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Title: EFFICIENT TRANSIENT EXPRESSION OF HUMAN GM-CSF PROTEIN IN NICOTIANA BENTHAMIANA USING POTATO VIRUS X VECTOR

Author
item ZHOU, FENGYONG - HARC
item WANG, MING-LI - HARC
item Albert, Henrik
item Moore, Paul
item ZHU, YUN - HARC

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/16/2005
Publication Date: 4/20/2006
Citation: Zhou, F., Wang, M., Albert, H.H., Moore, P.H., Zhu, Y.J. 2006. Efficient transient expression of human gm-csf protein in nicotiana benthamiana using potato virus x vector. Applied Microbiology and Biotechnology 72:756:762.

Interpretive Summary: A human cytokine, granulocyte macrophage colony stimulating factor (GMCSF) was produced in Nicotiana benthamiana (a tobacco relative) after infection with a plant virus (PVX) engineered to express GMCSF. Infected plants accumulated GMCSF at up to 2% of total soluble protein, and this protein showed native protein activity in a cell proliferation bioassay. GMCSF has important medical applications, including treatment of bone marrow transplant recipients. Production of GMCSF in plants may offer a safe and economical alternative means of producing pharmaceutical proteins while providing increased income opportunities for farmers.

Technical Abstract: Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a glycoprotein with important clinical applications for the treatment of neutropenia and aplastic anemia and reducing infections associated with bone marrow transplants. We evaluated the potential for using a potato virus X (PVX) viral vector system for efficient expression of the biologically functional GM-CSF protein in Nicotiana benthamiana leaves. The GM-CSF gene was cloned into PVX viral expression vector (PVX201) driven with the CaMV 35S promoter and followed with a 6 ' His tag at 3' end for improved protein purification. Gene transfer was accomplished by inoculating plants with the plasmid DNA of PVX vector containing GM-CSF gene. The expression level and size of recombinant GM-CSF protein were determined with ELISA and confirmed by Western blot analysis. The results showed that: 1) Leaf age significantly affects GM-CSF protein productivity with younger leaves expressing higher amounts of recombinant protein. 2) Recombinant protein accumulation is temporally regulated within a given leaf so that the protein level reaches a maximum at 7 days post-inoculation and subsequently declines. 3) The two leaves immediately above the inoculated leaves play an important role for systemic GM-CSF accumulation in higher younger leaves. Protein extract of infected N. benthamiana leaves contained recombinant human GM-CSF protein, in concentrations of up to 2% of total soluble protein that actively stimulated the growth of human TF-1 cells suggesting that the recombinant GM-CSF expressed via PVX viral vector was biological active. Thus, the plant PVX viral vector system has potential for eventual production of biologically active GM-CSF protein.