Expression and purification experiments of glutathione S-transferase fusion protein
The pGEX vectors can be used to express and purify peptides (including short peptides) used as immunogens and biochemical and biological reagents, or to construct expression libraries from cDNA. Each pGEX vector has an open reading frame encoding glutathione S-transferase, followed by a single restriction endonuclease site and then a stop codon corresponding to the three reading frames.
Operation method
basic program
Materials and Instruments
Escherichia coli Move 1. Subclone the DNA fragments into the appropriate pGEX vector according to the correct reading frame, transform the E. coli receptor cells, screen the transformants on LB/amphenicol dishes, and use the self-contained vector without insertion of exogenous DNA as a control, and incubate the dishes at 37℃ for 12-15 hours.2. Pick the transformed colonies to be inoculated in 2 ml LB/aminobenzylpenicillin medium and streak the culture on the ampicillin master plate. At the same time, the transformed bacteria containing the parent pGEX vector were inoculated as control. The main plate was incubated at 37℃ for 12~15 h. The liquid culture was incubated in a shaker at 37℃ with shaking until turbid. For more product details, please visit Aladdin Scientific website.
Ampicillin LB PBS SDS Glycerol Glutathione
Centrifuge Shaker Rotor Ultrasonic Generator
3. Add 100 mmol/l IPTG to a final concentration of 0.1 mmol/l to induce the expression of fusion proteins, and continue to incubate for 1~2 hours.4. Transfer the liquid culture into a labeled microcentrifuge tube and centrifuge at maximum speed for 5 s at room temperature, discard the supernatant, and resuspend the precipitate in 300 μl of pre-cooled PBS solution in an ice bath. Dispense 10 μl into another labeled tube.5. The cells are broken with an ultrasonic generator with a 2 mm diameter probe, centrifuged at high speed for 5 min at 4°C to remove insoluble material, and the supernatant is transferred to a new centrifuge tube.6. Add 50 μl of 50% glutathione-agarose bead suspension to the supernatant of each tube, mix gently at room temperature, add 1 ml of PBS and spin up with a vortex mixer with brief oscillation, centrifuge at high speed for 5 s, collect the agarose beads, discard the supernatant, and repeat the washing with PBS twice.7. Add an equal volume of 1×SDS sample buffer to the agarose beads, add another 30 μl to 10 μl of whole cell resuspension, heat at 100℃ for 3 min, and then add samples onto a 10% SDS-polyacrylamide gel after briefly oscillating and spinning with a vortex mixer, electrophoresis for an appropriate period of time, and then stain with Caulmers Brilliant Blue to make the GST proteins and fusion proteins visible.
8. Pick a pGEX transformed colony and inoculate it in 100 ml LB/aminobenzylpenicillin medium, and incubate it at 37℃ in a shaker for 12~15 h. The colony was then incubated for 12~15 h in a shaker.9. Dilute this culture in 1 L of fresh LB/aminobenzylpenicillin medium at a ratio of 1:10 into two 2 L flasks and incubate for 1 h at 37°C.
10. Add 100 mmol/l IPTG to a final concentration of 0.1 mmol/l, and continue incubation for 3-7 h.
11. Centrifuge the cells at 5000 g for 10 min at room temperature, collect the cells and discard the supernatant. The precipitate was resuspended in 10-20 ml of pre-cooled PBS in an ice bath.12. Place the centrifuge tube in an ice bath and lyses the cells with an ultrasonic generator with a 5 mm diameter probe, adjusting the frequency and intensity to avoid foaming and slowing down so that the lysis is completed within 30 s.
13. Add 10% Triton X-100 to a concentration of 1% and mix well. Centrifuge at 10,000 g for 5 min at room temperature to remove insoluble components and unlysed cells. Alternatively, take a 1.5 ml portion of the sample and centrifuge at maximum speed for 5 min at 4°C in a microcentrifuge and collect the supernatant (be careful to avoid aspiration of precipitate).
14. Add the supernatant to 1 ml of 50% glutathione-agarose bead suspension and mix gently at room temperature for ≥2 min. add 50 ml of ice-bath pre-cooled PBS solution, wash, mix, and centrifuge in a benchtop centrifuge at 500 g for 10 s. Repeat the wash two times, resuspend the agarose beads with a small amount (1-2 ml) of ice-cooled PBS, and transfer to a 1.5 ml microcentrifuge tube.
15. Centrifuge at 500 g for 10 s at room temperature, collect the agarose beads and discard the supernatant. Elute the fusion protein with 1 ml of 50 mmol/l Tris-Cl (pH 8.0)/5 mmol/l reduced glutathione. Gently mix for 2 min, centrifuge at 500 g for 10 s, collect the supernatant, repeat the elution 2~3 times, and analyze the samples collected from each section by SDS-PAGE. The eluted proteins were divided into small portions and stored at -70°C with 10% glycerol. The absorbance at 280 nm was measured to determine the fusion protein yield, and for GST vector, A280=1 corresponds to a protein concentration of 0.5 mg/ml.