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Title: PRODUCTION OF BIOLOGICALLY ACTIVE GM-CSF IN SUGARCANE: A SECURE BIOFACTORY

Author
item WANG, MING-LI - HI AG RESEARCH CENTER
item GOLDSTEIN, CINDY - HI AG RESEARCH CENTER
item SU, WINSTON - UNIV OF HAWAII
item Moore, Paul
item Albert, Henrik

Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/13/2004
Publication Date: 5/20/2005
Citation: Wang, M., Goldstein, C., Su, W., Moore, P.H., Albert, H.H. 2005. Production of biologically active gm-csf in sugarcane: a secure biofactory. Transgenic Research. V14:P167-178.

Interpretive Summary: Over 300 transgenic sugarcane lines producing the human cytokine GM-CSF were analyzed for recombinant protein accumulation and activity levels. Accumulation of GM-CSF protein ranged from undetectable to 0.02% of total soluble protein. Human bone marrow cells (TF-1), which require GM-CSF for cell division, proliferated when growth media was supplemented with transgenic sugarcane extracts. Comparison to purified commercially produced GM-CSF indicated the sugarcane-produced protein had essentially identical activity levels. In a 14-month field trial, accumulation levels remained stable. During this period, no flowering of the trial plants occurred; no pollen or seed was produced. Drying, burning, and burial of the test plants effectively blocked possible routes for the transgenic sugarcane to enter the environment or food supply. Sugarcane may provide a highly secure system for biofactory production of pharmaceutical proteins. In addition to providing an important medical product, sugarcane engineered to produce pharmaceutical proteins to provide an important new source of income for farmers in tropical climates.

Technical Abstract: Over 300 transgenic sugarcane lines producing the human cytokine GM-CSF were analyzed for recombinant protein accumulation and activity levels. Expression constructs differed in use of the maize polyubiquitin 1, Mubi-1, or the sugarcane polyubiquitin 9, SCubi9, promoters, presence or absence of a C terminal HDEL tag for ER retention, and presence or absence of a poly-H tag for metal ion affinity purification. Accumulation of GM-CSF protein ranged from undetectable to 0.02% of total soluble protein. No significant difference was observed between the two promoters; however, the ER retention tag was required for higher accumulation levels. Human bone marrow cells (TF-1), which require GM-CSF for cell division, proliferated when growth media was supplemented with transgenic sugarcane extracts. Comparison to purified commercially produced GM-CSF indicated the sugarcane-produced protein had essentially identical activity levels. In a 14-month field trial, accumulation levels remained stable. During this period, no flowering of the trial plants occurred; no pollen or seed was produced. Drying, burning, and burial of the test plants effectively blocked possible routes for the transgenic sugarcane to enter the environment or food supply. Sugarcane may provide a highly secure system for biofactory production of pharmaceutical proteins.