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.

Tobacco as biofactory for biologically active hPL production: a human hormone with potential applications in type-1 diabetes

“…This effect could be due to the noxious nature of tobacco, or any compound from the protein buffer, as previously reported by other authors (Panahi et al 2003;Panahi et al 2004). Further investigation is required to confirm that the hPL produced in tobacco plants has an enhanced proliferation activity when used at higher doses, and to clarify the effect of wt sample on cell growth of INS-1 cells.…”

Expression of biologically active human insulin‐like growth factor 1 in Arabidopsis thaliana seeds via oleosin fusion technology

“…The expression of hIGF-1 in transformed A. thaliana seeds was approximately seven times higher than previously reported in plants. In an earlier report, recombinant hIGF-1 was expressed in transgenic rice and tobacco [30]; the expression of hIGF-1 in transgenic rice and tobacco was 113 ± 24 and 70 ± 13 ng of rthIGF-1/mg total protein, respectively. Reaction time and temperature were tested for TEVP cleavage in our study.…”

“…Use of transgenic plants as a vehicle for protein expression has many benefits considering its cost-efficiency, scaling-up capacity, high-product quality, and low-contamination risks (see reviews in [26][27][28][29]). Transgenic plants producing recombinant human insulin-like growth factor-1 (hIGF-1) were recently obtained by Panahi et al [30]; the plant-derived recombinant hIGF-1 caused differentiation of human neuroblastoma cell line SH-SY5Y, indicating its biological activity. However, the expression level was relatively low.…”

Human CD14 Expressed in Seeds of Transgenic Tobacco Displays Similar Proteolytic Resistance and Bioactivity with its Mammalian-produced Counterpart

“…Nearly 80% of the world population still depends on traditional herbal medicine for primary health care, while close to one quarter of prescription drugs used in developed countries are of plant origin (WHO, 2002). In recent years, biotechnology has extended the medicinal use of plants by improving the expression of endogenous medicinal compounds and also by introducing foreign genes with health enhancing properties, such as human insulin-like growth factor-1 (Panahi et al, 2004), granulocyte-macrophage colony stimulating factor (Sardana et al, 2002) and hepatitis B vaccine antigens (Thanavala et al, 2005). Compared to other organisms, plants offer several advantages for producing important medicinal proteins.…”