Figure 1
From: A strategy to discover decoy chemokine ligands with an anti-inflammatory activity
Production and purification of recombinant human CCL22 and CCL17.
(a) E. coli BL21 (DE3) cells were transformed with pET32-TRX-(His)6-CCL17 or pET32-TRX-(His)6-CCL22 expression vectors. Cultures (1 ml) were collected and saved for SDS-PAGE (12%) analysis (lanes 2 and 5). Then, the production of TRX-(His)6 fusion CCL17 and CCL22 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) (lanes 3, 4, 6 and 7). « S » and « I » refer to the soluble (lanes 3 and 6) and insoluble (lanes 4 and 7) fractions, respectively. « NI » refers to the non-induced protein fraction (lanes 2 and 5). Lane 1: molecular weight standards. The arrow indicates the expected fused CCL17 or CCL22 band at ≈26 kDa corresponding to CCL17 or CCL22 + TRX + (His)6. (b) Purification steps of the human CCL22 on Ni2+-nitrilotriacetic acid (Ni-NTA) column. Samples from the « washing steps » were analysed on a 12% SDS-PAGE. Lane 1: molecular weight standards. Lane 2: non-induced fraction “NI”. Lane 3: soluble fraction “S”. Lane 4: insoluble fraction “I”. Lane 5: flow-through after incubating the insoluble fraction with the column. Lane 6: flow-through after denaturation. Lane 7: flow-through after β-cyclodextrin lavage. Lane 8: flow-through after oxidation. Lane 9: peak fractions eluted with imidazole. (c) Samples from enterokinase-digested chemokines were analysed on a 20% SDS-PAGE. Lane 1: molecular weight standards. Lanes 2 and 3: CCL22 and CCL17 flow-through fractions after enterokinase digestion and a second round Ni-NTA. CK: chemokine; (His)6: hexahistidin; TRX: thioredoxin.
