Purification and Identification of Natural Chemokines

A novel family of chemotactic cytokines or chemokines, essential for the directed migration of leukocytes to sites of inflammation, has been identified during the past decade. To obtain microgram amounts of natural chemokines, normal (e.g., freshly isolated leukocytes, connective tissue cell cultures) or malignant cell lines have to be selectively induced with endogenous (cytokines) or exogenous (bacterial, viral, or plant) products. We have developed a four-step procedure that allows for the complete purification of active C-C (MCP-1, MCP-2, MCP-3, RANTES, MIP-1alpha and MIP-1beta) and C-X-C (IL-8, GRO-alpha, GRO-beta, GRO-gamma, GCP-2, ENA-78, IP-10, PF-4, and CTAPIII/betaTG/NAP-2) chemokines from bulk volumes of culture supernatant. This method is applicable for the isolation of recombinant chemokines. Conditioned medium was first concentrated and partially purified on silicic acid or controlled pore glass beads. Further purification to homogeneity was achieved using heparin-Sepharose or antibody affinity chromatography, cation exchange FPLC, and reverse-phase HPLC. Purification of chemokines was monitored by testing column fractions for biological (chemotaxis) or immuno (RIA, ELISA) activity and protein content (SDS-PAGE). Homogeneous proteins were identified by amino-terminal or internal protein sequence analysis.