Expression and purification of the epidermal growth factor receptor extracellular domain utilizing a polycistronic expression system.
High level expression of the epidermal growth factor receptor ectodomain (EGFR-ED) has been achieved using a polycistronic expression system. The expression vector was designed such that EGFR-ED was at the 5' end of a tricistron followed by luciferase and dihydrofolate reductase (dhfr). Following transfection into Chinese hamster ovary cells, clones were isolated under selective conditions for dhfr expression and monitored for luciferase activity and EGFR-ED expression using immunofluorescence microscopy. A 100-kDa protein corresponding to EGFR-ED was efficiently secreted from expressing cells. Two purification schemes were used to obtain protein at least 95% pure. Glutaraldehyde crosslinking was used to show that EGFR-ED specifically binds EGF and TGF alpha and that the affinity for EGF is 5.5 x 10(-7) M.
Department of Medicine, University of California, San Diego, La Jolla 92093-0650.
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D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D016764:Cell Polarity; D006224:Cricetinae; D066246:ErbB Receptors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis; D014162:Transfection
D010446:Peptide Fragments; D011994:Recombinant Proteins; D008156:Luciferases; D013762:Tetrahydrofolate Dehydrogenase; D066246:ErbB Receptors