Study On Gene Cloning, Expression And Biological Activity Of Fibroblast Growth Factor23and Its Antagonist Protein

Fibroblast growth factors (FGFs) was first found in the brain and the extract ofpituitary, which is a kind of active peptide that can promote fibroblast cell growthand can combine with the cell membrane specific receptors to regulate cell growth.From the mid-1970s to the present, a lot of extensive research has been made. To date,we have known FGFs include at least23factors, which have homology in an aminoacid sequence and have similar biological functions, and widespread in a variety oftissues in the body. FGFs is very significant in promoting cell derived frommesodermal and neuroectodermal proliferation and growth and plays an importantrole in differentiation of embryonic development in the body, scar tissue healing andtumor development.Fibroblast growth factor23(FGF23) is a secreted polypeptide hormone involvedin phosphorus metabolism, a cytokines of FGF19subfamily of the fibroblast growthfactor family. The discovery of FGF23has expanded our understanding of phosphateand vitamin D homeostasis and provided new insights into the pathogenesis ofhereditary hypophosphatemic and hyperphosphatemic disorders, as well as acquireddisorders of phosphate metabolism, such as chronic kidney disease. FGF23is secretedby osteoblasts and osteocytes in bone and principally targets the kidney to regulate thereabsorption of phosphate, the production and catabolism of1,25-dihydroxyvitamin Dand the expression of α-Klotho, an anti-ageing hormone. Secreted FGF23plays acentral role in complex endocrine networks involving local bone derived factors thatregulate mineralization of extracellular matrix and systemic hormones involved inmineral metabolism. Inactivating mutations of PHEX, DMP1and ENPP1, whichcause hereditary hypophosphatemic disorders and primary defects in bonemineralization, stimulate FGF23gene transcription in osteoblasts and osteocytes, atleast in part, through canonical and intracrine FGF receptor pathways. These FGF23regulatory pathways may enable systemic phosphate and vitamin D homeostasis to becoordinated with bone mineralization. FGF23also functions as a counterregulatory hormone for1,25-dihydroxyvitamin D in a bone–kidney endocrine loop. FGF23,through regulation of additional genes in the kidney and extrarenal tissues, probablyhas broader physiological functions beyond regulation of mineral metabolism thataccount for the association between FGF23and increased mortality and morbidity inchronic kidney disease. Furthermore, FGF23is associated with vascular dysfunction,atherosclerosis, and left ventricular hypertrophy.As a key humoral regulator of phosphate homeostasis and its involvement in thepathogenesis of human disease, human fibroblast growth factor23(hFGF23) has madeit a particularly attractive therapeutic target; Because the C-terminal71amino acids(ie,180-251) of FGF23can compete with full-length ligand for binding to theFGFR-Klotho complex, and hence can antagonize the phosphaturic activity of FGF23in vivo, both in healthy rats and in a mouse model of phosphate wasting disorders.blocking excess FGF23pathogenic action with its C-terminal peptides holds promisein combating human hypophosphataemic diseases.To prepare biological and soluble recombinant human FGF23and its antagonistprotein to meet the increasing demand in its pharmacological application. The genesequence encoding227-residue protein and his6-tagged SUMO (smallubiquitin-related modifier) fragment were cloned, then constructed four kinds ofprokaryotic expression vectors; pET3c-FGF23, pET3c-SUMO-FGF23,pET22b-FGF23, pET22b-SUMO-FGF23, then they were transformed intoEscherichia coli Rosetta (DE3) and BL21(DE3), respectively. The best combinationof plasmid and host strain was screened, only pET-SUMO-FGF23plasmid and theRosetta (DE3) host strain is optimal for rhFGF23protein expressed. An analysis wasperformed to determine the effects of the three factors (IPTG concentration,temperature, and time after induction) on the expression yield and productivity ofsoluble rFGF23in Rosetta(DE3)/pET22b-SUMO-FGF23. The fusion proteinaccounted solubly for about29±1%of the total bacterial proteins after induced by0.4mMIPTG for19h at16℃and dissolved in the20mmol/LTris-HCl+2M urea buffer,then purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Oncecleaved by the SUMO protease, the result of western-blotting showed that theexpressed protein could react specifically with anti-hFGF23antibody. Theauthenticity of recombinant FGF23(rFGF23) was carried out by MALDI-TOF/TOFmass spectroscopy analysis. The purity of rFGF23by high performance liquid chromatography (HPLC) was shown to be higher than90%, with a retention time of14.770min. The antagonist protein of FGF23(rFGF23^180-251) was expressed in thesame exprssion program which we have screened for rFGF23, that was,FGF23^180-251gene fused with SUMO were ligated into pET22b and transformed into competentcells of strains Rosetta(DE3). After recombinant bacterial cells was induced by0.4mM IPTG for6h at30℃, the soluble fraction oftarget protein was as much as39±1%of the total cellular proteins. The purification steps was almost identical withrFGF23, except for buffer solution without2M urea. To evaluate the rFGF23biological activity, two candidate cell lines, which is human kindney(HK) cell lineand glioma U251cell line, were screened by western blot screening through inducingphosphorylation of ERK1/2using anti-phospho-ERK1/2(p-ERK1/2) antibodies. Theresults showed, among the two candidate cell lines, glioma U251cell line wassuccessfully screened, which overexpressed FGFR1, better response is obtainedalthough in the case of the absence of Klotho receptor. Then, we took commercialFGF23as control,which may be expressed in the inclusion body in E. coli, the resultshowed that the ratio of phosphorylation of ERK induced by the rFGF23produced bySUMO fusion method was141±1.8%activation (compared with negative-treatedgroup); while the ratio of commercial FGF23was116±1.6%activation (comparedwith negative-treated group). The subsequent results of in vivo animal experimentsfurther confirmed that the hypophosphatemic effect of rFGF23produced by fusionwith SUMO was better than commercial FGF23in normal rats fed with a fixedformula diet; the animal experimental results of rFGF23^180-251showed good inhibitoryeffect on endogenous FGF23.In the study, we have successfully prepared high bilogical and soluble protein ofrFGF23and rFGF23^180-251, the successful preparation of rFGF23laid a basis for theirclinical pharmacology and mechanism investigation; the successful preparation ofrFGF23^180-251not only laid the foundation for its further study in treatment of lowphosphorus hyperlipidemia disease caused by excess FGF23, but also provided thenecessary data for its pre-clinical studies.