Expression, purification and characterization of recombinant murine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast.
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.
Price
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Immunex Corporation, Seattle, WA 98101.
Mochizuki
D
D
March
C J
CJ
Cosman
D
D
Deeley
M C
MC
Klinke
R
R
Clevenger
W
W
Gillis
S
S
Baker
P
P
Urdal
D
D
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
D003115:Colony-Stimulating Factors; D007376:Interleukin-2; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins