Biologically active recombinant rat granulocyte macrophage colony-stimulating factor produced in Escherichia coli.
Rat granulocyte 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.
Holowachuk
E W
EW
Research Institute, Mary Imogene Bassett Hospital, Cooperstown, New York 13326, USA.
Ruhoff
M S
MS
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001854:Bone Marrow Cells; D002352:Carrier Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015951:Factor Xa; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009995:Osmosis; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013917:Thrombin / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; C461599:maltose transport system, E coli; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013917:Thrombin; D015951:Factor Xa