Fusion Protein Enzymolysis Assay
Fusion Protein Enzymolysis Assay
The use of gene fusion expression systems to express exogenous genes in E. coli has become increasingly popular. The reason for this is largely attributed to the ability of fusion systems to produce large amounts of soluble fusion proteins. Glutathione transferase as well as thioredoxin have been shown to be very successful in producing correctly folded, biologically active proteins. Each of these has a convenient purification method to separate the fusion protein from cellular contaminants. The resulting proteins are suitable for conducting studies of their biological activity or (and) interactions. As fusion expression methods become more commonly used, the ability to cleave the N-terminal portion of the carrier protein from the C-terminal target protein becomes even more important. Source: Compact Laboratory Guide to Molecular Biology (5th Edition)
Operation method
Basic Program With Xa Factor
Materials and Instruments
1 mg/ml fusion protein Move 1. Prepare two small pre-test reactions to determine the optimal incubation time. Reaction 1: 20 μl of 1 μl of 1 mg/ml fusion protein containing 1 μl of 200 μg/ml Xa factor Reaction 2: 5 μl of 1 mg/ml fusion protein without Xa factor (mock digestion) React at room temperature. 2. 2. At 2, 4, 8 and 24 h, remove 5 μl of reaction solution, add 5 μl of 2 × SDS sample buffer, and freeze at -20°C. 3. At 24 h, remove 5 μl of reaction solution, add 5 μl of 2 × SDS sample buffer, and freeze at -20°C. 3. At 24 h, mix 5 μl of Reaction 2 (analog digestion) solution with 5 μl of 2 × SDS sample buffer. 4. 4. 5 μl of the original fusion protein solution is mixed with 5 μl of 2 × SDS sample buffer (uncleaved control). 5. 5. Heat all samples in a boiling water bath for 10 min, add samples to an SDS-polyacrylamide gel for electrophoresis, and determine the appropriate incubation time based on the degree of cleavage. If only partial cleavage is evident, increase the amount of enzyme and/or extend the incubation time. If no cleavage occurs, proceed on the basis of the auxiliary protocol. 6. After determining the appropriate cleavage conditions, the remaining fusion protein samples are scaled up for the reaction, leaving only a small amount of uncleaved fusion protein for use as a control. The extent of cleavage is monitored by SDS polyacrylamide gel electrophoresis. Caveat Fusion proteins purified by glutathione-agarose gel can be subjected to factor Xa digestion in PBS buffer. Alternatively, the proteins can be dissolved in 20 mmol/L Tris - Cl (pH 8.0)/ 1 mmol/L CaCl2/ 100 mmol/L NaCl. Although most fusion proteins can be stored at 4°C, they should be stored in 10% glycerol at -70°C until step 6. For more product details, please visit Aladdin Scientific website.
200 μg Xa factor/ml reaction buffer 2 × SDS sample buffer
Boiling water bath