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Enhanced in vitro refolding of fibroblast growth factor 15 with the assistance of SUMO fusion partner.

Kong B, Guo GL - PLoS ONE (2011)

Bottom Line: However, when expressed in Escherichia coli (E. coli), the recombinant Fgf15 protein was insoluble and found only in inclusion bodies.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

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.

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Biological activity of the recombinant Fgf15 proteins.(A) Hepatic Cyp7a1 mRNA levels in mice injected intravenously with saline, fusion protein SUMOtFgf15, or tFgf15 without SUMO fusion tag. (B) Dose-dependency in suppressing hepatic Cyp7a1 gene expression in mice by recombinant tFgf15. * P<0.05, compared to saline-treated group.
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pone-0020307-g006: Biological activity of the recombinant Fgf15 proteins.(A) Hepatic Cyp7a1 mRNA levels in mice injected intravenously with saline, fusion protein SUMOtFgf15, or tFgf15 without SUMO fusion tag. (B) Dose-dependency in suppressing hepatic Cyp7a1 gene expression in mice by recombinant tFgf15. * P<0.05, compared to saline-treated group.

Mentions: Fgf15 is known to reduce the mRNA expression of the Cyp7a1 gene in liver. To assess the biological activity of the recombinant Fgf15 protein in vivo, we analyzed the effect of Fgf15 on Cyp7a1 gene expression. Mice were administered SUMOtFgf15 or tFgf15 protein through tail-vein injection. Both SUMOtFgf15 and tFgf15 protein suppressed 90% of hepatic Cyp7a1 mRNA levels, and there was no difference in the extent of suppression between SUMOtFgf15 and tFgf15 protein (Figure 6A). In addition, SUMO protein alone did not suppress Cyp7a1 expression, indicating that the biological activity of the SUMOtFgf15 fusion protein comes from the tFgf15 protein and not the SUMO moiety. These results, in combination with those from Figure 4B, suggest that both the SUMO moiety and tFgf15 protein have kept their individual structures after refolding in vitro. With the assistance of SUMO fusion tag, tFgf15 was properly refolded and maintained its function, and assumedly has kept its tertiary structure after cleavage of the fusion tag.


Enhanced in vitro refolding of fibroblast growth factor 15 with the assistance of SUMO fusion partner.

Kong B, Guo GL - PLoS ONE (2011)

Biological activity of the recombinant Fgf15 proteins.(A) Hepatic Cyp7a1 mRNA levels in mice injected intravenously with saline, fusion protein SUMOtFgf15, or tFgf15 without SUMO fusion tag. (B) Dose-dependency in suppressing hepatic Cyp7a1 gene expression in mice by recombinant tFgf15. * P<0.05, compared to saline-treated group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105028&req=5

pone-0020307-g006: Biological activity of the recombinant Fgf15 proteins.(A) Hepatic Cyp7a1 mRNA levels in mice injected intravenously with saline, fusion protein SUMOtFgf15, or tFgf15 without SUMO fusion tag. (B) Dose-dependency in suppressing hepatic Cyp7a1 gene expression in mice by recombinant tFgf15. * P<0.05, compared to saline-treated group.
Mentions: Fgf15 is known to reduce the mRNA expression of the Cyp7a1 gene in liver. To assess the biological activity of the recombinant Fgf15 protein in vivo, we analyzed the effect of Fgf15 on Cyp7a1 gene expression. Mice were administered SUMOtFgf15 or tFgf15 protein through tail-vein injection. Both SUMOtFgf15 and tFgf15 protein suppressed 90% of hepatic Cyp7a1 mRNA levels, and there was no difference in the extent of suppression between SUMOtFgf15 and tFgf15 protein (Figure 6A). In addition, SUMO protein alone did not suppress Cyp7a1 expression, indicating that the biological activity of the SUMOtFgf15 fusion protein comes from the tFgf15 protein and not the SUMO moiety. These results, in combination with those from Figure 4B, suggest that both the SUMO moiety and tFgf15 protein have kept their individual structures after refolding in vitro. With the assistance of SUMO fusion tag, tFgf15 was properly refolded and maintained its function, and assumedly has kept its tertiary structure after cleavage of the fusion tag.

Bottom Line: However, when expressed in Escherichia coli (E. coli), the recombinant Fgf15 protein was insoluble and found only in inclusion bodies.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

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.

Show MeSH
Related in: MedlinePlus