Expression and purification of bioactive high-purity mouse monokine induced by IFN-γ in Escherichia coli

MIG (monokine induced by IFN-gamma) is a CXC-chemokine (CXCL9). It plays important roles in regulation of immune activities, and knowledge of the protein in areas of allograft transplants, autoimmune diseases, and cancer therapy is evolving quickly. The non-tagged recombinant murine MIG (rMuMIG) is therefore required to facilitate the functional studies of this important chemokine. Here we present the use of a bacteria expression system to produce non-tagged rMuMIG. The coding sequence for MIG was cloned into the pET28a (+) vector that was transformed into Escherichia coli BL21 bE3). Expression of rMuMIG was induced by IPTG. Bacteria inclusion bodies containing the protein were isolated and washed to remove contaminated bacteria proteins, and resolved in Urea buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose cation exchange chromatography. The final preparation of the rMuMIG was more than 99%,, pure as measured by capillary electrophoresis and SDS-PAGE analysis. The biological activity of rMuMIG was demonstrated in a murine spleen cell chemotaxis assay with ED50 30 rigy/ml. Further experiments showed that rMuMIG could inhibit proliferation of mouse bone marrow cells in vivo. (C) 2007 Elsevier Inc. All rights reserved.