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- W2000461990 abstract "EGFR subtypes have been found overexpressed in pancreatic cancer. Recently, a great number of inhibitory compounds acting on EGFR have reached clinical application. Our aim was to establish ELISA-based tyrosine kinase assays based on different molecular mutational conformants of the kinase domain. To meet the needs of high amount EGFR enzyme arising in these assay technologies, custom production of the recombinant enzymes is necessary. To establish a recombinant expression system, we isolated mRNA from EGFR-overexpressing human A431 cells and synthesized cDNAs. EGFR intracellular domain was amplified with PCR reaction using primers with Not1 cleavage sequences designed for subcloning into Baculovirus transfer vector. The 1564 bp PCR product was analyzed by agarose gel electrophoresis and inserted directly into pCR-Blunt II-TOPO vector. Constructs were subcloned into competent E. coli and transformants were spread on selective plates containing Kanamycin. For restriction analysis and DNA sequencing, we picked 8 colonies and isolated plasmid DNAs that were, first, digested with BamH1 endonuclease and fragments 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 (pAcGHLT) 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" @default.
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- W2000461990 date "2004-11-01" @default.
- W2000461990 modified "2023-09-27" @default.
- W2000461990 title "RECOMBINANT HUMAN EGFR PROTEIN-FRAGMENTS FOR SCREENING OF DRUG CANDIDATE COMPOUNDS TARGETING PANCREATIC CANCER." @default.
- W2000461990 doi "https://doi.org/10.1097/00006676-200411000-00113" @default.
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