Details
| Original language | English |
|---|---|
| Qualification | Doctor rerum naturalium |
| Awarding Institution | |
| Supervised by |
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| Thesis sponsors |
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| Date of Award | 11 Apr 2023 |
| Place of Publication | Hannover |
| Publication status | Published - 2023 |
Abstract
Sustainable Development Goals
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Hannover, 2023. 88 p.
Research output: Thesis › Doctoral thesis
}
TY - BOOK
T1 - Refolding, purification, and characterization of human-Smad8 produced in E. coli in form of inclusion bodies
AU - Segovia Trinidad, Carla Lizbeth
PY - 2023
Y1 - 2023
N2 - Smad8 is a transcriptional regulator involved in many cellular processes, such as cell differentiation. During tissue regeneration, cell differentiation takes place. Particularly, it has been reported before that the combined effect of a truncated form of Smad8 (hcSmad8) and BMP2 induces the differentiation of mesenchymal stem cells (MSCs) into tenocytes (Hoffmann et al.,2006). Smad8 is therefore considered a promising molecule that can be used to enhance tendon regeneration. This can be achieved, for example, by loading implants with hcSmad8. Hence, there is an interest in producing cSmad8 in a recombinant way. Herein, it is reported the production, purification, and characterization of human cSmad8 produced in E. coli. To allow the internalization of cSmad8, the Transactivator Sequence (TAT) of the HIV was genetically attached. TAT-hcSmad was refolded from inclusion bodies, purified by heparin chromatography, characterized by physicochemical methods, and finally, the internalization and biological activity were tested in MSCs. The purified TAT-hcSmad8 displayed a hydrophobic core consisting of a mixture of α-helixes and β-sheets. TAT-hcSmad8 was also able to efficiently internalize into C3H10T1/2 cells. Luciferase-based reporter assays suggested that TAT-hcSmad8 is active as it interferes with the BMP signaling pathway. More research needs to be done around Smad8 to understand better its role in cells and with this fully exploit Smad8 potential.
AB - Smad8 is a transcriptional regulator involved in many cellular processes, such as cell differentiation. During tissue regeneration, cell differentiation takes place. Particularly, it has been reported before that the combined effect of a truncated form of Smad8 (hcSmad8) and BMP2 induces the differentiation of mesenchymal stem cells (MSCs) into tenocytes (Hoffmann et al.,2006). Smad8 is therefore considered a promising molecule that can be used to enhance tendon regeneration. This can be achieved, for example, by loading implants with hcSmad8. Hence, there is an interest in producing cSmad8 in a recombinant way. Herein, it is reported the production, purification, and characterization of human cSmad8 produced in E. coli. To allow the internalization of cSmad8, the Transactivator Sequence (TAT) of the HIV was genetically attached. TAT-hcSmad was refolded from inclusion bodies, purified by heparin chromatography, characterized by physicochemical methods, and finally, the internalization and biological activity were tested in MSCs. The purified TAT-hcSmad8 displayed a hydrophobic core consisting of a mixture of α-helixes and β-sheets. TAT-hcSmad8 was also able to efficiently internalize into C3H10T1/2 cells. Luciferase-based reporter assays suggested that TAT-hcSmad8 is active as it interferes with the BMP signaling pathway. More research needs to be done around Smad8 to understand better its role in cells and with this fully exploit Smad8 potential.
U2 - 10.15488/13924
DO - 10.15488/13924
M3 - Doctoral thesis
CY - Hannover
ER -