TY - JOUR

T1 - Refolding, purification, and characterization of constitutive-active human-Smad8 produced as inclusion bodies in ClearColi® BL21 (DE3)

AU - Segovia-Trinidad, Carla Lizbeth

AU - Quaas, Bastian

AU - Li, Zhaopeng

AU - Lavrentieva, Antonina

AU - Roger, Yvonne

AU - Scheper, Thomas

AU - Hoffmann, Andrea

AU - Rinas, Ursula

N1 - Funding Information: This work was funded by the research group Graded Implants FOR 2180 (Deutsche Forschungsgemeinschaft, DFG). CST received support from the German Academic Exchange Service ( DAAD ).

PY - 2021/8

Y1 - 2021/8

N2 - Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-β (TGF-β) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and β-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

AB - Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-β (TGF-β) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and β-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

KW - BMP-2

KW - BRE-luc assay

KW - Inclusion bodies

KW - Protein refolding

KW - Smad8

KW - TAT HIV

UR - http://www.scopus.com/inward/record.url?scp=85104072173&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2021.105878

DO - 10.1016/j.pep.2021.105878

M3 - Article

AN - SCOPUS:85104072173

VL - 184

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

M1 - 105878

ER -