Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells.
Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic 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 CaMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence) was fused to the N-terminal end of the GM-CSF transgene. For ease of purification, a 6-His tag was added to the 3' end of the GM-CSF cDNA. Addition of the TEV leader sequence increased protein production more than twofold compared to non-TEV controls. Initial batch cultivation studies indicated a maximum of 250 microg/L extracellular and 150 microg/L intracellular GM-CSF. Western blot analysis detected multiple peptides with masses from 14 to 30 kDa in the extracellular medium. The plant-produced GM-CSF was biologically active and could be bound to a nickel affinity matrix, indicating that both the receptor-binding region and the 6-His tag were functional. The batch production of GM-CSF was compared with the production of other recombinant proteins secreted by transformed tobacco cells. The recovery of secreted GM-CSF was increased by the addition of stabilizing proteins and by increasing salt in the growth medium to physiological levels.
James
E A
EA
Department of Chemical Engineering, Washington State University, Pullman, Washington 99164-2710, USA.
Wang
C
C
Wang
Z
Z
Reeves
R
R
Shin
J H
JH
Magnuson
N S
NS
Lee
J M
JM
D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006801:Humans; D010947:Plants, Toxic; D010957:Plasmids; D017800:Potyvirus / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000166:cytology / Q000235:genetics; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D006639:Histidine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor