Abstract
Fibroblast growth factor 15 (Fgf15) is the mouse orthologue of human FGF19. Fgf15 is highly expressed in the ileum and functions as an endocrine signal to regulate liver function, including bile acid synthesis, hepatocyte proliferation and insulin sensitivity. In order to fully understand the function of Fgf15, methods are needed to produce pure Fgf15 protein in the prokaryotic system. However, when expressed in Escherichia coli (E. coli), the recombinant Fgf15 protein was insoluble and found only in inclusion bodies. In the current study, we report a method to produce recombinant Fgf15 protein in E. coli through the use of small ubiquitin-related modifier (SUMO) fusion tag. Even though the SUMO has been shown to strongly improve protein solubility and expression levels, our studies suggest that the SUMO does not improve Fgf15 protein solubility. Instead, proper refolding of Fgf15 protein was achieved when Fgf15 was expressed as a partner protein of the fusion tag SUMO, followed by in vitro dialysis refolding. After refolding, the N-terminal SUMO tag was cleaved from the recombinant Fgf15 fusion protein by ScUlp1 (Ubiquitin-Like Protein-Specific Protease 1 from S. cerevisiae). With or without the SUMO tag, the refolded Fgf15 protein was biologically active, as revealed by its ability to reduce hepatic Cyp7a1 mRNA levels in mice. In addition, recombinant Fgf15 protein suppressed Cyp7a1 mRNA levels in a dose-dependent manner. In summary, we have developed a successful method to express functional Fgf15 protein in prokaryotic cells.
Highlights
Fibroblast growth factor 15 (Fgf15) is mainly expressed in mouse ileum and colon and is not expressed in liver
Similar effects have been observed in mice treated intravenously with recombinant FGF19 [7], suggesting that Fgf15/FGF19 are important to physiology, in addition to regulating bile acid homeostasis
Fgf15 clones were characterized for their expression and solubility in E. coli by SDS-PAGE
Summary
Fibroblast growth factor 15 (Fgf15) is mainly expressed in mouse ileum and colon and is not expressed in liver. Fgf functions as an enterohepatic hormone in that it is produced in the small intestine, but travels to the liver via portal circulation, and binds to its receptor, FGFR4 that is expressed in hepatocytes [2]. Activation of FGFR4 leads to activation of downstream signaling pathways and results in suppression of gene transcription of Cyp7a1 gene that encodes cholesterol 7a-hydroxylase, the rate-limiting enzyme for bile acid synthesis in the liver [3,4]. Fgf15/FGF19 plays a critical role in regulating cholesterol and bile acid homeostasis. Similar effects have been observed in mice treated intravenously with recombinant FGF19 [7], suggesting that Fgf15/FGF19 are important to physiology, in addition to regulating bile acid homeostasis. The study of Fgf function in mice can be a valuable research tool because mouse models are widely used to study human physiology and pathology. In order to produce large quantities of biologically active Fgf protein to study its effects in vivo, it will be ideal to develop a prokaryotic system to produce biologically active Fgf protein
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