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1.2.2: Module 4 and 5 Overview

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  • 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.