« Previous AbstractBrassinosteroid-related transcription factor BIL1/BZR1 increases plant resistance to insect feeding    Next Abstract"GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction" »

EMBO J

Title:		Expression of murine and human granulocyte-macrophage colony-stimulating factors in S. cerevisiae: mutagenesis of the potential glycosylation sites
Author(s):		Miyajima A; Otsu K; Schreurs J; Bond MW; Abrams JS; Arai K;
Address:
Journal Title:		EMBO J
Year:		1986
Volume:		5
Issue:		6
Page Number:		1193 - 1197
DOI:		10.1002/j.1460-2075.1986.tb04346.x
ISSN/ISBN:		0261-4189 (Print) 1460-2075 (Electronic) 0261-4189 (Linking)
Abstract:		"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"
Keywords:		"Amino Acids/analysis Animals Antibodies, Monoclonal Base Sequence Colony-Stimulating Factors/*genetics/isolation & purification/pharmacology Glycosides/analysis Humans Mice Molecular Weight *Mutation Plasmids Saccharomyces cerevisiae/*genetics;"
Notes:		"MedlineMiyajima, A Otsu, K Schreurs, J Bond, M W Abrams, J S Arai, K eng England 1986/06/01 EMBO J. 1986 Jun; 5(6):1193-7. doi: 10.1002/j.1460-2075.1986.tb04346.x"