Soluble Expression of Disulfide Bond Containing Proteins FGF15 and FGF19 in the Cytoplasm of Escherichia coli

Fibroblast growth factor 19 (FGF19) is the human ortholog of mouse FGF15, and both proteins function as an endocrine signal to regulate various liver functions. FGF15/FGF19 protein contains two disulfide bonds. It is unfavorable to form disulfide bonds in Escherichia coli (E. coli) cytoplasm because of the bacterial cytoplasmic reducing environment. Modification of the cytoplasmic reducing environment and/or co-expression of protein chaperones are common strategies to express disulfide bond containing proteins in E. coli. In the current study, we report a method to produce soluble FGF15/FGF19 protein in cytoplasm of E. coli. Several commercial available strains with the disruption of thiol-redox pathways, and/or co-expression of redoxase or refolding chaperones were used to develop this novel method for expression of FGF15/FGF19 in E. coli. Mutation of the thiol-disulfide bond reducing pathway in E. coli or N-terminal fusion of thioredox (TRX) alone is not enough to support disulfide bond formation in FGF15/19 proteins. However, TRX fusion protein improved FGF19 solubility in strains of thiol-redox system mutants. In addition, DsbC co-expressed in thiol-redox system mutants alone improved and further enhanced FGF19 solubility with combination of TRX fusion tag. The soluble FGF19 proteins were easily purified through Ni-NTA affinity chromatography and anion exchange chromatography, and the purified protein maintained its biological activities, confirmed by suppressing hepatic Cyp7a1 gene transcription in mice and by activating ERK1/2 signaling pathway in HepG2 cells. In contrast, soluble FGF15 protein in cytoplasm remained very low using these strategies. In summary, we have successfully developed a method to express functional FGF19 protein in prokaryotic cells, and this strategy may be adapted for the expression of other disulfide-containing proteins.