A Study On The Prokaryotic And Eukaryotic Expression Of Human EGFR Intracellular Tyrosine Kinase Domain

Objective 1. To investigate the prokaryotic expression of the intracellular tyrosine kinase domain of human EGFR and the purification conditions. 2. To stably express human EGFR-TKD in human cancer cell line.Methods 1. Recombinant plasmid pGEX/GST-EGFR-TKD which can express human EGFR intracellular tyrosine kinase domain is constructed. The fusion protein is expressed in E.coli and then denatured to dissolve in urea solution, refold by dialysis in gradient urea solution and purified. Enterokinase is added to remove GST-tag to get human EGFR-TKD. 2. H1299 (human non-small cell lung cancer cell line) cells were co-transfected with recombinant plasmid pEF-BOS/GST-EGFR-TKD and pBabe-puro by calcium phosphate transfection. Puromycin was added for selection. The expressing level and localization of protein products were detected by immuno-flourescence.Results 1. Confirmed by enzyme restriction and sequencing, recombinant plasmid pGEX/GST-EGFR-TKD is successfully constructed. SDS-PAGE indicates fusion protein GST-EGFR-TKD is expressed under induction of IPTG in form of insoluble inclusion body. After denaturing in urea, gradient dialysis and cleavage of GST-tag, refolded human EGFR-TKD is obtained. 2. 8 days after puromycin had been added, all cells in the control group died whereas a few cells in transfection group survived and formed single colonies. After several passages, GST-EGFR-TKD could be detected in cell plasma in most of these colonies under fluorescence microscope.