Xk 16 40 column

IL-15 D8SQ108S, mhIL-15 and siIL-15 proteins were expressed in E. coli and purified following the procedure previously described for obtaining siIL-15 [27 (link)]. Briefly, the proteins expressed in the insoluble fraction were solubilized in an 8 M urea solution. Then, the insoluble material was eliminated by centrifugation and chaotrope was removed through Sephadex G-25 Fine packed on XK 16/40 column (GE Healthcare Life Sciences, USA). The sample collected from G-25 (without urea) was loaded onto Q Sepharose Fast Flow using a column of 1.6 × 10 cm (GE Healthcare, USA), which was operated at 5 mL/min. After a washing step, IL-15 containing fractions were eluted and the collected sample was applied to a C₄ column (1 × 25 cm, 10 µm, Vydac, USA) at a flow of 1 mL/min. Proteins were separated using a mobile phase containing solution A (0.1% TFA in water) and solution B (0.1% TFA in acetonitrile), using the same gradient as previously described [27 (link)]. Protein separations were monitored at 226 nm. The Bradford method was employed to determine the total protein concentration using BSA as standard [45 (link)].

Cell culture supernatant containing the RBD-CD protein was thawed and centrifuged at 4000× g for 10 min at 4 °C. The clarified cell culture supernatant was filtered through 0.45 µm and 0.2 µm cellulose nitrate filters (Sartorius). Afterward, protein purification was performed by metal affinity chromatography (IMAC). Briefly, filtered supernatant (250 mL) was equilibrated to a final concentration of PBS plus 2 mM imidazole (equilibrium buffer) by diluting in the same volume of PBS, and pH was adjusted to 7.4. Equilibrated supernatant was loaded at 0.5 mL/min at 4 °C onto an XK 16/40 column (GE Healthcare) packed with Ni-NTA agarose matrix (Qiagen, Hilden, Germany) previously equilibrated with 5 column volumes (CV) of equilibrium buffer. Then, the column was washed with 10 CV of washing buffer (PBS plus 10 mM imidazole, pH 7.4). The elution was performed with 5 CV of elution buffer (PBS plus 250 mM imidazole, pH 7.4). A buffer exchange to PBS was performed after purification with a PD10 desalting column (GE Healthcare, Chicago, IL, USA). The purified protein concentration was determined by measuring the absorbance at 280 nm (IMPLEN, Westlake Village, CA, USA). The purity of recombinant protein was assayed by densitometry scanning of protein gels, considering total protein concentration employing a BIO-RAD GS-800 scanner and the software Quantity One, version 4.6.9.