High yield bacterial expression of active c-Abl and c-Src tyrosine kinases.
The Abl and Src tyrosine kinases are key signaling proteins that are of considerable interest as drug targets in cancer and many other diseases. The regulatory mechanisms that control the activity of these proteins are complex, and involve large-scale conformational changes in response to phosphorylation and other modulatory signals. The success of the Abl inhibitor imatinib in the treatment of chronic myelogenous leukemia has shown the potential of kinase inhibitors, but the rise of drug resistance in patients has also shown that drugs with alternative modes of binding to the kinase are needed. The detailed understanding of mechanisms of protein-drug interaction and drug resistance through biophysical methods demands a method for the production of active protein on the milligram scale. We have developed a bacterial expression system for the kinase domains of c-Abl and c-Src, which allows for the quick expression and purification of active wild-type and mutant kinase domains by coexpression with the YopH tyrosine phosphatase. This method makes practical the use of isotopic labeling of c-Abl and c-Src for NMR studies, and is also applicable for constructs containing the SH2 and SH3 domains of the kinases.
Seeliger
Markus A
MA
Department of Molecular and Cell Biology, 16 Barker Hall, University of California, Berkeley, CA 94720-3202, USA.
Young
Matthew
M
Henderson
M Nidanie
MN
Pellicena
Patricia
P
King
David S
DS
Falick
Arnold M
AM
Kuriyan
John
J
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015007:Yersinia; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D016315:Proto-Oncogene Proteins c-abl; D019061:src-Family Kinases; D017027:Protein Tyrosine Phosphatases; C067571:yopH protein, Yersinia