Purification of an active EGF receptor kinase with monoclonal antireceptor antibodies.
A method is described for a rapid two-step purification of the membrane receptor for epidermal growth factor (EGF) from cultured human A-431 cells. After solubilization of the cells with Triton X-100, the receptor is immobilized on an immunoaffinity column containing a monoclonal antibody directed against the receptor. In the second step of purification, the receptor, eluted from the antibody column, is adsorbed and specifically eluted from a lectin-agarose column. The molecular species obtained is mainly the 170,000-dalton EGF receptor polypeptide. The activity of the pure receptor depends on the conditions used for the desorption from the immunoaffinity beads. High-yield elution is obtained with acidic buffer and the receptor so purified specifically binds EGF, but is devoid of the kinase activity. When the elution is done with alkaline buffers or with buffer containing urea, a fully active receptor kinase is purified (yield of 10%). The pure receptor binds 125I-EGF with a Kd of 4 X 10(-8) M and retains EGF-sensitive protein kinase activity which phosphorylates tyrosine residues on the receptor itself. An additional protocol is described for large-scale purification (yield of 55%) of EGF receptor for the analysis of its primary structure. In this procedure, the EGF receptor is first purified by immunoaffinity chromatography which is followed by preparative gel electrophoresis of the 32P internally labeled receptor to remove minor protein contaminants.
Yarden
Y
Y
Harari
I
I
Schlessinger
J
J
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D066246:ErbB Receptors; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D066246:ErbB Receptors