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 -