Expression, Purification And Identification Of Soluble Ectodomain Of Human Epidermal Growth Factor Receptor

Objeetive We successfully constructed recombinant soluble ectodomain of human epidermal growth factor receptor(s EGFR) plasmid and engineering baceteria. s EGFR protein was expressed after bacteria induction and broken. Purified s EGFR was not only able to inhibit EGF/EGFR signaling by binding to EGF, but also inhibit the promotion of proliferation of tumor cells by EGF.Methods Gene of s EGFR amplifying by PCR and p ET-28 a were digested by restriction endonuclease Nde I and Xho I, and are connected to construct the recombinant plasmid s EGFR-p ET-28 a. Then s EGFR protein was expressed in s EGFR-p ET-28 a BL21(+). The target protein was purified by Ni-NTA affinity chromatography, identified by western blot and its activity was detected by CCK-8.Results the results of PCR and sequencing of recombinant plasmid demonstrated that s EGFR-p ET-28 a was constructed correctly. SDS-PAGE results showed a single band in 58 KD after purification by Ni-NTA affinity chromatography. Western blot analysis showed that the target protein was identified by anti-EGFR. CCK-8 assay showed that recombinant s EGFR could inhibit the promotion of H1648 cell proliferation stimulating by EGF, and 320 ng/ml already had the greatest inhibitory effect.Conclutions s EGFR protein could be expressed by s EGFR-p ET-28 a BL21(+) engineering bacteria in our experiment, and 320 ng/ml recombinant s EGFR had the maxium inhibitory effect on the promotion of proliferation of H1648 cells by EGF.