Winthrop Initiative for STEM Educators

1. Winthrop Initiative for STEM Educators Chemistry 2011 WISE Summer Internship Amy Moore, Destinee Johnson, and Derion Reid

2. Introduction to Experiment

3. Content Research with Mentor TEV protease TEV protease- Enzyme that clips at known sequence: ENLYFQG/S The whole protein includes 3 parts, in the middle between the two pieces of protein domain is where TEV protease finds the clip site Link that includes TEV protease’s clip site MBP domain N-197 protein Clip Site ENLYFQG/S

4. Stable protein amount in each combination The experiment will place a constant amount of protein together with varying amounts of TEV protease to determine most efficient combination Variation in TEV protease amounts in each combination

5. Experiment Preparation

6. Uncleaved Protein Gel Electrophoresis confirming presence of protein The mass of the uncut protein is approx. 60 kDa(exactly 59.537 kDa) MBP and N197

7. Post Cleavage MBP Uncut N197 Illustrates all three portions of the protein and confirms cleavage is occurring N197 MBP Uncut

8. Gel of TEV Cleavage Uncut MBP N197 Confirms data from Mass Spec

9. How much is there of me(Protein)? Need to know concentrations to find the right ratio of protein to enzyme. Absorbance of Protein with Standard Protein Assay Final concentration determined for protein =0.6313 using Spectroscopy ● .7757

10. Final Experiment

11. Time Dependent Concentration 3 hours 3 hours 2 hours 2 hours 3 hours 1 hour 1 hour 2 hours 1 hour Controlled No TEV protease Low High Medium Electrophoresis gel used to show the relationship between the concentration and time.

12. Concentration Dependent Controlled 0.00009765 mg/mL 0.25 mg/mL Concentration dependent This is a three hours digestion comparing the high concentration of TEV protease to the low concentration of TEV protease.