Optimized expression and purification of a soluble BMP2 variant based on in-silico design

  • Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of L-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency.
Metadaten
Verfasserangaben:Tobias Heinks, Anette Hettwer, Christian Hiepen, Christoph Weise, Marcel Gorka, Petra Knaus, Thomas D. Müller, Angelika Loidl-Stahlhofen
URN:urn:nbn:de:hbz:1010-opus4-40229
DOI:https://doi.org/10.1016/j.pep.2021.105918
ISSN:1046-5928
Titel des übergeordneten Werkes (Englisch):Protein Expression and Purification
Dokumentart:Wissenschaftlicher Artikel
Sprache:Englisch
Datum der Veröffentlichung (online):14.12.2021
Jahr der Erstveröffentlichung:2021
Veröffentlichende Institution:Westfälische Hochschule Gelsenkirchen Bocholt Recklinghausen
Datum der Freischaltung:14.12.2021
Freies Schlagwort / Tag:Aggregation-prone; Bone morphogenetic protein 2; E. coli SHuffle® T7; Hydrophilicity enhanced hBMP2 variant; In-silico-design; pH-shift elution
Jahrgang:186
Ausgabe / Heft:October
Seitenzahl:12
Erste Seite:105918
Fachbereiche / Institute:Fachbereiche / Wirtschaftsingenieurwesen
Lizenz (Deutsch):License LogoEs gilt das Urheberrechtsgesetz

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