Preparation of human EGFR intracellular domain-GST fusion protein with recombinant technique

EGFR subtypes have been found overexpressed in many GI tumours. Recently, a great number of inhibitory compounds acting on EGFR have been identified.

Our aim is to screen large numbers of inhibitory compounds with an ELISA-based tyrosine kinase assay established in our laboratory. To meet the needs of high amount EGFR enzyme arising in this assay technology, custom production of the recombinant enzyme is necessary. To establish a recombinant expression system, we isolated mRNA from human A431 cells and EGFR intracellular domain was amplified with PCR reaction using primers with Not1 cleavage sequences. PCR product was analyzed by agarose gel electrophoresis and inserted into pCR-Blunt II-TOPO vector. Constructs were subcloned into competent E. coli and transformants were spread on selective plates. For restriction analysis and DNA sequencing, we picked 8 colonies and isolated plasmid DNAs. First fragments after digestion by BamH1 were analyzed by agarose gel electrophoresis. Then plasmid DNAs were sequenced using sequencing vector designed for pCR-Blunt II-TOPO vector. The cDNA fragment is now ready for subcloning into Baculovirus transfer vector with a GST Tag. In 6 of our 8 samples BamH1 cleavage resulted in a 146 and a 539 bp DNA fragment as we expected, showing that the vectors in these samples contain the insert. DNA sequencing will determine whether the sequence of the insert and the EGFR intracellular domain is identical. Mass production of recombinant proteins will enable scale up in the testing processes with future potential of full automation