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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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Related in: MedlinePlus

Expression of Fgf15 with or without N-terminal signal peptide in E. coli.M: protein molecular weight marker, lane 1: soluble lysate fraction from E. coli containing pET28a(+), lane 2: insoluble lysate fraction from E. coli containing pET28a(+), lane 3: soluble lysate fraction from E. coli containing pET/Fgf15, lane 4: insoluble lysate fraction from E. coli containing pET/Fgf15, lane 5: soluble lysate fraction from E. coli containing pET/tFgf15, lane 6: insoluble lysate fraction from E. coli containing pET/tFgf15.
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pone-0020307-g002: Expression of Fgf15 with or without N-terminal signal peptide in E. coli.M: protein molecular weight marker, lane 1: soluble lysate fraction from E. coli containing pET28a(+), lane 2: insoluble lysate fraction from E. coli containing pET28a(+), lane 3: soluble lysate fraction from E. coli containing pET/Fgf15, lane 4: insoluble lysate fraction from E. coli containing pET/Fgf15, lane 5: soluble lysate fraction from E. coli containing pET/tFgf15, lane 6: insoluble lysate fraction from E. coli containing pET/tFgf15.

Mentions: The full length cDNA of Fgf15, including the predicted N-terminal signal peptide, was cloned from mouse intestine and inserted into a pET28a(+) plasmid to construct pET/Fgf15. Fgf15 contains 218 aa with a predicted 25-aa signal peptide and produces a 25 kDa protein if the full-length protein is expressed. Fgf15 clones were characterized for their expression and solubility in E. coli by SDS-PAGE. Figure 2 shows proteins in lysed cell supernatant and pellet (lanes 3 and 4, respectively). There was no major band at the expected molecular weight in either the supernatant or pellet fraction compared to the control E. coli lysate (lanes 1 and 2). A more sensitive western blot analysis was then used to detect Fgf15 expression by His-tag specific antibody, but Fgf15 protein was not detected (data not shown).


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

Kong B, Guo GL - PLoS ONE (2011)

Expression of Fgf15 with or without N-terminal signal peptide in E. coli.M: protein molecular weight marker, lane 1: soluble lysate fraction from E. coli containing pET28a(+), lane 2: insoluble lysate fraction from E. coli containing pET28a(+), lane 3: soluble lysate fraction from E. coli containing pET/Fgf15, lane 4: insoluble lysate fraction from E. coli containing pET/Fgf15, lane 5: soluble lysate fraction from E. coli containing pET/tFgf15, lane 6: insoluble lysate fraction from E. coli containing pET/tFgf15.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020307-g002: Expression of Fgf15 with or without N-terminal signal peptide in E. coli.M: protein molecular weight marker, lane 1: soluble lysate fraction from E. coli containing pET28a(+), lane 2: insoluble lysate fraction from E. coli containing pET28a(+), lane 3: soluble lysate fraction from E. coli containing pET/Fgf15, lane 4: insoluble lysate fraction from E. coli containing pET/Fgf15, lane 5: soluble lysate fraction from E. coli containing pET/tFgf15, lane 6: insoluble lysate fraction from E. coli containing pET/tFgf15.
Mentions: The full length cDNA of Fgf15, including the predicted N-terminal signal peptide, was cloned from mouse intestine and inserted into a pET28a(+) plasmid to construct pET/Fgf15. Fgf15 contains 218 aa with a predicted 25-aa signal peptide and produces a 25 kDa protein if the full-length protein is expressed. Fgf15 clones were characterized for their expression and solubility in E. coli by SDS-PAGE. Figure 2 shows proteins in lysed cell supernatant and pellet (lanes 3 and 4, respectively). There was no major band at the expected molecular weight in either the supernatant or pellet fraction compared to the control E. coli lysate (lanes 1 and 2). A more sensitive western blot analysis was then used to detect Fgf15 expression by His-tag specific antibody, but Fgf15 protein was not detected (data not shown).

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