Expression of murine and human granulocyte-macrophage colony-stimulating factors in S. cerevisiae: mutagenesis of the potential glycosylation sites.
Murine (m) and human (h) granulocyte--macrophage colony-stimulating factors (GM-CSF) have been expressed in large quantities in Saccharomyces cerevisiae using a secretion vector containing the promoter and leader sequences of the mating pheromone alpha-factor. Functionally active mGM-CSF was identified by a proliferation assay with a factor-dependent cell line and by a granulocyte--macrophage colony formation assay using bone marrow cells. The activity of hGM-CSF was confirmed by stimulation of granulocyte--macrophage colony formation using human cord blood cells. Murine GM-CSF with various apparent mol. wts (13, 18, 24, 34 and 40 kd, as well as a smear of higher mol. wts) was detected in yeast culture medium by protein blotting using a rat monoclonal antibody specific for the mGM-CSF N-terminal region peptide. Protein blotting using a rat monoclonal antibody specific for the hGM-CSF N-terminal region demonstrated that a 15.6-kd and higher mol. wt heterogeneous species were secreted. Mutations introduced at each of the two potential N-linked glycosylation sites in mGM-CSF showed that the 13-kd protein is not glycosylated and the major 18-kd protein is mainly glycosylated at the more C-terminal site, whereas the heterogeneous higher mol. wt species were not affected by the mutations. The N-terminal amino acid of the 13-kd protein was shown to be Ser which was four amino acids in the C-terminal direction from the fusion point.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003115:Colony-Stimulating Factors / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D006027:Glycosides / Q000032:analysis; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D003115:Colony-Stimulating Factors; D006027:Glycosides