Human fibroblast growth factor 21 (hFGF21) is a promising candidate for metabolic diseases. In this study, a tobacco chloroplast transformation vector, pWYP21406, was constructed. In the vector, the codon optimized encoding gene hFGF21 fused with GFP at its 5’ terminal was driven by the promoter of plastid rRNA operon (Prrn) and terminated by the terminator of plastid rps16 gene (Trps16). Spectinomycin resistant gene (aadA) as the selectable marker was placed in the same cistron between hFGF21 and the terminator Trps16. Transplastomic plants were generated by biolistic bombardment method and proven to be homoplastic by Southern Blotting analyses. The expression of GFP was detected under UV light and laser confocal microscope. The expression of GFP-hFGF21 was confirmed by immunoblotting and quantified by ELISA. The accumulation of was GFP-hFGF21confirmed to be 12.44 ± 0.45% of total soluble protein (i.e., 1.9232 ± 0.0673 g kg− 1 of fresh weight). The GFP-hFGF21 was confirmed to be capable of promoting the proliferation of hepatoma cell line HepG2, inducing the expression of glucose transporter 1 in hepatoma HepG2 cells and improve the glucose uptakes. The above results suggested that chloroplast expression is a promising approach for bioactive recombinant hFGF21 production.
Background
Chloroplast transformation is a robust technology for the expression of recombinant proteins. Various types of pharmaceutical proteins including growth factors have been reported in chloroplasts via chloroplast transformation approach at high expression levels. However, high expression of epidermal growth factor (EGF) in chloroplasts with the technology is still unavailable.
Results
The present work explored the high-level expression of recombinant EGF, a protein widely applied in many clinical therapies, in tobacco chloroplasts. In this work, homoplastic transgenic plants expressing fusion protein GFP-EGF, which was composed of GFP and EGF via a linker, were generated. The expression of GFP-EGF was confirmed by the combination of green fluorescent observation and Western blotting. The achieved accumulation of the recombinant fusion GFP-EGF was 10.21 ± 0.27% of total soluble proteins (1.57 ± 0.05 g kg− 1 of fresh leaf). The chloroplast-derived GFP-EGF was capable of increasing the cell viability of the NSLC cell line A549 and enhancing the phosphorylation level of the EGF receptor in the A549 cells.
Conclusion
The expression of recombinant EGF in tobacco chloroplasts via chloroplast transformation method was achieved at considerable accumulation level. The attempt gives a good example for the application of chloroplast transformation technology in recombinant pharmaceutical protein production.
Brain-derived neurotrophic factor (BDNF) is a factor with many functions such as acceleration of cell proliferation and differentiation and is therefore widely used in clinical applications. In this study, an expression vector named pWYP23402 having a codon-optimized BDNF gene was constructed and transferred into chloroplasts of tobacco by gene-gun. After three or four rounds of selection with proper spectinomycin, BDNF was integrated into the chloroplast genome of homoplastomic plants confirmed by PCR and Southern hybridization. ELISA assay indicated that BDNF fused with GFP represented approximately 15.72%±0.33% of total soluble protein in the leaves of transplastomic plants. Moreover, the chloroplast-derived BDNF displayed similar biological activity to the commercial product. This is the first case report of BDNF expression by chloroplast transformation in this model plant, supplying an additional pathway for the production of chloroplast-expressed therapeutic proteins.
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