Human interleukin-3 and granulocyte-macrophage colony stimulating factor: site-specific mutagenesis and expression in yeast.
The two human colony stimulating factors, interleukin-3 and granulocyte-macrophage colony stimulating factor, have been molecularly cloned and expressed as secreted proteins in yeast. In both cases, non-glycosylated and glycosylated forms of the molecules were produced. Removal of N-linked glycosylation sites from the genes by site-directed mutagenesis prevented addition of most of the sugar residues, but revealed a low level of residual O-linked glycosylation on a portion of the molecules. No difference in specific biological activity was found between the different forms of the proteins. It was found that a significant proportion of human granulocyte-macrophage colony stimulating factor was degraded by the yeast KEX2 protease that was cleaving after the dibasic sequence Arg-Arg at positions 23-24 of the mature protein. Site-specific mutagenesis was employed to change this sequence to Leu-Arg, and this change resulted in greatly increased expression levels of full length protein and biological activity.
Cosman
D
D
Immunex Corporation, Seattle, Washington 98101.
Deeley
M C
MC
Price
V
V
Klinke
R
R
Clevenger
W
W
Hemenway
T
T
Anderson
D
D
Sassenfeld
H
H
Urdal
D L
DL
D001853:Bone Marrow / Q000187:drug effects; D001854:Bone Marrow Cells; D003001:Cloning, Molecular; D003115:Colony-Stimulating Factors / Q000235:genetics* / Q000502:physiology; D005796:Genes; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics* / Q000502:physiology; D009154:Mutation; D011994:Recombinant Proteins / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D003115:Colony-Stimulating Factors; D006133:Growth Substances; D007377:Interleukin-3; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor