Purification and properties of bacterially synthesized human granulocyte-macrophage colony stimulating factor.
Human granulocyte-macrophage colony stimulating factor (GM-CSF) has been synthesized in high yield using a temperature inducible plasmid in Escherichia coli. The human GM-CSF is readily isolated from the bacterial proteins because of its differential solubility and chromatographic properties. The bacterially synthesized form of the human GM-CSF contains an extra methionine residue at position 1, but otherwise it is identical to the polypeptide predicted from the cDNA sequence. The specific activity of 2.9 X 10(7) units/mg of protein for purified bacterially synthesized human GM-CSF indicates that despite the lack of glycosylation, the molecule is substantially in its native conformation. This molecule stimulated the same number and type of both seven- and 14-day human bone marrow colonies as the CSF alpha preparation from human placental conditioned medium. Human GM-CSF had no activity on murine bone marrow or murine leukemic cells. There was no detectable, direct stimulation of adult human erythroid burst forming units (BFU-E) by the bacterially synthesized human GM-CSF. Although impure preparations containing native human GM-CSF (eg, human placental conditioned medium) stimulated the formation of mixed colonies, even in the presence of erythropoietin, the bacterially synthesized human GM-CSF failed to stimulate the formation of mixed colonies from adult human bone marrow cells. The bacterially synthesized human GM-CSF increased N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced superoxide production and lysozyme secretion. Antibody-dependent cytotoxicity and phagocytosis by human neutrophils was stimulated by the bacterially synthesized human GM-CSF and eosinophils were also activated in the antibody-dependent cytotoxicity assay.
D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects; D001854:Bone Marrow Cells; D002455:Cell Division / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004920:Erythropoiesis / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006098:Granulocytes / Q000187:drug effects; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D008970:Molecular Weight; D009113:Muramidase / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism
D007377:Interleukin-3; D011994:Recombinant Proteins; D013481:Superoxides; D004921:Erythropoietin; D009113:Muramidase