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

Heinks T, Hettwer A, Hiepen C, Weise C, Gorka M, Knaus P, Mueller TD, Loidl-Stahlhofen A (2021)
Protein Expression and Purification 186: 105918.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Autor*in
Heinks, TobiasUniBi; Hettwer, Anette; Hiepen, Christian; Weise, Christoph; Gorka, Marcel; Knaus, Petra; Mueller, Thomas D.; Loidl-Stahlhofen, Angelika
Abstract / Bemerkung
Bone morphogenetic protein 2 (BMP2(1)) 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 (R) 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.
Stichworte
Bone morphogenetic protein 2; Aggregation-prone; In-silico-design; Hydrophilicity enhanced hBMP2 variant; E. coli SHuffle (R) T7; pH-shift; elution
Erscheinungsjahr
2021
Zeitschriftentitel
Protein Expression and Purification
Band
186
Art.-Nr.
105918
ISSN
1046-5928
eISSN
1096-0279
Page URI
https://pub.uni-bielefeld.de/record/2957065

Zitieren

Heinks T, Hettwer A, Hiepen C, et al. Optimized expression and purification of a soluble BMP2 variant based on in-silico design. Protein Expression and Purification. 2021;186: 105918.
Heinks, T., Hettwer, A., Hiepen, C., Weise, C., Gorka, M., Knaus, P., Mueller, T. D., et al. (2021). Optimized expression and purification of a soluble BMP2 variant based on in-silico design. Protein Expression and Purification, 186, 105918. https://doi.org/10.1016/j.pep.2021.105918
Heinks, Tobias, Hettwer, Anette, Hiepen, Christian, Weise, Christoph, Gorka, Marcel, Knaus, Petra, Mueller, Thomas D., and Loidl-Stahlhofen, Angelika. 2021. “Optimized expression and purification of a soluble BMP2 variant based on in-silico design”. Protein Expression and Purification 186: 105918.
Heinks, T., Hettwer, A., Hiepen, C., Weise, C., Gorka, M., Knaus, P., Mueller, T. D., and Loidl-Stahlhofen, A. (2021). Optimized expression and purification of a soluble BMP2 variant based on in-silico design. Protein Expression and Purification 186:105918.
Heinks, T., et al., 2021. Optimized expression and purification of a soluble BMP2 variant based on in-silico design. Protein Expression and Purification, 186: 105918.
T. Heinks, et al., “Optimized expression and purification of a soluble BMP2 variant based on in-silico design”, Protein Expression and Purification, vol. 186, 2021, : 105918.
Heinks, T., Hettwer, A., Hiepen, C., Weise, C., Gorka, M., Knaus, P., Mueller, T.D., Loidl-Stahlhofen, A.: Optimized expression and purification of a soluble BMP2 variant based on in-silico design. Protein Expression and Purification. 186, : 105918 (2021).
Heinks, Tobias, Hettwer, Anette, Hiepen, Christian, Weise, Christoph, Gorka, Marcel, Knaus, Petra, Mueller, Thomas D., and Loidl-Stahlhofen, Angelika. “Optimized expression and purification of a soluble BMP2 variant based on in-silico design”. Protein Expression and Purification 186 (2021): 105918.

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