Purification of Recombinant hLF Protein

The yeast strain Komagaetella phaffii X-33 transformed with the plasmid previously characterized for the expression of hLF was used, and its production was induced in the YPD selective culture medium (Sigma-Aldrich, St. Louis, MO, USA), incubating at 30 °C for 48 h with stirring at 150 RPM and with aeration. Recombinant proteins were purified from the culture medium using a Probond^TM affinity chromatography system (Thermo Fisher Scientific, Waltham, MA, USA), which uses a polymeric matrix activated with iminodiacetic acid and nickel (nickel is a di- and trivalent metal that allows us to selectively separate recombinant proteins tagged with polyhistidine residues, His Tag). Simple purification of secreted recombinant proteins is possible due to the relatively low levels of native secreted proteins.