For construction of human LIF expression plasmid, a nucleic acid fragment encoding the full‐length human LIF with a poly His‐tag at the C terminus was synthesized and inserted in pcDNA3.1(+) vector at NotI/XbaI site by GenScript USA, Inc. The Expi293 expression system (Life Technologies, A14524) was used to generate the conditioned media for purification. Pyrogen‐free reagents were employed. To inactivate endotoxin, columns and equipment (Akta Explorer System, GE Healthcare) were sanitized prior to use by exposure to 0.5 N NaOH for approximately 40 min, as described (Xin et al, 2021 (link)). LPS levels were measured by ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript, Cat. No. L00350).
Conditioned medium was adjusted to 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 20 mM imidazole, and 0.01% Tween 20 before applying to HisTrap™ HP 1 ml column (GE Healthcare). The column was then washed with buffer containing 50 mM imidazole, and the elution of protein was achieved in 100 mM imidazole. Eluted protein was buffer exchanged into PBS containing 0.01% Tween 20. Recombinant protein was more than 95% pure, with LPS levels of approximately 0.02 EU/mg protein.
Conditioned medium was adjusted to 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 20 mM imidazole, and 0.01% Tween 20 before applying to HisTrap™ HP 1 ml column (GE Healthcare). The column was then washed with buffer containing 50 mM imidazole, and the elution of protein was achieved in 100 mM imidazole. Eluted protein was buffer exchanged into PBS containing 0.01% Tween 20. Recombinant protein was more than 95% pure, with LPS levels of approximately 0.02 EU/mg protein.
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