Course Meeting Times

Lectures: 6 sessions over 8 weeks, 1 hour / session Labs: 2 sessions / week, 4 hours / session

Overview
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 structureviewing 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.

Grading
ACTIVITIES Lab preparation and participation Lecture attendance Laboratory work / lab report Total POINTS 40 points 10 points 50 points 100 points

Calendar
The calendar below provides information on the course's lecture (Lec) and lab (Lab) sessions.

SES # Lec 1 Lab 1

H396P ABL PROTEIN EXPRESSION/ KINASE INHIBITION ASSAYS

DNA SITE-DIRECTED MUTAGENESIS

Introduction. Kinases in healthy and cancerous cells Grow a starter culture of cells with the H396P Abl and Yop-encoding vectors. Express the H396P Abl protein. (Spin down cells on the following day.) DNA digestion and PCR Digest isolated DNA to check for the wt Abl insert. Run DNA agarose gel. Design primers for an Abl kinase domain mutant. Affinity tags for protein purification / detection Prepare protein purification buffers. Create a BSA standard curve for future protein quantification. Lyse cells and isolate the H396P Abl kinase domain. Dialyze protein into TBS. Prepare an SDS-PAGE protein gel. Conserved and variable structural features of kinase domains Run SDS protein gel. Concentrate protein and quantify final protein concentration. Grow a starter culture of cells with the wild type Abl vector. Isolate wt-Abl vector DNA through a miniprep. Quantify DNA concentration by UV-Vis.

Lab 2 Lec 2

Lab 3

Lec 3 Lab 4 Lab 5 Lab 6 Lec 4 Lab 7 / Lab8

SES # Lec 5 Lab 9 Lab 10

H396P ABL PROTEIN EXPRESSION/ KINASE INHIBITION ASSAYS

DNA SITE-DIRECTED MUTAGENESIS

Site directed mutagenesis (Quickchange method) Set up PCR for DNA mutagenesis. Digest template DNA, and transform storage cells with mutant DNA. Pour LB/agar plates. Isolate (by miniprep) and quantify DNA. Prepare mutant DNA samples for sequencing. Detecting kinase activity Prepare buffers and reagents for the coupled kinase activity assay. Complete kinase assays: wt Abl kinase domain and the H396P mutant domain in the absence and presence of inhibitors. Complete crystal structure viewing exercises. Analyze DNA sequencing results.

Lab 11 Lec 6 Lab 12 Lab 13 / Lab 14 Lab 15