Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells.
Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coli. The E. coli produced-protein--lacking any carbohydrate--had by far the highest specific activity observed for the recombinant hGM-CSFs.
Moonen
P
P
Mermod
J J
JJ
Ernst
J F
JF
Hirschi
M
M
DeLamarter
J F
JF
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003115:Colony-Stimulating Factors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D006801:Humans; D008970:Molecular Weight; D014411:Neoplastic Stem Cells / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D003115:Colony-Stimulating Factors; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases