Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells

Human. granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoi etic growth factor, was produced and secreted from tobacco cell suspensions . The GM-CSF cDNA was carried by a binary vector under the control of the C aMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated reg ion from the tobacco etch virus (TEV leader sequence) was fused to the N-te rminal end of the GM-CSF transgene, For ease of purification, a g-His tag w as added to the 3' end of the GM-CSF cDNA. Addition of the TEV leader seque nce increased protein production more than twofold compared to non-TEV cont rols, Initial batch cultivation studies indicated a maximum of 250 mu g/L e xtracellular and 150 mu g/L intracellular GM-CSF. Western blot analysis det ected multiple peptides with masses from 14 to 30 kDa in the extracellular medium. The plant-produced GM-CSF was biologically active and could be boun d to a nickel affinity matrix, indicating that both the receptor-binding re gion and the g-His tag were functional. The batch production of GM-CSF was compared with the production of other recombinant proteins secreted by tran sformed tobacco cells. The recovery of secreted GM-CSF was increased by the addition of stabilizing proteins and by increasing salt in the growth medi um to physiological levels. (C) 2000 Academic Press.