Biological activity of human granulocyte-macrophage colony stimulating factor is maintained in a fusion with seed glutelin peptide.
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.
Sardana
Ravinder K
RK
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ontario, Canada.
Alli
Zaman
Z
Dudani
Anil
A
Tackaberry
Eilleen
E
Panahi
Mitra
M
Narayanan
Muthukrishnan
M
Ganz
Peter
P
Altosaar
Illimar
I
D015139:Blotting, Southern; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D018506:Gene Expression Regulation, Plant; D005983:Glutens / Q000235:genetics*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D012275:Oryza / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018749:RNA, Plant / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000502:physiology*; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*
D017931:DNA Primers; D010446:Peptide Fragments; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005983:Glutens; D016178:Granulocyte-Macrophage Colony-Stimulating Factor