title: 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-β-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.
authors.affiliation: School of Public Health, Jilin University, Changchun 130021, China.
authors.forename: Xiaoju
authors.initials: X
authors.lastname: Liu
authors.forename: Yubin
authors.initials: Y
authors.lastname: Chen
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Wu
authors.forename: Haiyan
authors.initials: H
authors.lastname: Li
authors.forename: Chao
authors.initials: C
authors.lastname: Jiang
authors.forename: Haishan
authors.initials: H
authors.lastname: Tian
authors.forename: Lu
authors.initials: L
authors.lastname: Tang
authors.forename: Dezhong
authors.initials: D
authors.lastname: Wang
authors.forename: Ting
authors.initials: T
authors.lastname: Yu
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Li
mesh_terms: D001709:Biotechnology / Q000379:methods; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D058207:Sumoylation; D013678:Technology, Pharmaceutical / Q000379:methods; D014169:Transformation, Bacterial
chemical_list: C000717427:FGF23 protein, human; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D005346:Fibroblast Growth Factors; D000089703:Fibroblast Growth Factor-23; D020928:Mitogen-Activated Protein Kinases