BIOLOGICALLY-ACTIVE RECOMBINANT RAT GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR PRODUCED IN ESCHERICHIA-COLI

Rat granuloycte macrophage colony-stimulating factor (rGM-CSF) cDNA was amplified and cloned, and recombinant-rGM-CSF (R-rGM-CSF) was expressed and isolated from Escherichia coli. The synthesis of R-rGM-CSF was directed by a modified, inducible maltose binding protein (MBP) gene fusion expression vector, pMTR-23, and secreted to the periplasm. The vector pMTR-23 contains a new multiple cloning site encoding a unique thrombin-sensitive cleavage site and short spacer arm which facilitates separation of the MBP from the foreign protein domain of hybrid proteins. Biologically active R-rGM-CSF was rapidly purified by a combination of affinity and ion exchange chromatography, with a yield of 1.5 mg of R-rGM-CSF per liter of cultured cells. The purified R-rGM-CSF, like the native molecule, exhibits potent biological activity in two rGM-CSF-specific assays, considerably enhancing the accessory activity of rat dendritic cells and stimulating the differentiation of dendritic cells from fresh cultures of rat bone marrow cells. Although dendritic cells are difficult to isolate from, tissues, the availability of R-rGM-CSF should now facilitate the development of large numbers of dendritic cells and the understanding of their regulatory role in the immune response. (C) 1995 Academic Press, Inc.