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{"title"=>"Soluble expression of disulfide bond containing proteins FGF15 and FGF19 in the cytoplasm of Escherichia coli", "type"=>"journal", "authors"=>[{"first_name"=>"Bo", "last_name"=>"Kong", "scopus_author_id"=>"26025129400"}, {"first_name"=>"Grace L.", "last_name"=>"Guo", "scopus_author_id"=>"7402768177"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"372852918", "sgr"=>"84924841477", "issn"=>"19326203", "pmid"=>"24465767", "scopus"=>"2-s2.0-84924841477", "doi"=>"10.1371/journal.pone.0085890", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)"}, "id"=>"c6f6d2db-a56f-374f-8c59-448f1395449d", "abstract"=>"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.", "link"=>"http://www.mendeley.com/research/soluble-expression-disulfide-bond-containing-proteins-fgf15-fgf19-cytoplasm-escherichia-coli", "reader_count"=>51, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>5, "Student > Ph. D. Student"=>17, "Student > Master"=>13, "Other"=>2, "Student > Bachelor"=>11}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>5, "Student > Ph. D. Student"=>17, "Student > Master"=>13, "Other"=>2, "Student > Bachelor"=>11}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>19, "Agricultural and Biological Sciences"=>29, "Medicine and Dentistry"=>1, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>29}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>19}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"South Korea"=>1, "Spain"=>1}, "group_count"=>0}

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• http://doi.org/10.1016/j.sjbs.2015.04.017
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• http://doi.org/10.1002/hep.30041
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• http://doi.org/10.1007/s00449-018-2050-9

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Figshare

• {"files"=>["https://ndownloader.figshare.com/files/1351557"], "description"=>"<p>A): Schematic maps of plasmids. Truncated FGF15 or FGF19 gene (without signal peptide) was located downstream from the T7 promoter, coding in frame with the TRX tag. <i>Nde</i>I and <i>Xho</i>I restriction enzyme sites are used to subclone the PCR fragments. His6 tag for purification and TEV protease site for fusion tag cleavage are indicated in the figure. B): SDS-PAGE analysis of FGF15 and FGF19 in <i>E. coli</i> strain BL21(DE3); non-induced total cell lysates (N), and the soluble (S) and insoluble fractions (P) were prepared as described in Methods. The molecular weight markers from Pierce (Rockford, IL) are also shown in gels. C): SDS-PAGE analysis of TRX-FGF15/19 in BL21(DE3) strain; the soluble (S) and insoluble fractions (P) before (Lane 3, 4, 7, and 8) and after IPTG induction (lane 5, 6, 9 and 10) were determined. Total cell lysates of BL21(DE3) strain (BL) were loaded as the control.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Recombinant proteins", "Model organisms", "Prokaryotic models", "Escherichia coli", "Molecular cell biology", "Signal transduction", "Nuclear receptor signaling", "Gastroenterology and hepatology", "Biliary disorders", "Cholecystitis and biliary colic", "biophysics", "Protein chemistry", "Protein folding", "fgf15", "fgf19"], "article_id"=>904273, "categories"=>["Physics", "Biological Sciences", "Medicine"], "users"=>["Bo Kong", "Grace L. Guo"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085890.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Construction_and_expression_analyses_of_FGF15_and_FGF19_Plasmids_/904273", "title"=>"Construction and expression analyses of FGF15 and FGF19 Plasmids.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-20 03:17:17"}
• {"files"=>["https://ndownloader.figshare.com/files/1351558"], "description"=>"<p>Total cell lysates without IPTG induction (N), the soluble (S) and insoluble fractions (P) from IPTG induced cell lysates prepared as described in Methods. The arrows indicate the predicted positions according to the protein size. Lane 1: protein markers; Lane 2: FGF15 without induction; lane 3: soluble FGF15 fraction after induction; lane 4: insoluble FGF15 fraction after induction; Lane 5: TRXtFGF15 without induction; lane 6: soluble TRXtFGF15 fraction after induction; lane 7: insoluble TRXtFGF15 fraction after induction; Lane 8: FGF19 without induction; lane 9: soluble FGF19 fraction after induction; lane 10: insoluble FGF19 fraction after induction; Lane 11: TRXtFGF19 without induction; lane 12: soluble TRXtFGF19 fraction after induction; lane 13: insoluble TRXtFGF19 fraction after induction.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "Recombinant proteins", "Model organisms", "Prokaryotic models", "Escherichia coli", "Molecular cell biology", "Signal transduction", "Nuclear receptor signaling", "Gastroenterology and hepatology", "Biliary disorders", "Cholecystitis and biliary colic", "biophysics", "Protein chemistry", "Protein folding", "strains", "rosetta-gami", "shuffle", "t7", "trx", "fusion", "co-expression", "dsb", "disruption", "cytosolic", "reducing", "soluble"], "article_id"=>904274, "categories"=>["Physics", "Biological Sciences", "Medicine"], "users"=>["Bo Kong", "Grace L. Guo"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0085890.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SDS_PAGE_analysis_of_FGF15_19_and_TRXtFGF15_19_expression_in_E_coli_strains_of_Rosetta_gami_2_A_and_SHuffle_T7_B_showing_the_effect_of_TRX_fusion_protein_co_expression_of_Dsb_and_disruption_of_cytosolic_reducing_environment_on_the_expression_of_soluble_F/904274", "title"=>"SDS-PAGE analysis of FGF15/19 and TRXtFGF15/19 expression in <i>E. coli</i> strains of Rosetta-gami 2 (A) and SHuffle T7 (B), showing the effect of TRX fusion protein, co-expression of Dsb and disruption of cytosolic reducing environment on the expression of soluble FGF15/19 in the cytoplasm.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-20 03:17:17"}
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PMC Usage Stats | Further Information

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Relative Metric

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F1000Prime