Recombinant Asc l 5 Protein Expression

The cDNA sequence coding for Asc l 5 protein was subcloned without signal peptide into the expression vector pET-45b+ (GenScript, USA) and E. coli Origami (DE3) competent cells were transformed by electroporation. The E. coli cultures were grown overnight in Luria–Bertani medium containing 100 mg/L ampicillin at 37 °C. Expression of the recombinant protein was induced by adding IPTG to a final concentration of 1 mM at an OD₆₀₀ of 0.5. After cultivation for additional 5 h at 37 °C, E. coli cultures were centrifuged (30 min, 3500 rpm, 4 °C). Induced cultures were re-suspended in native buffer (50 mM NaH₂PO₄, 300 mM NaCl), incubated with lysozyme (1 mg/mL, 30 min on ice) and sonicated. Lysates were incubated with Nickel-Nitrilotriacetic Acid (Ni-NTA) resin (Invitrogen) for one hour, washed with native buffer plus 20 mM imidazole, and eluted with native buffer plus 250 mM imidazole as a 6xHis-tagged protein [8 (link)]. The purified recombinant was directly subjected to SDS-PAGE analysis.