4.1. Protein Expression and Purification

Expression of CC0, CC3, CC5, and WT XCL1 was performed as previously described [15,19,29]. In brief, proteins were expressed recombinantly with a His₆-SUMO tag (pET28a expression vector, BL21 DE3 E. coli.). Cultures (grown in terrific broth with 50 mg/mL Kanamycin at 37 °C) were induced with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) once they reached an optical density of 0.5–0.7, after which they were grown for an additional 5 h at 37 °C. Cells were harvested by centrifugation (8000× g, 10 min) and stored at −80 °C. For protein purification, 50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, 10 mM imidazole, 0.1% (v/v) β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride (PMSF) was used to resuspend cell pellets, which were then lysed using a French press or by sonication. Centrifugation was used to collect inclusion bodies from cell lysates (12,000× g, 20 min) which were then resuspended and further purified along with the soluble fractions. Nickel column chromatography (nickel resin, Qiagen, Hilden, Germany) was used as an initial purification step. Elution fractions from the nickel columns underwent infinite dilution refolding into 20 mM Tris (pH 8.0), 200 mM sodium chloride, 10 mM cysteine, and 0.5 mM cystine. Refolding solutions were incubated at room temperature with gentle stirring overnight, then concentrated. ULP1 protease cleavage was used to remove the His₆-SUMO fusion tag, which was then separated from the protein of interest using either cation exchange chromatography (SP Sepharose Fast Flow resin, GE Healthcare, Chicago, IL, USA) or reverse nickel column chromatography (nickel resin, Qiagen). High-performance liquid chromatography was performed with a C18 column as a final purification step. Proteins were then frozen and lyophilized. Sample purities, identities, and homogeneities were checked with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectroscopy.