Cloning, Expression And Purification Of Fibroblast Growth Factor Receptor

Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor, which was found later in the fibroblast growth factor receptor family. It is a single chain glycoprotein, which is constructed by extracellular domain, a single transmembrane domain and cytoplasmic tyrosine kinase domain. FGFR4 could transmit extracellular signal into cell from switching on multiple signal transduction pathways, such as PLC, Ras, JAK/STAT signal transduction pathway, while associating with the fibroblast growth factor. It is highly expressed in embryonic tissues, mainly expressed in actively growing tissues of adults, also mainly takes effect on fetal development, healing of wound, vascularization, tissue differentiation and rehabilitation, and other important physiological processes, it plays an important role in the development and aggressiveness of some cancers, these make FGFR4 to be an excellent predictor for pre-cancer and an efficient target point for therapeutics.Objective:To obtain an interest gene fragment in the hepatoma carcinoma cells and shift it in E. coli to be expressed, then the expressed condition will be explored, the stable denaturation and refolding and purification process conditions of inclusion bodies will be found and progress. Identifying FGFR4 and mensuring the activity of it.Methods:Extracted the total RNA from HepG2 cells, reverse transcripted of it to generate cDNA which will be a template to amplify ligand binding fragment in FGFR4(D2-D3), and then transformed into clone recombinant strain DH5a for identification after linking the pET22b vector, the correct identification of prokaryotic expression vector pET22b-FGFR4 will be shift into the expression strain BL21(DE3). IPTG induced product is identified by SDS-PAGE and Western-blot, through explorating the induction time, time after induction, concentration of IPTG and other conditions, the high-density fermentor to be used for fermentation. The precipitate is obtained by centrifugation after broking cells by sonication, then washing it and dissolveing it by 3M urea solution with a strong alkality. Slowly diluting and dialyzing by refolding buffer made the density of urea drop to 0.25mol/L, then use G25 to remove the denaturant and otiose solute.The unwanted proteins and DNA in the protein solution was removed by anion-exchange chromatography, then the purity of the purified FGFR4 protein will be known by high efficiency liquid chromatography, the protein activity will be assayed by MTT measurement.Results:DNA sequencing results showed that high expressed recombinant expression strain BL21(DE3)/ pET22b-FGFR4 was successfully constructed. In terms of optimized expression, the engineering bacteria was shaking culture at 37℃until bacterial concentration was added to A600=0.8-1.0, then added final concentration of 0.6mM of IPTG to induced more than 6h, the interest protein expression was higher than 25%. The A600 absorbance of fermentation broth was harvested top to 30, and the per liter of fermentation broth obtain bacterial sludge over 62g, the quantity of interest protein expression was higher than 20%. The precipitate which was obtained from broken thalli was washed twice, the purity of interst protein in this harvested inclusion body is higher than 80%. The inclusion body was dissolved by 3M urea solution with a strong alkality. this process can effectively keep FGFR4 protein folding correctly and promote some mismatch protein folding to be correct configuration. Using G25 to remove denaturant when the urea solution reduced to 0.25mol/L is a good method to resolve the problem of the inclusion body refolding flocculent precipitate. Refolded protein solution was purified by anion-exchange chromatography, then analyzed by high performance liquid chromatography, the purity is above 95%. Live cell measurements showed that the purified protein had protein activity and could promote hepatoma cells growing, suggesting that FGFR4 on hepatoma cells surface themselves can promote apoptosis. Conclusion:The recombinant cloning and expression engineering bacteria strain were successfully constructed, then optimum expression and high-density fermentation conditions was defined, while the denaturation and refolding conditions of extracellular domain proteins were innovated, the high-purity bioactive FGFR4 ligand Binding protein was also obtained so that it established experimental basis for better exploited and utilized of it in the future.