Studies On The Ameliorate Of Prokaryotic Expression Of MsFGFR2c Ectodomain And Its Refolding Technique

Purpose The stability of pET-3c-msFGFR2c recombinant plasmids was assayed.The cultivating conditions of recombinant bacteria to improve the expression level ofmsFGFR2c were optimized. In order to obtain recombinant msFGFR2c with highactivity, the refolding conditions are improved.Methods The pET-3c-msFGFR2c plasmids were extracted and transferred to BL21(DE3) bacteria, positive clones were chosen, and the recombinant msFGFR2c wasidentified by SDS-PAGE. The stability of pET-3c-msFGFR2c plasmids was tested.The culture temperature, IPTG concentration, induction time were optimized. Thenwe tried to improve sonication conditions, such as the cell concentration andsonication time. The refolding through stepwise dialysis and gel filtrationchromatography were compared. The refolding protein was identified by WB. And theprotein molecular weight was identified by mass spectrometry. To compare theactivities of two kinds of refolded recombinant msFGFR2c, CCK-8assay of H1648cells (lung carcinoma cells) was done. We also analyzed the inhibitory effect ofmsFGFR2c on the proliferations of PC-9cells and gifitinib-resistant PC-9cells.Results pET-3c-msFGFR2c recombinant plasmids were transferred to bacteriasuccessfully, and the stability of plasmids sustained to15passages. ExpressedmsFGFR2c was45%of bacteria proteins in37℃, induced by0.8mM IPTG for4h.100mg/ml bacteria were entirely lysed under the sonicating condition of working3s,stopping5s, total working time is15min. After centrifuge, the inclusion bodies werecollected. Then they were washed by2%Triton X-100until purified.27mgmsFGFR2c were produced by stepwise dialysis refolding, the inhibitory rate ofmsFGFR2c on H1648cells was21%. And20mg msFGFR2c were produced by gelfiltration chromatography refolding, the inhibitory rate of msFGFR2c on H1648cellswas17.5%. Furthermore the inhibition of PC-9and gifitinib-resistant PC-9cellsinduced by msFGFR2c were significant.Conclusion The yield and activity of msFGFR2c refolded by stepwise dialysis ishigher. Stepwise dialysis refolding is a better production process which can be scaled up.