Content Research with Mentor TEV protease- Enzyme that clips at known sequence: ENLYFQG/S TEV protease MBP domain N-197 protein Link that includes TEV protease’s clip site The whole protein includes 3 parts, in the middle between the two pieces of protein domain is where TEV protease finds the clip site Clip Site ENLYFQG/S
The experiment will place a constant amount of protein together with varying amounts of TEV protease to determine most efficient combination Stable protein amount in each combination Variation in TEV protease amounts in each combination
Uncleaved Protein MBP and N197 The mass of the uncut protein is approx. 60 kDa (exactly 59.537 kDa) Gel Electrophoresis confirming presence of protein
Post Cleavage Uncut MBP N197 Illustrates all three portions of the protein and confirms cleavage is occurring N197 MBP Uncut
Gel of TEV Cleavage Uncut MBP N197 Confirms data from Mass Spec
Absorbance of Protein with Standard Protein Assay ●.7757 Final concentration determined for protein =0.6313 using Spectroscopy How much is there of me(Protein)? Need to know concentrations to find the right ratio of protein to enzyme.
Time Dependent Concentration Controlled 1 hour2 hours 3 hours 1 hour2 hours3 hours 1 hour 2 hours 3 hours High Low Electrophoresis gel used to show the relationship between the concentration and time. Medium No TEV protease
Concentration Dependent Controlled 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. 0.00009765 mg/mL