Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector.
The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X-ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155-kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(-9) M in intact infected insect cells which falls to 2 X 10(-7) M upon detergent solubilization. The recombinant protein exhibits an EGF-stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large-scale production.
Greenfield
C
C
Ludwig Institute for Cancer Research, London, UK.
Patel
G
G
Clark
S
S
Jones
N
N
Waterfield
M D
MD
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007700:Kinetics; D007915:Lepidoptera / Q000235:genetics*; D009036:Moths / Q000235:genetics*; D010449:Peptide Mapping; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D004247:DNA; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases