Expression of human granulocyte-macrophage colony-stimulating factor gene in insect cells by a baculovirus vector.
A plasmid pAc373GM-CSF was constructed and co-transfected into Spodoptera frugiperda (Sf9) cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The recombinant virus vAc373GM-CSF was identified and purified by several rounds of plaque hybridization. By assaying the culture medium, we demonstrated recombinant virus infected Sf9 cells expressing hGM-CSF. Recombinant hGM-CSFs with apparent molecular masses of 14.5, 15.5 and 16.5 kDa were detected by the Western blot method. All 3 forms have biological activity of hGM-CSF. Following N-glycanase treatment, a single band of 14.5-15.5 kDa appeared in SDS-PAGE. Western blot analysis of expression in Sf9 cell treated with tunicamycin revealed only the presence of the 14.5 kDa species. Thus, the signal sequence of recombinant hGM-CSF could be recognized and cleaved by infected insect cell and the resultant molecule secreted into the media.
Department of Biochemistry, University of North Texas/Texas College of Osteopathic Medicine, Denton 76203.
C J
CJ
Chiou
M C
MC
Wu
D000818:Animals; D002460:Cell Line; D003115:Colony-Stimulating Factors / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D008970:Molecular Weight; D009036:Moths; D010957:Plasmids
D003115:Colony-Stimulating Factors; D006133:Growth Substances; D016178:Granulocyte-Macrophage Colony-Stimulating Factor