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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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Expression of SUMOtFgf15 in E. coli.The solubility of fusion proteins was analyzed on 12% SDS-PAGE gel and stained with Coomassie Brilliant blue. M: protein molecular weight marker, lane 1: total cellular lysate from E. coli containing pET/SUMO, lane 2: soluble lysate fraction from E. coli containing pET/SUMO, lane 3: insoluble lysate fraction from E. coli containing pET/SUMO, lane 4: total cellular lysate from E. coli containing pET/SUMOtFgf15, lane 5: soluble lysate fraction from E. coli containing pET/SUMOtFgf15, lane 6: insoluble lysate fraction from E. coli containing pET/SUMOtFgf15.
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pone-0020307-g003: Expression of SUMOtFgf15 in E. coli.The solubility of fusion proteins was analyzed on 12% SDS-PAGE gel and stained with Coomassie Brilliant blue. M: protein molecular weight marker, lane 1: total cellular lysate from E. coli containing pET/SUMO, lane 2: soluble lysate fraction from E. coli containing pET/SUMO, lane 3: insoluble lysate fraction from E. coli containing pET/SUMO, lane 4: total cellular lysate from E. coli containing pET/SUMOtFgf15, lane 5: soluble lysate fraction from E. coli containing pET/SUMOtFgf15, lane 6: insoluble lysate fraction from E. coli containing pET/SUMOtFgf15.

Mentions: The truncated Fgf15 protein was expressed in an insoluble form and was therefore retained in the inclusion bodies. We have performed pilot experiments in an attempt to fuse tFgf15 with GST and Trx, two well-known solubility enhancers, to improve the solubility of Fgf15 protein. However, neither of these tags enhanced Fgf15 protein solubility, even at various culture conditions (data not shown). The SUMO tag has been shown to improve expression levels and solubility, as well as to promote proper folding of many proteins that are difficult to be solubilizied. Therefore, the SUMO tag has been suggested to better enhance solubility than GST, Trx, or MBP tags. We expressed constructs of SUMO and SUMOtFgf15 in E. coli (Figure 3) and found that most SUMO tags were expressed in the soluble form (lane 2) with less SUMO tag expressed in the insoluble fraction (lane 3). Note that SUMO migrated on the SDS-PAGE gel as more than 20 kDa in size even though it has a molecular mass of 11.5 kDa [18]. Figure 3 shows that an apparent band of 40 kDa was observed (lane 4, consistent with the calculated molecular mass of recombinant SUMOtFgf15), but there was no obvious SUMOtFgf15 protein expression in the supernatant (lane 5). In addition, all fusion proteins were expressed in the centrifuged pellet fraction, which indicates that all SUMOtFgf15 proteins expressed were insoluble and retained within inclusion bodies. Therefore, the fusion tag SUMO could not improve tFgf15 protein solubility. These results were further confirmed by western blot analysis against the His-tag (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 SUMOtFgf15 in E. coli.The solubility of fusion proteins was analyzed on 12% SDS-PAGE gel and stained with Coomassie Brilliant blue. M: protein molecular weight marker, lane 1: total cellular lysate from E. coli containing pET/SUMO, lane 2: soluble lysate fraction from E. coli containing pET/SUMO, lane 3: insoluble lysate fraction from E. coli containing pET/SUMO, lane 4: total cellular lysate from E. coli containing pET/SUMOtFgf15, lane 5: soluble lysate fraction from E. coli containing pET/SUMOtFgf15, lane 6: insoluble lysate fraction from E. coli containing pET/SUMOtFgf15.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020307-g003: Expression of SUMOtFgf15 in E. coli.The solubility of fusion proteins was analyzed on 12% SDS-PAGE gel and stained with Coomassie Brilliant blue. M: protein molecular weight marker, lane 1: total cellular lysate from E. coli containing pET/SUMO, lane 2: soluble lysate fraction from E. coli containing pET/SUMO, lane 3: insoluble lysate fraction from E. coli containing pET/SUMO, lane 4: total cellular lysate from E. coli containing pET/SUMOtFgf15, lane 5: soluble lysate fraction from E. coli containing pET/SUMOtFgf15, lane 6: insoluble lysate fraction from E. coli containing pET/SUMOtFgf15.
Mentions: The truncated Fgf15 protein was expressed in an insoluble form and was therefore retained in the inclusion bodies. We have performed pilot experiments in an attempt to fuse tFgf15 with GST and Trx, two well-known solubility enhancers, to improve the solubility of Fgf15 protein. However, neither of these tags enhanced Fgf15 protein solubility, even at various culture conditions (data not shown). The SUMO tag has been shown to improve expression levels and solubility, as well as to promote proper folding of many proteins that are difficult to be solubilizied. Therefore, the SUMO tag has been suggested to better enhance solubility than GST, Trx, or MBP tags. We expressed constructs of SUMO and SUMOtFgf15 in E. coli (Figure 3) and found that most SUMO tags were expressed in the soluble form (lane 2) with less SUMO tag expressed in the insoluble fraction (lane 3). Note that SUMO migrated on the SDS-PAGE gel as more than 20 kDa in size even though it has a molecular mass of 11.5 kDa [18]. Figure 3 shows that an apparent band of 40 kDa was observed (lane 4, consistent with the calculated molecular mass of recombinant SUMOtFgf15), but there was no obvious SUMOtFgf15 protein expression in the supernatant (lane 5). In addition, all fusion proteins were expressed in the centrifuged pellet fraction, which indicates that all SUMOtFgf15 proteins expressed were insoluble and retained within inclusion bodies. Therefore, the fusion tag SUMO could not improve tFgf15 protein solubility. These results were further confirmed by western blot analysis against the His-tag (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