Hyper Expression of Active, GFP-fused HFGF21 in Tobacco Chloroplasts

作者：Yunpeng Wang, Jieying Fan, Nuo Xu, Xin Wen, Zhengyi Wei, Shaochen Xing · 发表于：Research Square · 年份：2022 · DOI：10.21203/rs.3.rs-1299734/v1 · 研究领域：Cancer, Hypoxia, and Metabolism、Fibroblast Growth Factor Research、Microtubule and mitosis dynamics

Abstract 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.