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.

• PMID: 3066674
• PubMed
• PubTator

Reference Type

Journal Article

Authors

Cosman D, Deeley MC, Price V, Klinke R, Clevenger W, Hemenway T, Anderson D, Sassenfeld H, Urdal DL

Primary Lit For

Additional Lit For

Review For