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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

SUMOtFgf15 cleavage and tFgf15 purification by Ni-NTA resin.The samples were separated on 15% SDS-PAGE gel, and stained with Coomassie Brilliant Blue (A) or undergone western blot analysis (B) with anti-His6 tag antibody. M: protein molecular weight marker, lane 1: purified SUMOtFgf15 inclusion bodies, Lane 2: refolded SUMOtFgf15, lane 3: SUMOtFgf15 digested by ScUlp1, lane 4: purified tFgf15 flow through Ni-NTA column, lane 5: eluate from Ni-NTA column using 200 mM imidazle.
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pone-0020307-g005: SUMOtFgf15 cleavage and tFgf15 purification by Ni-NTA resin.The samples were separated on 15% SDS-PAGE gel, and stained with Coomassie Brilliant Blue (A) or undergone western blot analysis (B) with anti-His6 tag antibody. M: protein molecular weight marker, lane 1: purified SUMOtFgf15 inclusion bodies, Lane 2: refolded SUMOtFgf15, lane 3: SUMOtFgf15 digested by ScUlp1, lane 4: purified tFgf15 flow through Ni-NTA column, lane 5: eluate from Ni-NTA column using 200 mM imidazle.

Mentions: Many biological and biomedical applications require protein fusion tag removal from the target protein because the tag may alter the biological activity of the target protein. There is a Gly-Gly motif between SUMO and tFgf15 that can be specifically recognized and cleaved by ScUlp1. Figure 5A shows the protein analysis by SDS-PAGE gel after protease cleavage. Fusion protein identity was also assessed by western blot analysis using an antibody against the His tag (Figure 5B).


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

Kong B, Guo GL - PLoS ONE (2011)

SUMOtFgf15 cleavage and tFgf15 purification by Ni-NTA resin.The samples were separated on 15% SDS-PAGE gel, and stained with Coomassie Brilliant Blue (A) or undergone western blot analysis (B) with anti-His6 tag antibody. M: protein molecular weight marker, lane 1: purified SUMOtFgf15 inclusion bodies, Lane 2: refolded SUMOtFgf15, lane 3: SUMOtFgf15 digested by ScUlp1, lane 4: purified tFgf15 flow through Ni-NTA column, lane 5: eluate from Ni-NTA column using 200 mM imidazle.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020307-g005: SUMOtFgf15 cleavage and tFgf15 purification by Ni-NTA resin.The samples were separated on 15% SDS-PAGE gel, and stained with Coomassie Brilliant Blue (A) or undergone western blot analysis (B) with anti-His6 tag antibody. M: protein molecular weight marker, lane 1: purified SUMOtFgf15 inclusion bodies, Lane 2: refolded SUMOtFgf15, lane 3: SUMOtFgf15 digested by ScUlp1, lane 4: purified tFgf15 flow through Ni-NTA column, lane 5: eluate from Ni-NTA column using 200 mM imidazle.
Mentions: Many biological and biomedical applications require protein fusion tag removal from the target protein because the tag may alter the biological activity of the target protein. There is a Gly-Gly motif between SUMO and tFgf15 that can be specifically recognized and cleaved by ScUlp1. Figure 5A shows the protein analysis by SDS-PAGE gel after protease cleavage. Fusion protein identity was also assessed by western blot analysis using an antibody against the His tag (Figure 5B).

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