1.2.2: Module 4 and 5 Overview
- Page ID
- 25816
Gleevec is a blockbuster small molecule drug for chronic myelogenous leukemia (CML) that functions as a potent inhibitor of Bcr-Abl, an aberrant kinase implicated in the disease. While most of patients treated with Gleevec in the chronic stage of CML experience remission, a significant population eventually develops resistance to the drug. A number of point mutations in the gene that encodes the Bcr-Abl protein have been identified in patients with Gleevec-resistant CML. Over the semester you will develop and execute a research plan to 1) determine whether a selected mutation (H396P) in the BCR-ABL gene confers Gleevec resistance using an in-vitro kinase assay, 2) explore the efficacy of an alternative Bcr-Abl inhibitor, Dasatinib, on the wild-type and mutant kinases, 3) evaluate crystal structures to understand the mechanism(s) by which Bcr-Abl mutations block drug activity, and 4) use site-directed mutagenesis to create an another Gleevec-resistant Abl mutant of your choice. A brief description of the 15 lab sessions is provided below.
H396P Abl protein expression/ kinase inhibition assays | DNA site-directed mutagenesis | |
Session 1 | Grow a starter culture of cells with the H396P Abl and Yop-encoding vectors. | Grow a starter culture of cells with the wild type Abl vector. |
Session 2 | Express the H396P Abl protein. (Spin down cells on the following day.) | Isolate wt-Abl vector DNA through a miniprep. Quantify DNA concentration by UV-Vis. |
Session 3 | Digest isolated DNA to check for the wt Abl insert. Run DNA agarose gel. Design primers for an Abl kinase domain mutant. | |
Session 4 | Prepare protein purification buffers. Create a BSA standard curve for future protein quantification. | |
Session 5 | Lyse cells and isolate the H396P Abl kinase domain. Dialyze protein into TBS. | |
Session 6 | Prepare an SDS-PAGE protein gel. | |
Session 7/8 | Run SDS protein gel. Concentrate protein and quantify final protein concentration. | |
Session 9 | Set up PCR for DNA mutagenesis. | |
Session 10 | Complete the DPN digest and transform storage cells with mutant DNA. Pour LB/agar plates. | |
Session 11 | Isolate (by miniprep) and quantify DNA. Prepare mutant DNA samples for sequencing. | |
Session 12 | Prepare buffers and reagents for the coupled kinase activity assay. | |
Session 13 & 14 | Complete kinase assays: wt Abl kinase domain and the H396P mutant domain in the absence and presence of inhibitors. | |
Session 15 | Complete crystal structure viewing exercises. | Analyze DNA sequencing results. |