Hyper-expression of GFP-fused active hFGF21 in tobacco chloroplasts

Human fibroblast growth factor 21 (hFGF21) is a promising candidate for metabolic diseases. In this study, a tobacco chloroplast transformation vector, pWYP21406, was constructed that consisted of codon-optimized encoding gene hFGF21 fused with GFP at its 5' terminal; it was driven by the promoter of plastid rRNA operon (Prrn) and terminated by the terminator of plastid rps16 gene (Trps16). Spectinomycin-resistant gene (aadA) was the marker and placed in the same cistron between hFGF21 and the terminator Trps16. Transplastomic plants were generated by the biolistic bombardment method and proven to be homoplastic by Southern blotting analysis. The expression of GFP was detected under ultraviolet light and a laser confocal microscope. The expression of GFP-hFGF21 was confirmed by immunoblotting and quantified by enzyme-linked immunosorbnent assay (ELISA). The accumulation of GFP-hFGF21 was confirmed to be 12.44 ± 0.45% of the total soluble protein (i.e., 1.9232 ± 0.0673 g kg^-1 of fresh weight). GFP-hFGF21 promoted the proliferation of hepatoma cell line HepG2, inducing the expression of glucose transporter 1 in hepatoma HepG2 cells and improving glucose uptake. These results suggested that a chloroplast expression is a promising approach for the production of bioactive recombinant hFGF21.