title: Expression, purification and characterization of recombinant murine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast.
abstract: Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.
authors.affiliation: Immunex Corporation, Seattle, WA 98101.
authors.forename: V
authors.initials: V
authors.lastname: Price
authors.forename: D
authors.initials: D
authors.lastname: Mochizuki
authors.forename: C J
authors.initials: CJ
authors.lastname: March
authors.forename: D
authors.initials: D
authors.lastname: Cosman
authors.forename: M C
authors.initials: MC
authors.lastname: Deeley
authors.forename: R
authors.initials: R
authors.lastname: Klinke
authors.forename: W
authors.initials: W
authors.lastname: Clevenger
authors.forename: S
authors.initials: S
authors.lastname: Gillis
authors.forename: P
authors.initials: P
authors.lastname: Baker
authors.forename: D
authors.initials: D
authors.lastname: Urdal
mesh_terms: D000818:Animals; D002417:Cattle; D003115:Colony-Stimulating Factors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D006098:Granulocytes; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008264:Macrophages; D051379:Mice; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D003115:Colony-Stimulating Factors; D007376:Interleukin-2; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins