High yield bacterial expression of active c-Abl and c-Src tyrosine kinases
Markus A. Seeliger, Matthew Young, M. Nidanie Henderson, Patricia Pellicena, David S. King, Arnold M. Falick and John Kuriyan
Abstract: 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.
Figure 1: Expression and purification of c-Src kinase domain and c-Abl SH3-SH2 kinase domain in bacteria.
A. Coomassie-stained SDS-PAGE of c-Src kinase domain. (Lane 1) Whole-cell lysates; (lane 2) soluble protein; (lane 3) flow-throught of Ni-affinity resin; (lane 4) elution from Ni-affinity resin after overnight TEV digest; (lane 5) elution from anion exchange resin. Src kinase domain expresses in hight levels in bacteria but only a small fraction of the total expressed protein is soluble, as indicated by the small fraction of protein in lane 2. The His-tagged kinase binds to the Ni-affinity resin, and no kinase is found in the flow-through. After overnight dialysis and TEV digest, the major impurity is undigested kinase, still containing the His tag. Anion exchange chromatography yields homogenous and pure kinase protein.
B. Coomassie-stained SDS-PAGE of c-Abl three-domain construct (SH3-SH2-kinase; residues 46 to 515). (Lane 1) Whole-cell lysates; (lane 2) soluble protein; (lane 3) flow-through Ni-affinity resin; (lane 4) elution Ni-affinity resin. Larger Abl and Src kinase domain constructs express with high yield in bacteria but are only fractionally soluble. The His-tagged kinase binds to the Ni-affinity resin and elutes with high purity from it.
C. Representative mass spectrum (electrospray-ion trap) of a Src kinase domain preparation. The calculated mass from the DNA sequence is 32,633.5 Da, the experimental mass is 32,633 Da (standard deviation 1 Da), indicating that the protein is not posttranslationally modified.
Figure 2: Characterization of the interaction between Abl kinase domain and imatinib.
A. Kinase activity was monitored in a continuous spectrophotometric assay against a peptide substrate and varying concentrations of imatinib. Imatinib inhibited the activity of the Abl kinase domain with an IC50 of 0.8 uM.
B. Isothermal titration calorimetry of imatinib to Abl kinase domain. Abl kinase domain binds imatinib with an affinity of 50 nM and a 1:1 stoichiometry, indicating that the protein is folded and that the concentration is determined accurately.