TEV Cleavage Sample Clauses

TEV Cleavage. The FG Loop, Alpha Helix and Distal Active Site Mutants originally contained a His-Tag which helped with purification of the enzymes. However, once the enzyme is purified, there is no need for the His-Tag, so it must be removed to leave one attachment site, the cysteine, for the site-specific binding of AuNPs. TEV protease is used remove the His-Tag by recognizing the seven-amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly and cleaves between Gln and Gly. Initially, the uncleaved enzymes were stored in a -80C freezer until the TEV cleavage was required. Once the enzymes were removed from the freezer, their concentrations were checked using a UV/Vis spectrometer, and were diluted to 250 M using 50 mM sodium phosphate buffer. 20% by volume of TEV protease was added to the reaction solution and was left to react at 4C for 24 to 48 hours. After the reaction time was over, the solution was run through a Nickel-nitrilotriacetic acid (Ni-NTA) column. A Ni-NTA column was used to purify cleaved product because the His-Tags from uncleaved protein and TEV protease bind to it, while the cleaved protein flows through. To clean the resin, 150 mM imidazole buffer was run through the column to remove residual enzyme bound with His-Tag from the column. 20% ethanol was run thorough to store the resin. The collected sample was then buffer exchanged in an Amicon ® Ultra – 15 Centrifugal filter, and was centrifuged six times at 5,000xg for 20 minutes each spin. The protein concentration was determined using the extinction coefficient of 3.11 x 104 M-1 cm-1 at 280 nm29 with UV/Vis analysis with a Nanodrop 2000, then diluted to 50 M enzyme concentration, and aliquoted into single use tubes. The samples were lyophilized and then stored at -20C for up to three months. To determine whether the TEV cleavage was successful, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was run. To prepare the samples of protein, the samples were rehydrated with 1.42 mg/mL of Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and DI water and left to incubate at room temperature for approximately an hour, to prevent disulfide bonds from forming between the individual mutant enzymes.30 A 50:50 ratio of protein and 2x Laemmli sample buffer was added. The samples were heated to boiling at 100C for 5 minutes, then immediately transferred to ice for 5 minutes. The samples were loaded into a polyacrylamide gel, placed in a Biorad CiterionTM Vertical Electrophoresis Cell with Tris...

TEV Cleavage. TEV protease is used to cleave the His-Tag from the single cysteine mutants to leave a single binding site on the enzyme to bind to AuNPs. SDS-PAGE was used to determine the difference in mass of the cleaved and uncleaved protein and showed that cleaved protein had a lower molecular weight that the uncleaved protein (Figure 2.1). The mass difference represented the loss of the His-Tag which is about 1-2 kDa. Since the mass of the cleaved protein is less than that of the uncleaved protein, it travels further in the gel. Both the cleaved Alpha Helix and FG Loop mutants have a lower mass due to the removal of the His-Tag. The Distal Active Site mutant is not shown, but due to the similarities and characteristics to that of the other two mutants, it is fair to assume that it ideally runs through the gel in a similar manner as cleaved and uncleaved protein.

TEV Cleavage. The conjugates were run in a similar manner to that of the free protein.