title: Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants.
abstract: Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been reported to express an endogenous insulin-like protein, we also looked for any effect of the human growth factor in transgenic plants, but no developmental or morphological differences with wild type tobacco or rice plants were detected. Since insulin and hIGF-1 share some overlapping roles, hIGF-1 may become a substitute therapeutic agent in subjects with certain defects in their insulin receptor signaling. Hence, if the full beneficial potential of rthIGF-1 is achieved, it is expected that in the future the demand will likely increase significantly.
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada.
authors.forename: Mitra
authors.initials: M
authors.lastname: Panahi
authors.forename: Zaman
authors.initials: Z
authors.lastname: Alli
authors.forename: Xiongying
authors.initials: X
authors.lastname: Cheng
authors.forename: Loubaba
authors.initials: L
authors.lastname: Belbaraka
authors.forename: Jaafar
authors.initials: J
authors.lastname: Belgoudi
authors.forename: Ravinder
authors.initials: R
authors.lastname: Sardana
authors.forename: Jenny
authors.initials: J
authors.lastname: Phipps
authors.forename: Illimar
authors.initials: I
authors.lastname: Altosaar
mesh_terms: D001425:Bacterial Outer Membrane Proteins; D015153:Blotting, Western; D003062:Codon / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000378:metabolism*; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000378:metabolism*; D018272:Porins; D011401:Promoter Regions, Genetic / Q000235:genetics; D011991:Receptors, Virus / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism*; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*; D014407:Tumor Cells, Cultured
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003062:Codon; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; D007334:Insulin-Like Growth Factor I