title: Generation of recombinant cytoplasmic domain of epidermal growth factor receptor with intrinsic protein tyrosine kinase activity.
abstract: We have generated a recombinant baculovirus using the high expression vector pVL941 containing the complementary DNA encoding the intracellular domain of the human epidermal growth factor receptor (EGFR-IC). Upon infection of Spodoptera frugiperda insect cells, protein tyrosine kinase-active EGFR-IC was produced. The expressed protein has a molecular weight of 61,000 and is specifically recognized by antibodies directed against peptides representing different regions of human EGFR-IC. Upon sonication of infected cells, EGFR-IC was detected in both the soluble and insoluble fractions of the cell lysate. About 20-50% of the expressed EGFR-IC was soluble. Metabolic labeling and protein analyses showed that EGFR-IC comprised 7% of newly synthesized proteins in the cytoplasmic lysate and 0.1-0.2% of the total soluble protein. We have used a three-step purification procedure (fast-Q-Sepharose and phenyl-Sepharose column chromatographies and 30% ammonium sulfate precipitation) to purify EGFR-IC to 85% purity with 15-20% recovery from the initial soluble lysate. A yield of 3-4 mg of purified EGFR-IC has been consistently produced from 20 roller bottles with 2-4 x 10(8) infected cells/bottle. The tyrosine kinase activity was retained through purification. The enzyme demonstrated much higher autophosphorylation activity in the presence of Mn2+ than Mg2+. Phosphopeptide mapping revealed the same autophosphorylation sites utilized by EGFR-IC as those identified in wild-type EGFR. EGFR-IC-catalyzed phosphorylation of either a synthetic peptide representing the major autophosphorylation site or angiotensin II showed that the baculovirus-expressed EGFR-IC exhibits similar enzymatic kinetic characteristics to the intact activated EGFR kinase.
authors.affiliation: Rorer Biotechnology, Inc., King of Prussia, Pennsylvania 19406.
authors.forename: C Y
authors.initials: CY
authors.lastname: Hsu
authors.forename: M
authors.initials: M
authors.lastname: Mohammadi
authors.forename: M
authors.initials: M
authors.lastname: Nathan
authors.forename: A
authors.initials: A
authors.lastname: Honegger
authors.forename: A
authors.initials: A
authors.lastname: Ullrich
authors.forename: J
authors.initials: J
authors.lastname: Schlessinger
authors.forename: D R
authors.initials: DR
authors.lastname: Hurwitz
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009036:Moths; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D004247:DNA; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases