Expression, Purification And Biological Functional Analyses Of Single-chain Fv Against Human FGFR3and Its Fusion Protein Containing Truncated Protamine

Fibroblast growth factor receptor3(FGFR3) is a member of fibroblast growth factorreceptors (FGFRs) family, which plays a significant role in Cell growth, differentiationand other biological process after binding to its ligands, fibroblast growth factors.Recently, emerging researches have reported the close association between FGFR3activating mutation or overexpression and the initiation and progression of cancer. Thisalso makes FGFR3a potential therapeutic target in a wide spectrum of solid tumors. Inthis study, two novel proteins were designed: anti-FGFR3single chain fragment variable(ScFv) and a fusion protein (ScFv-tp) consisting of ScFv and a truncated protamine (tp),which aimed at:1. ScFv can bind to FGFR3on cell membrane, and therefore attenuatethe phosphorylation of FGFR3and the downstream signaling cascades;2. fusion proteinScFv-tp can deliver siRNA into target cancer cells and silent target genes related to cancer,and eventually kill cancer cells.To achieve the high level production and soluble proteins, ScFv was fused to a smallubiquitin-related modifier (Sumo) by overlap PCR. Then the expression vector,pET-20b-Sumo-ScFv was constructed which expressed in Escherichia coli. Anti-FGFR3ScFv proteins were obtained after Ni-NTA chromatography purification, cleavage bySumo protease and re-purification, with the purity over90%and their yield reached4mgper liter of bacterial culture. The results of in vitro experiment showed that ScFv couldbind to FGFR3specifically and inhibit the phosphorylation of FGFR3.In order to solve the problem that fusion protein ScFv-tp could bind to the nucleicacid of the bacteria, we decided to express the protein in the insoluble inclusion bodyform. In the same way, another expression vector, pET-20b-ScFv-tp was constructed andexpressed the fusion protein in Escherichia coli. The target fusion protein ScFv-tp wasobtained after washing repeatedly, denaturation and renaturation, with the purity over90%and their yield reached10mg per liter of bacterial culture. Gel retardation assayshowed that1μg ScFv-tp could efficiently bind to about2pmol siRNA. Flow cytometryand immunofluorescence detection showed that ScFv-tp could deliver siRNA intoFGFR3+K562and RT-112cells specifically and efficiently, but failed deliver siRNA intoFGFR3-THP-1cells. After that, RT-PCR and western blotting were employed to detectwhether the siRNA ScFv-tp delivered into target cells will silent target gene effectively.The data indicated that the given siRNA which delivered by ScFv-tp into RT-112cells significantly inhibited the expression of amplified in breast cancer1(AIB1) gene in bothmRNA and protein level through RNAi mechanism.In conclusion, anti-FGFR3ScFv was obtained in a soluble expression system andScFv could bind to FGFR3specifically and inhibit the phosphorylation of FGFR3triggered by FGF9; later, fusion protein ScFv-tp was obtained in inclusion body formScFv-tp could effectively deliver siRNA into FGFR3+cells and significantly decrease theexpression of AIB1gene.