Purification and properties of bacterially synthesized human granulocyte-macrophage colony stimulating factor

Abstract

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.

Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy

“…rhGM-CSF with the identical amino acid sequence to the naturally occurring protein has been produced in mammalian chinese hamster ovary (CHO) cells and is variably glycosylated on both O-linked and Nlinked sites, which gives rise to a heterogeneous population of proteins with molecular weights of 18-30 kD [19]. The expression of GM-CSF in Escherichia coli yields a non-glycosylated product with a molecular weight of 14 kD [20]. The yeast-expressed GM-CSF differs from the native protein in the substitution of a leucine residue for arginine at position 23 of the amino acid sequence (A. Troutt, Immunex Corporation, Seattle, WA; personal communication).…”

Mechanisms of the in vitro fungicidal effects of human neutrophils against Penicillium marneffei induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

“…Defensins, rich in the azurophilic granules, were shown to express a potent killing activity against C. neoformans [36]. Interestingly, several investigators demonstrated that GM‐CSF induced lysozyme secretion by neutrophils [13], promoted cell–cell adhesion through enhanced expression of β 2 ‐integrins on their surface [15] and enhanced the neutrophil fungicidal activity against T. glabrata [16]. In our study the fungicidal activity of granular lysate was significantly abrogated by heat treatment, but was partially resistant to this treatment, indicating that the granular fungicidal materials consisted largely of heat‐labile molecules.…”

“…It specifically binds to neutrophil plasma membranes, and stimulates the development of both granulocyte and macrophage colonies and activates neutrophils [12]. In previous studies, GM‐CSF was demonstrated to induce superoxide production, lysozyme release, phagocytosis of heat‐killed yeast cells by neutrophils and their antibody‐dependent cell‐mediated cytotoxicity [13], enhance survival [14], promote cell–cell adhesion and alter surface receptor expression of neutrophils [15]. GM‐CSF also enhances the killing of Torulopsis glabrata by neutrophils [16].…”

Haemopoietic growth factors induce human basophil migration in vitro

“…The following reagents were purchased as indicated: PBS and FCS (GIBCO, Grand Island, New York, U.S.A.); Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden); anti-Leu-5b (CD2), anti-Leu-12 (CD19) and anti-Leu-M3 (CD14) (Becton Dickinson & Co., Mountain View, California, U.S.A.); anti-CD-16 (Immunotech, Marceille, France); goat anti-mouse IgG (affinity purified, Tago, BUrlingame, California, U.S.A.); recombinant (r) human C5a (Sigma Chemical Co., St Louis, Missouri, U.S.A.); and human rIL-4 [8] (Genzyme, Boston, Massachusetts, U.S.A.). Purified human rGM-CSF [9] was donated by Sumitomo Pharmaceutical Co. Ltd (Tokyo, Japan). Purified human rG-CSF [10] was provided by Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan).…”