Expression and purification of the intact cytoplasmic domain of the human ephrin receptor A2 tyrosine kinase in Escherichia coli.
The ephrin receptor A2 (EphA2) is an integral membrane protein tyrosine kinase and a member of the Eph family, the largest known family of receptor tyrosine kinases. EphA2 overexpression is sufficient to transform normal epithelial cells into an aggressive, metastatic phenotype. In normal cells, EphA2 negatively regulates cell growth and invasiveness. Here we report expression of the intact cytoplasmic domain (juxtamembrane linker, tyrosine kinase, and sterile alpha motif domains) of the human EphA2 receptor in an Escherichia coli system. The expressed protein was purified to near homogeneity by use of metal chelation chromatography combined with removal of vector-encoded tags by specific proteolysis. The cytoplasmic domains of EphA2 are expressed as an active kinase, with the expressed protein found to contain phosphorylated tyrosine residues. In addition, protein tyrosine phosphorylation appears only after EphA2 expression is induced and is removable with alkaline phosphatase treatment. The enzyme was purified 5-fold in yields that average 10-30 mg/L of active EphA2 cytoplasmic domains, which will now be used for further biophysical and structural characterization.
Zabell
Kathryn M
KM
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Laurence
Jennifer S
JS
Kinch
Michael S
MS
Knapp
Deborah W
DW
Stauffacher
Cynthia V
CV
D020816:Amino Acid Motifs / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D017434:Protein Structure, Tertiary / Q000235:genetics; D036104:Receptor, EphA2 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D036104:Receptor, EphA2