Fusion proteins are generally used as antigens to stimulate antibody production and in many cases for biochemical analysis. Two widely used E. coli expression systems are the lacZ fusion system utilizing the pUR vector family and the trpE fusion system utilizing the pATH vector family.
Operation method
Basic methods
Materials and Instruments
Escherichia coli Move Expression of lacZ fusion proteins using the pUR vector:1a. The target gene was subcloned into the pUR vector according to the correct reading frame, E. coli receptor cells were transformed, and transformants were selected on LB/aminobenzylpenicillin flat dishes. For more product details, please visit Aladdin Scientific website.
Ampicillin IAA IPTG PBS Guanidine hydrochloride Tris-Cl
Ultrasonic generator Dialysis bag Rotor Centrifuge
2a. Inoculate single colonies containing the expression vector in 2~5 ml LB/aminobenzylpenicillin medium and incubate overnight at 37°C with shaking.
3a. Take 1 ml of overnight culture and add it to 400 ml LB/aminobenzylpenicillin medium contained in a 2 L bottle, and incubate at 37℃ with shaking until OD600 reaches 0.5.
4a. Add 1.6 ml of 100 mmol/l IPTG (final concentration of 0.4 mmol/l) and continue the incubation for 2 h. The incubator was then incubated for 2 h with the addition of 1.6 ml of 100 mmol/l IPTG.This trpE fusion protein was epitomized using the pATH vector:
1b. The target gene was subcloned into the pATH vector according to the correct reading frame, transcribed to E. coli receptor cells, and the transformants were selected on M9 medium flat dishes supplemented with tryptophan.
2b. Inoculate 2~5 ml of M9 medium with tryptophan in a single colony containing the expression plant and incubate overnight as in step "2a".3b. Take 1 ml of the overnight culture and add it to 400 ml of tryptophan-free M9 medium and incubate at 37°C with vigorous shaking until OD600=0.5.
4b. 1.6 ml of 2.5 mg/ml IAA (final concentration 10 μg/ml) was added and the incubation was continued for 2 h. The incubation was continued for 2 h. The incubation was continued for 2 h. The incubation was continued for 2 h.5. Pour the bacterial culture into two 200 ml centrifuge flasks and centrifuge at 4,000 g for 10 min.6. Add 5 ml of PBS to the precipitate, resuspend by blowing up and down, and transfer the precipitate from each vial to a 15 ml conical centrifuge tube and centrifuge at 3,000 g for 10 min.
7. Resuspend the bacteria in 2 ml of HEMGN buffer containing protease bet preparation, add 20 μl of 50 mg/ml lysozyme and leave on an ice bath for 5-30 min.8. Break the cells with a microprobe ultrasonic generator for 15 s each time, 2 times in total, and place the sample in an ice bath between sonication sessions. Pour the lysate into a 50 ml centrifuge tube.9. Centrifuge the cell lysate at 27,000 g for 15 min, pour off the supernatant and retain. The precipitate, which contains almost all of the insoluble induced protein, is retained. Soluble proteins are prepared in the next step.10. Resuspend the precipitate in 2 ml of HEMGN buffer containing protease inhibitors.11. Add 2 ml HEMGN buffer/8 mol/l guanidine hydrochloride (final concentration of guanidine is 4 mol/l) for 30 min at 4°C with gentle shaking.12. Centrifuge at 27,000 g for 30 min in a pre-cooled ultracentrifuge or at 27,000 g for 20 min at 4 °C.
13. Transfer the supernatant into a dialysis bag and dialyze in 2 steps, each over 3 h or even overnight: first in 500 ml HEMGN buffer/1 mol/l guanidine hydrochloride, followed by 1 L of HEMGN buffer without guanidine.
14. Transfer all material to a centrifuge tube and centrifuge at 12 000 g for 5 min, retaining the supernatant, which is a clear, colorless liquid with a protein concentration of approximately 1 mg/ml. (Fusion proteins typically make up 1% to 10% of the total proteins.) Insoluble precipitates, which usually also contain fusion proteins, can be used for gel purification and should be retained.
15. Protein concentrations in the supernatant and precipitate of cell lysates and in the final supernatant and precipitate after guanidine extraction are determined by the Bradford method. The results of the induction were examined by SDS-PAGE, and 5~10 μg of protein was spiked per well.