TY  - JOUR
A1  - Heinks, Tobias
A1  - Hettwer, Anette
A1  - Hiepen, Christian
A1  - Weise, Christoph
A1  - Gorka, Marcel
A1  - Knaus, Petra
A1  - Müller, Thomas D.
A1  - Loidl-Stahlhofen, Angelika
T1  - Optimized expression and purification of a soluble BMP2 variant based on in-silico design
T2  - Protein Expression and Purification
N2  - 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.
KW  - Bone morphogenetic protein 2
KW  - Aggregation-prone
KW  - In-silico-design
KW  - Hydrophilicity enhanced hBMP2 variant
KW  - E. coli SHuffle® T7
KW  - pH-shift elution
Y1  - 2021
UR  - https://whge.opus.hbz-nrw.de/frontdoor/index/index/docId/4022
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:1010-opus4-40229
SN  - 1046-5928
VL  - 186
IS  - October
SP  - 105918
ER  -