Expression and purification experiments of thioredoxin fusion protein
Expression and purification experiments of thioredoxin fusion protein
A gene fusion expression system using the E. coli trxA gene product-thioredoxin as a fusion partner is particularly suitable for the expression of high yield soluble fusion proteins in the cytoplasm of E. coli.
Operation method
basic program
Materials and Instruments
Fusion Proteins Move 1. A DNA fragment encoding the target sequence is cloned into the 3' end of the trxA gene on the pTRXFUS or hpTRXFUS plasmid to construct a fusion gene that conforms to the reading frame or a short peptide coding sequence is inserted at a single Rsr 2 site of pALtrxA-781. For more product details, please visit Aladdin Scientific website.
Ampicillin Glycerol Tryptophan
Electrophoresis apparatus Incubator Shaking table Test tube
2. Receptor GI724 cells were transformed with a ligation mix containing a plasmid containing a recombinant thioredoxin fusion protein. Transformed cells were seeded into IMC dishes containing 100 μg/ml ampicillin to select transformants, and the dishes were warmed in a convection incubator at 30°C until colonies appeared.
3. Candidate colonies were inoculated in 5 ml of CAA/glycerol/aminobenzylpenicillin 100 culture medium and incubated at 30°C overnight. A small amount of plasmid DNA was prepared and the gene was checked for correct insertion into pTRXFUS by restriction enzyme digestion.
4. Sequencing of plasmid DNA from candidate clones to confirm the linkage region between the thioredoxin and the gene or sequence to be studied.5. Cryopreserved GI724 bacteria containing thioredoxin expression plasmid were streak inoculated onto IMC medium containing 100 μg/ml ampicillin to grow single colonies. Growth was carried out at 30°C for 20 h.
6. Newly grown, well-separated colonies were picked from petri dishes and inoculated into 5 ml of IMC medium containing 100 μg/ml ampicillin in 18 mm × 150 mm culture tubes. The culture was incubated on a shaker at 50°C overnight.
7. 0.5 ml of the overnight culture was inoculated into 50 ml of fresh IMC medium containing 100 μg/ml ampicillin in 250 ml culture flasks. Incubate at 30°C with full aeration until its 550 nm absorption value reaches 0.4-0.6 OD/ml.
8. Remove a small 1 ml tube from the culture (uninduced cells). Measure the absorption at 550 nm and when the density reaches 0.4-0.6 OD/ml, harvest the cells by microcentrifugation at maximum speed for 1 min at room temperature. Carefully remove all the used medium with a pipette and store the cell pellet at -80℃.
9. 0.5 ml of 10 mg/ml tryptophan (to bring the final concentration to 100 μg/ml) was immediately added to the remaining cells to induce PL transcription.10. incubate at 37°C for 4 h. During this period, remove a small portion of 1 ml of culture at 1 h intervals and harvest the cells according to step 8.
11. 4 h after induction, centrifuge (e.g., using a Becjman J6 rotor) at 3 000 r/min for 10 min at 4°C and harvest the remaining cells from the culture. Cell face blocks were stored at -80°C.
12. Resuspend the cell clusters obtained during induction (from steps 8 and 10) with SDS-PAGE sample buffer at 200 μl/OD550 cells. Heat at 70°C for 5 min to completely lyse the cells and denature the proteins.
13. On an SDS-polyacrylamide gel, a sample equivalent to 0.15 OD650 cells was added to each lane, and the gel was stained with Caumas Brilliant Blue for 1 h. The gel was decolorized and checked for expression.