) and SHuffle T7 Express. Non-reducing SDS-PAGE analysis revealed that rhBMP-2 was present within IBs in both strains as monomer and in form of disulfide-linked dimers. Purified dimeric disulfide-linked rhBMP-2 was obtained from IBs by two different methods. The first method involved classical solubilization using strong denaturants and subsequent refolding. The second method involved mild extraction without refolding. Both rhBMP-2 dimer variants were purified by Heparin-affinity chromatography. Mildly extracted rhBMP-2 was further purified by size-exclusion chromatography. The resulting dimeric rhBMP-2 variants were studied regarding bioactivity and folding status. In contrast to the rhBMP-2 dimer obtained by classical refolding it was shown that the disulfide-linked dimer obtained by mild extraction was not correctly folded e.g. the hydrophobic core not correctly formed. Moreover, refolded dimeric rhBMP-2 was bioactive and the mildly extracted dimeric rhBMP-2 did not show any bioactivity. Disulfide-bond analysis revealed that the intricate disulfide-bond pattern of the complex cystine-knot scaffold was not present in the IB embedded disulfide-linked rhBMP-2 dimer but was formed later during classical refolding of the reduced protein under appropriate redox conditions.
Keywords
- BRE-Luc assay, Cystine-knot, Disulfide-bond, Heparin-affinity chromatography, Mild extraction, Purification, Recombinant human bone morphogenetic protein-2, Refolding
ASJC Scopus subject areas
- Chemical Engineering(all)
- Bioengineering
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Process Biochemistry, Vol. 67, 04.2018, p. 80-87.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Properties of dimeric, disulfide-linked rhBMP-2 recovered from E. coli derived inclusion bodies by mild extraction or chaotropic solubilization and subsequent refolding
AU - Quaas, Bastian
AU - Burmeister, Laura
AU - Li, Zhaopeng
AU - Nimtz, Manfred
AU - Hoffmann, Andrea
AU - Rinas, Ursula
N1 - Funding Information: The authors gratefully acknowledge funding through the Forschergruppe “Gradierte Implantate” FOR2180 and the Exzellenzcluster “Rebirth” EXC62 , both Deutsche Forschungsgemeinschaft (DFG). In addition, the authors acknowledge Thilo Flörkemeier, Department of Orthopaedic Surgery (Annastift), for providing bone marrow for isolation of the human mesenchymal stem cells.
PY - 2018/4
Y1 - 2018/4
N2 - Recombinant human bone morphogenetic protein-2 (rhBMP-2), a cystine-knot containing disulfide-linked homodimer, was produced in form of inclusion bodies (IBs) using E. coli BL21 (DE3) and SHuffle T7 Express. Non-reducing SDS-PAGE analysis revealed that rhBMP-2 was present within IBs in both strains as monomer and in form of disulfide-linked dimers. Purified dimeric disulfide-linked rhBMP-2 was obtained from IBs by two different methods. The first method involved classical solubilization using strong denaturants and subsequent refolding. The second method involved mild extraction without refolding. Both rhBMP-2 dimer variants were purified by Heparin-affinity chromatography. Mildly extracted rhBMP-2 was further purified by size-exclusion chromatography. The resulting dimeric rhBMP-2 variants were studied regarding bioactivity and folding status. In contrast to the rhBMP-2 dimer obtained by classical refolding it was shown that the disulfide-linked dimer obtained by mild extraction was not correctly folded e.g. the hydrophobic core not correctly formed. Moreover, refolded dimeric rhBMP-2 was bioactive and the mildly extracted dimeric rhBMP-2 did not show any bioactivity. Disulfide-bond analysis revealed that the intricate disulfide-bond pattern of the complex cystine-knot scaffold was not present in the IB embedded disulfide-linked rhBMP-2 dimer but was formed later during classical refolding of the reduced protein under appropriate redox conditions.
AB - Recombinant human bone morphogenetic protein-2 (rhBMP-2), a cystine-knot containing disulfide-linked homodimer, was produced in form of inclusion bodies (IBs) using E. coli BL21 (DE3) and SHuffle T7 Express. Non-reducing SDS-PAGE analysis revealed that rhBMP-2 was present within IBs in both strains as monomer and in form of disulfide-linked dimers. Purified dimeric disulfide-linked rhBMP-2 was obtained from IBs by two different methods. The first method involved classical solubilization using strong denaturants and subsequent refolding. The second method involved mild extraction without refolding. Both rhBMP-2 dimer variants were purified by Heparin-affinity chromatography. Mildly extracted rhBMP-2 was further purified by size-exclusion chromatography. The resulting dimeric rhBMP-2 variants were studied regarding bioactivity and folding status. In contrast to the rhBMP-2 dimer obtained by classical refolding it was shown that the disulfide-linked dimer obtained by mild extraction was not correctly folded e.g. the hydrophobic core not correctly formed. Moreover, refolded dimeric rhBMP-2 was bioactive and the mildly extracted dimeric rhBMP-2 did not show any bioactivity. Disulfide-bond analysis revealed that the intricate disulfide-bond pattern of the complex cystine-knot scaffold was not present in the IB embedded disulfide-linked rhBMP-2 dimer but was formed later during classical refolding of the reduced protein under appropriate redox conditions.
KW - BRE-Luc assay
KW - Cystine-knot
KW - Disulfide-bond
KW - Heparin-affinity chromatography
KW - Mild extraction
KW - Purification
KW - Recombinant human bone morphogenetic protein-2
KW - Refolding
UR - http://www.scopus.com/inward/record.url?scp=85044017732&partnerID=8YFLogxK
U2 - 10.1016/j.procbio.2018.02.001
DO - 10.1016/j.procbio.2018.02.001
M3 - Article
AN - SCOPUS:85044017732
VL - 67
SP - 80
EP - 87
JO - Process Biochemistry
JF - Process Biochemistry
SN - 1359-5113
ER -