Prokaryotic expression and protein purification

An in-frame insertion of the cDNA of the full human Smad1 gene with a glutathione S-transferase (GST) tag into the plasmid pGEX-4 T-1 was performed, and the resulting recombinant plasmid was transformed into Escherichia coli strain BL21. GST-tagged Smad1 protein expression was induced using 1 mM isopropyl-β-d-thiogalactopyranoside, and the resulting protein was purified with glutathione Sepharose 4B beads (GE Healthcare) according to the manufacturer’s instructions. Protein was digested by thrombin and dialyzed against buffer I [10 mM tris-HCl (pH 7.5), 1 mM dithiothreitol (DTT), and 0.2 M NaCl] at 4°C overnight. Proteins were brought to a final concentration of 200 μg/ml.

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