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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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Purification of SUMOtFgf15 inclusion bodies (A), confirmation of SUMOtFgf15 protein refolding following ScUlp1 digestion (B).Panel A lane 1: soluble cell lysate from pET/SUMOtFgf15, lane 2: insoluble inclusion bodies, lane 3: unbound protein after Ni-NTA resin, lane 4: elutes from Ni-NTA by 200 mM imidazole, lane 5: soluble protein after refolding. Panel B lane 1: purified SUMOtFgf15 for starting refolding, lane 2: soluble SUMOtFgf15 protein after refolding, lane 3: refolded SUMOtFgf15 digested by ScUlp1 for 30 mins. Panel C shows the expression and purification of protease ScUlp1. Lane 1–2: lysate from E. coli containing pET28a(+) (lane 1, soluble fraction, lane 2: insoluble fraction), lane3–4: lysate from E. coli containing pET/ScUlp1 (lane 3: soluble fraction, lane 4: insoluble fraction), lane 5: unbound protein after Ni-NTA resin, lane 6: eluted ScUlp1 by 100 mM imidazole, lane 7: eluted ScUlp1 by 200 mM imidazole.
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pone-0020307-g004: Purification of SUMOtFgf15 inclusion bodies (A), confirmation of SUMOtFgf15 protein refolding following ScUlp1 digestion (B).Panel A lane 1: soluble cell lysate from pET/SUMOtFgf15, lane 2: insoluble inclusion bodies, lane 3: unbound protein after Ni-NTA resin, lane 4: elutes from Ni-NTA by 200 mM imidazole, lane 5: soluble protein after refolding. Panel B lane 1: purified SUMOtFgf15 for starting refolding, lane 2: soluble SUMOtFgf15 protein after refolding, lane 3: refolded SUMOtFgf15 digested by ScUlp1 for 30 mins. Panel C shows the expression and purification of protease ScUlp1. Lane 1–2: lysate from E. coli containing pET28a(+) (lane 1, soluble fraction, lane 2: insoluble fraction), lane3–4: lysate from E. coli containing pET/ScUlp1 (lane 3: soluble fraction, lane 4: insoluble fraction), lane 5: unbound protein after Ni-NTA resin, lane 6: eluted ScUlp1 by 100 mM imidazole, lane 7: eluted ScUlp1 by 200 mM imidazole.

Mentions: Fusion proteins of SUMOtFgf15 were extracted from inclusion bodies under denaturing conditions and were purified by Ni-NTA chelating affinity chromatography (Qiagen) before renatured to native state. The purification profile of SUMOtFgf15 is shown in Figure 4A. The majority of the fusion protein bound to the Ni-NTA resin, leaving a small amount of the SUMOtFgf15 in solution (lane 3). The SUMOtFgf15 protein was eluted by 200 mM imidazole and purification was efficient as shown by the distinct band in lane 4. After purification, the Fgf15 protein was refolded by stepwise dialysis in the presence of reducing agents to allow for the formation of two native disulfide bridges in the protein. The fusion protein SUMOtFgf15 became soluble after removal of denaturants and reducing reagents by dialysis against PBS buffer (Figure 4A, lane 5). Refolding of the purified tFgf15 protein without the SUMO fusion tag was also performed in parallel to compare the effect of SUMO tag on refolding. All tFgf15 protein without SUMO tag precipitated out after removal of denaturing reagent (data not shown). These results suggest that SUMO moiety functions as a chaperone to assist its fusion partners in refolding into correct structure.


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

Kong B, Guo GL - PLoS ONE (2011)

Purification of SUMOtFgf15 inclusion bodies (A), confirmation of SUMOtFgf15 protein refolding following ScUlp1 digestion (B).Panel A lane 1: soluble cell lysate from pET/SUMOtFgf15, lane 2: insoluble inclusion bodies, lane 3: unbound protein after Ni-NTA resin, lane 4: elutes from Ni-NTA by 200 mM imidazole, lane 5: soluble protein after refolding. Panel B lane 1: purified SUMOtFgf15 for starting refolding, lane 2: soluble SUMOtFgf15 protein after refolding, lane 3: refolded SUMOtFgf15 digested by ScUlp1 for 30 mins. Panel C shows the expression and purification of protease ScUlp1. Lane 1–2: lysate from E. coli containing pET28a(+) (lane 1, soluble fraction, lane 2: insoluble fraction), lane3–4: lysate from E. coli containing pET/ScUlp1 (lane 3: soluble fraction, lane 4: insoluble fraction), lane 5: unbound protein after Ni-NTA resin, lane 6: eluted ScUlp1 by 100 mM imidazole, lane 7: eluted ScUlp1 by 200 mM imidazole.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105028&req=5

pone-0020307-g004: Purification of SUMOtFgf15 inclusion bodies (A), confirmation of SUMOtFgf15 protein refolding following ScUlp1 digestion (B).Panel A lane 1: soluble cell lysate from pET/SUMOtFgf15, lane 2: insoluble inclusion bodies, lane 3: unbound protein after Ni-NTA resin, lane 4: elutes from Ni-NTA by 200 mM imidazole, lane 5: soluble protein after refolding. Panel B lane 1: purified SUMOtFgf15 for starting refolding, lane 2: soluble SUMOtFgf15 protein after refolding, lane 3: refolded SUMOtFgf15 digested by ScUlp1 for 30 mins. Panel C shows the expression and purification of protease ScUlp1. Lane 1–2: lysate from E. coli containing pET28a(+) (lane 1, soluble fraction, lane 2: insoluble fraction), lane3–4: lysate from E. coli containing pET/ScUlp1 (lane 3: soluble fraction, lane 4: insoluble fraction), lane 5: unbound protein after Ni-NTA resin, lane 6: eluted ScUlp1 by 100 mM imidazole, lane 7: eluted ScUlp1 by 200 mM imidazole.
Mentions: Fusion proteins of SUMOtFgf15 were extracted from inclusion bodies under denaturing conditions and were purified by Ni-NTA chelating affinity chromatography (Qiagen) before renatured to native state. The purification profile of SUMOtFgf15 is shown in Figure 4A. The majority of the fusion protein bound to the Ni-NTA resin, leaving a small amount of the SUMOtFgf15 in solution (lane 3). The SUMOtFgf15 protein was eluted by 200 mM imidazole and purification was efficient as shown by the distinct band in lane 4. After purification, the Fgf15 protein was refolded by stepwise dialysis in the presence of reducing agents to allow for the formation of two native disulfide bridges in the protein. The fusion protein SUMOtFgf15 became soluble after removal of denaturants and reducing reagents by dialysis against PBS buffer (Figure 4A, lane 5). Refolding of the purified tFgf15 protein without the SUMO fusion tag was also performed in parallel to compare the effect of SUMO tag on refolding. All tFgf15 protein without SUMO tag precipitated out after removal of denaturing reagent (data not shown). These results suggest that SUMO moiety functions as a chaperone to assist its fusion partners in refolding into correct structure.

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