Expression of murine GM-CSF in recombinant Aspergillus niger

Recombinant Aspergillus niger was constructed to express and secrete a biologically active murine granulocyte macrophage-colony stimulating factor (mGM-CSF). A 500 bp fragment encoding the signal peptide and mature mGM-CSF was cloned between the promoter and terminator of glyceraldehyde-3-phosphate dehydrogenase (gpd). The hygromycin phosphotransferase gene (hph) was used as a selection marker for the fungal transformants. An expression vector was introduced into A. niger ATCC 9642, and a Northern blot analysis indicated the presence of a considerable amount of transcripts from the introduced mGM- CSF. The biological activity of recombinant mGM-CSF (rmGM-CSF) isolated from the culture filtrate was confirmed by measuring the proliferation of the GM- CSF dependent FDC-P1 cell line. It appeared that rmGM-CSF was amenable to the proteolytic activity produced by A. niger, since biological activity was only observed when the transformants were grown in a protease-repressing medium, and the activity of rmGM CSF dramatically decreased with an increase of age of the culture. The yield of rmGM-CSF, as determined by ELISA, was 640 ng/1 of culture filtrate. Accordingly, its specific activity is estimated to be approximately two-and-a-half times higher than that of a commercial preparation from E. coli.