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SUMO fusion system facilitates soluble expression and high production of bioactive human fibroblast growth factor 23 (FGF23).

ABSTRACT: As a key humoral regulator of phosphate homeostasis and its involvement in the pathogenesis of human disease, human fibroblast growth factor 23 (hFGF23) has become a particularly attractive therapeutic target. To prepare soluble and bioactive recombinant human FGF23 to meet the increasing demand in its pharmacological application, small ubiquitin-related modifier (SUMO)-FGF23 fusion gene and FGF23 non-fusion gene were amplified by standard PCR methods and cloned into vector pET-22b and pET-3c, then transformed into Escherichia coli Rosetta (DE3) and BL21 (DE3). The best combination of plasmid and host strain was screened, and only Rosetta (DE3)/pET-SUMO-FGF23 was screened for rhFGF23 protein expressed. The average bacterial yield and the soluble expression level of recombinant hFGF23 of three batches attained 687 ± 18 g and 30 ± 1.5%, respectively, after treatment with 0.4 mM isopropyl-thio-?-galactopyranoside for 19 h at 16 °C in a 30-L fermentor, after which it was purified by DEAE Sepharose FF and nickel nitrilotriacetic acid affinity chromatography. Once cleaved by the SUMO protease, the recombinant human FGF23 was released from the fusion protein. The purity of rFGF23 was shown by high performance liquid chromatography to be greater than 90% and the yield was 60 ± 1.5 mg/L. In vitro data showed that the purified rFGF23 can induce the phosphorylation of mitogen-activated protein kinases in the glioma U251 cell. The results of in vivo animal experiments also showed that rFGF23 could decrease the concentration in the plasma of normal rats fed with a fixed formula diet.

SUBMITTER: Liu X

PROVIDER: S-EPMC7080044 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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SUMO fusion system facilitates soluble expression and high production of bioactive human fibroblast growth factor 23 (FGF23).

Liu Xiaoju X Chen Yubin Y Wu Xiaoping X Li Haiyan H Jiang Chao C Tian Haishan H Tang Lu L Wang Dezhong D Yu Ting T Li Xiaokun X

Applied microbiology and biotechnology 20120117 1

As a key humoral regulator of phosphate homeostasis and its involvement in the pathogenesis of human disease, human fibroblast growth factor 23 (hFGF23) has become a particularly attractive therapeutic target. To prepare soluble and bioactive recombinant human FGF23 to meet the increasing demand in its pharmacological application, small ubiquitin-related modifier (SUMO)-FGF23 fusion gene and FGF23 non-fusion gene were amplified by standard PCR methods and cloned into vector pET-22b and pET-3c, t ...[more]

PMID: 22249722

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Project description:Fibroblast growth factor 23 (FGF23) is a hormone mainly secreted by bone cells. Its most prominent effects are the regulation of renal phosphate reabsorption and calcitriol (active vitamin D, 1,25(OH)2D3) formation, effects dependent on its co-receptor αKlotho. Besides these actions, further paracrine and endocrine effects exist. The production of FGF23 is regulated by 1,25(OH)2D3, parathyroid hormone, dietary phosphate intake, iron status, as well as inflammation. Glucocorticoids are hormones with anti-inflammatory properties and are, therefore, widely used for acute and chronic inflammatory diseases, autoimmune disorders, and malignancies. The present study explored whether glucocorticoids influence the production of FGF23 in vitro as well as in mice. Fgf23 transcription was analyzed by semi-quantitative real-time PCR. Serum concentrations of FGF23 and 1,25(OH)2D3 were measured by ELISA. Urinary phosphate and Ca2+ excretion were determined in metabolic cages. As a result, in UMR106 rat osteoblast-like cells and in MC3T3-E1 cells, both, dexamethasone and prednisolone, downregulated Fgf23 transcription and FGF23 protein synthesis. Dexamethasone increased Dmp1 and Phex (encoding FGF23-regulating genes) as well as Nfkbia (encoding NFκB inhibitor IκBα) transcription in UMR106 cells. In mice, a single injection of dexamethasone or prednisolone was followed by a significant decrease of serum C-terminal and intact FGF23 concentration and bone Fgf23 mRNA expression within 12 h. These effects were paralleled by increased renal phosphate excretion and enhanced 1,25(OH)2D3 formation. We conclude that a single glucocorticoid treatment strongly downregulates the FGF23 plasma concentration. KEY MESSAGES: Glucocorticoids dexamethasone and prednisolone suppress the formation of bone-derived hormone fibroblast growth factor 23 (FGF23) in vitro. The effect is accompanied by an upregulation of Dmp1, Phex, and IκBα, negative regulators of FGF23, in UMR106 osteoblast-like cells. Glucocorticoid receptor antagonist RU-486 attenuates the effect of dexamethasone on FGF23, Dmp1, and Phex. In mice, a single glucocorticoid dose suppresses FGF23 and enhances 1,25(OH)2D3 (active vitamin D).

Project description:Rationale & Objective Patients with chronic kidney failure have markedly elevated fibroblast growth factor 23 (FGF-23) levels and decreased soluble Klotho levels. However, no studies have examined the effects of hemodialysis initiation on the levels of these hormones and other parameters of mineral metabolism. Study Design Prospective single-arm study. Setting & Participants 20 individuals with incident kidney failure initiating hemodialysis. Exposure Initiation of hemodialysis. Dose adjustments of phosphate binders and vitamin D receptor activators and use of calcimimetics, erythropoiesis-stimulating agents, and intravenous iron were prohibited. Outcomes Changes in serum levels of FGF-23, soluble Klotho, and other biochemical parameters of mineral metabolism, measured before and after each hemodialysis session, for a total of 4 sessions over 5 days. Analytical Approach Repeated-measures analysis of variance. Results At baseline, participants had 18-fold higher median FGF-23 levels and 1.6-fold lower mean soluble Klotho levels compared with age- and sex-matched healthy individuals. Initiation of hemodialysis led to progressive reductions in serum phosphorus, intact parathyroid hormone, and FGF-23 levels, with dialysis-related fluctuations. No reductions were observed in levels of ?1-microglobulin, which has molecular weight comparable to FGF-23. The magnitude of the FGF-23 level reductions was strongly associated with concomitant changes in serum phosphorus levels but not with the changes in intact parathyroid hormone levels. Soluble Klotho levels did not change after the initiation of hemodialysis. Limitations Single-arm design, small sample size, short follow-up period. Conclusions Initiation of hemodialysis in patients with chronic kidney failure led to progressive reductions in FGF-23 levels in association with reductions in serum phosphorus levels. These results suggest that phosphorus is a strong inducer of FGF-23 production and that regulation of FGF-23 production is a rapid process.

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SUMO fusion system facilitates soluble expression and high production of bioactive human fibroblast growth factor 23 (FGF23).

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SUMO fusion system facilitates soluble expression and high production of bioactive human fibroblast growth factor 23 (FGF23).

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