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Abstract

Human granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with many applications in clinical medicine, was produced specifically in the seeds of transgenic tobacco plants. Two rice endosperm-specific glutelin promoters of different size and sequence, Gt1 and Gt3, were used to direct expression. Also in the Gt3 construct, the GM-CSF coding region was in fusion with the first 24 nucleotides of the mature rice glutelin sequence at its 5' end. With the Gt1 construct plants, seed extracts contained the recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein. Transgenic seed extracts actively stimulated the growth of human TF-1 cells suggesting that the seed-produced GM-CSF alone and in fusion with the rice glutelin peptide was stable and biologically active. Furthermore, native tobacco seed extracts inhibited the activity of E. coli-derived GM-CSF in this cytokine-dependent cell line. The seeds of F1 generation plants retained the biological activity of human GM-CSF protein indicating that the human coding sequence was stably inherited. The feasibility of oral delivery of such stable seed-produced cytokines is discussed.

Improved Production of the Human Granulocyte-Macrophage Colony Stimulating Factor in Transgenic Arabidopsis thaliana Seeds Using a Dual Sorting Signal Peptide

“…1) from KD and AF transgenic plants. This was similar to level of rhGM-CSF detected in transgenic tobacco seeds (Sardana et al 2002). A clearly observed strong band of similar molecular weight was observed by Western blotting in both KD and AF transformants.…”

“…The human GM-CSF has been expressed in a number of plants, with the highest accumulation reaching 0.03% of TSP (Sardana et al 2002;Wang et al 2005). In this study, a novel approach is used to enhance the accumulation of recombinant proteins in transgenic plants.…”

“…A recombinant human granulocytemacrophage colony stimulating factor (hGM-CSF) protein has been produced in Escherichia coli (Libby et al 1987), yeast (Cantrell et al 1985), COS-7 monkey cells (Lee et al 1985), human lymphoblastoid Namalwa cells (Okamoto et al 1990), mouse bladder uroepithelium (Ryoo et al 2001), insect cells (Au et al 1996), tomato , rice (Kim et al 2008a, b;Lee et al 2002;Shin et al 2003), tobacco (Sardana et al 2002), and sugarcane (Wang et al 2005). It is postulated that in plants, a four-helix cytokine GM-CSF utilizes alpha-helical-rich structural domains for compact folding and stable deposition, although its actual status remains unknown (Sardana et al 2002). Different plant expression systems vary in their accumulation of recombinant hGM-CSF (rhGM-CSF).…”

“…Different plant expression systems vary in their accumulation of recombinant hGM-CSF (rhGM-CSF). For example, the rhGM-CSF protein reaches up to 2% of total soluble protein (TSP) of young leaves inoculated with a potato virus X viral vector in a Nicotiana benthamiana transient expression system (Zhou et al 2006), while this reaches 0.03% of TSP in transgenic tobacco seeds (Sardana et al 2002). The high production cost of rhGM-CSF remains a limiting factor in mass production of this protein in using plants, particularly due to the low deposition of this protein in harvestable organs.…”

Rapid transient expression of human granulocyte-macrophage colony-stimulating factor in two industrial cultivars of tobacco (Nicotiana tabacum L.) by agroinfiltration

“…The active form of the protein is found extracellulary as a homodimer. To date, production of GM-CSF in plant-based expression systems has been achieved in cell cultures of tobacco [11], [12], [13], [14], [15], [16] and rice [17]. Further, whole tobacco plants transfected with viral vectors have also been employed [18].…”