Course Meeting Times
Lectures: 6 sessions over 8 weeks, 1 hour / session
Labs: 2 sessions / week, 4 hours / session
The course covers biochemistry laboratory techniques in the context of investigating resistance to the cancer drug Gleevec.
Gleevec is a small molecule drug for chronic myelogenous leukemia (CML) that functions as a potent inhibitor of the protein Bcr-Abl, an aberrant kinase implicated in CML. While many CML patients treated with Gleevec experience remission, a significant population develops resistance to the drug. Point mutations in the gene that encodes the Bcr-Abl protein have been identified in patients with Gleevec-resistant CML.
In this course, students express a Gleevec-resistant mutant of the Bcr-Abl kinase domain and investigate in-vitro inhibition of kinase activity by Gleevec and a second kinase inhibitor, Dasatinib. The mutant Abl domain is expressed in E. coli and then purified and analyzed using nickel affinity chromatography, polyacrylamide gel electrophoresis, UV-Vis spectroscopy, and BSA quantification. Students use a coupled phosphorylation assay to determine the specific activity of their expressed mutant kinase domain and the wild type kinase domain in the absense and presence of Gleevec and Dasatinib. Students use a structure-viewing program to explore the mechanistic basis of Bcr-Abl inhibition and Gleevec-resistance.
Students also use site-directed mutagenesis to create the DNA for another Gleevec-reistance mutant of their choice.
Pre-Labs and Lab Participation
Prior to each laboratory session (except for session 1), you should outline the procedures you will be carrying out and complete any relevant calculations. Prelabs should include a clear outline, but need not repeat detailed procedures that you will refer to in your lab manual (for example, the step by step procedure for a miniprep). Your TA will assign you a grade of 0, 1 or 2 points for each lab session based on your preparation and participation in lab for that day.
|Lab preparation and participation||40 points|
|Lecture attendance||10 points|
|Laboratory work / lab report||50 points|
The calendar below provides information on the course’s lecture (Lec) and lab (Lab) sessions.
|SES #||H396P ABL PROTEIN EXPRESSION/ KINASE INHIBITION ASSAYS||DNA SITE-DIRECtED MUTAGENESIS|
|Lec 1||Introduction. Kinases in healthy and cancerous cells|
|Lab 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.|
|Lab 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.|
|Lec 2||DNA digestion and PCR|
|Lab 3||Digest isolated DNA to check for the wt Abl insert. Run DNA agarose gel. Design primers for an Abl kinase domain mutant.|
|Lec 3||Affinity tags for protein purification / detection|
|Lab 4||Prepare protein purification buffers. Create a BSA standard curve for future protein quantification.|
|Lab 5||Lyse cells and isolate the H396P Abl kinase domain. Dialyze protein into TBS.|
|Lab 6||Prepare an SDS-PAGE protein gel.|
|Lec 4||Conserved and variable structural features of kinase domains|
|Lab 7 / Lab8||Run SDS protein gel. Concentrate protein and quantify final protein concentration.|
|Lec 5||Site directed mutagenesis (Quickchange method)|
|Lab 9||Set up PCR for DNA mutagenesis.|
|Lab 10||Digest template DNA, and transform storage cells with mutant DNA. Pour LB/agar plates.|
|Lab 11||Isolate (by miniprep) and quantify DNA. Prepare mutant DNA samples for sequencing.|
|Lec 6||Detecting kinase activity|
|Lab 12||Prepare buffers and reagents for the coupled kinase activity assay.|
|Lab 13 / Lab 14||Complete kinase assays: wt Abl kinase domain and the H396P mutant domain in the absence and presence of inhibitors.|
|Lab 15||Complete crystal structure viewing exercises.||Analyze DNA sequencing results.|