Purification of recombinant C-terminal Smp76 protein

In order to express the fusion protein Thioredoxine-C-terminal, pET22b-Thio-C-terminal plasmid was transformed into E. coli BL21 (DE3) using electroporation. The pellet was harvested and the fusion protein was purified using the Ni–NTA agarose resin columns (QIAGEN) as previously described (Vargas-Jaimes et al. 2017 (link)). HPLC purification was further performed using a C18 RP-HPLC column (250 × 10 mm, 5 µm; Vydac, California, United States). The purified fusion protein Thioredoxine-C-terminal was digested with enterokinase (New England Biolabs) in 200 mM Tris HCl (pH 8.0), 500 mM NaCl, 20 mM CaCl₂ for 16 h at 25 °C. Then, the pure recombinant C-terminal of Smp76 was finally isolated and purified by HPLC as described above.