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

authored by

Carla Lizbeth Segovia-Trinidad, Bastian Quaas, Zhaopeng Li, Antonina Lavrentieva, Yvonne Roger, Thomas Scheper, Andrea Hoffmann, Ursula Rinas

Abstract

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.

Organisation(s)

Institute of Technical Chemistry

External Organisation(s)

Hannover Medical School (MHH)
Helmholtz Centre for Infection Research (HZI)
NIFE - Lower Saxony Centre for Biomedical Engineering, Implant Research and Development

Type

Article

Journal

Protein Expression and Purification

Volume

184

ISSN

1046-5928

Publication date

08.2021

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Biotechnology

Sustainable Development Goals

SDG 3 - Good Health and Well-being

Electronic version(s)

https://doi.org/10.1016/j.pep.2021.105878 (Access: Closed)