Studies On Mutant Ectodomains Of Human Fibroblast Growth Factor Receptor 2IIIc: Prokaryotic Expression, In Vitro Renaturation And Activity Assay

The S252W, P253R and S252W+ P253R mutations were introduced into the gene of FGFR2IIIc , wildtype and mutant FGFR2IIIc were expressed in E.coli and refolded in vitro. Ectodomain of FGFR2IIIc with AS mutations had increased ligands affinity. Mutant soluble ectodomain of FGFR2IIIc can be considered as a potential antitumor drug.Gene of α FGFR2IIIc was amplified by RT-PCR and β FGFR2IIIc was synthesized by full-length gene synthesis. S252W, P253R, and S252W+ P253R point mutations were introduced into the FGF-binding portion of human β FGFR2IIIc and α FGFR2IIIc by the overlapping extension PCR. Wild-type and mutant β FGFR2IIIc genes were cloned into pET3c, recombinant protein was expressed in inclusion body. Different refolding conditions were studied. Wild-type and mutant β FGFR2IIIc were refolded by gel filtration chromatography.The refolded recombinant protein were purified by heparin affinity chromatography, activity of refolded recombinant protein were studied by co-IP assay and MTT assay.Wild type α FGFR2IIIc and β FGFR2IIIc were synthesized and S252W, P253R, and S252W+ P253R point mutations were introduced successfully. Our refolding procedure had higher renaturation yield and suitable for large-scale production. Co-IP result showed that Both wild-type and mutant β FGFR2IIIc could bind FGF-2 with specific affinity. The results of MTT assay indicated that mutant 3 FGFR2IIIc had greater affinity towards FGF-2 than wild type.In conclusion , mitogenic activity of FGFs can be inhibited by ectodomain of FGFR2IIIc, and the three mutant had greater inhibition activity than wild type. The effect of S252W+P253R mutant should be characterized by futher study.