Expression of soluble proteins

Operation Procedure

1. Inoculate 50ul of the strain into 200ml of resistant medium and incubate overnight at 37℃.

2. The next day, fresh resistant medium was added to 800 ml and incubated for 1-2 hours until bacterial viability peaked.

3. Induction was induced by the addition of 200ul of 1M IPTG (28°C or 37°C) for 3.5 hours.

4. Collect the bacterial body (66 rpm×15min), discard the supernatant, add 30 ml of extraction solution to suspend the bacterial body, add the final concentration of 1 mM PMSF and the final concentration of 0.5 mM EDTA (its fusion protein was extracted without EDTA), and ultrasonic crush 200W for 6min under the condition of ice bath.

5. Incubate 1 hour at 4°C on an orbital shaker.

6. Centrifuge at 133 rpm for 15 minutes, remove supernatant and add 400ul of beads at 4°C to bind overnight.

7. Collect the beads (33 rpm×5min, while storing the supernatant at 4℃), wash the beads with washing solution to remove the heterogeneous proteins (1ml×3 times). Add 300ul of eluent and allow the eluent and beads to fully bind for 1h, then centrifuge to collect the supernatant. Add 300ul of eluent to the beads again, elute for 1h, centrifuge to collect the supernatant, and combine the two eluents in one tube.

8. The beads were washed 3 times with PBS, resuspended in the retained supernatant, bound for 2 hours at 4°C and the procedure of 4.2.7 was repeated.

9. The eluents obtained from 4.2.7 and 4.2.8 were combined in a tube, mixed well, 10ul were taken and 10ul of sample preparation buffer was added to boil the sample, then 8.3ug/10ul of Maker (BSA 67kd; GST 26kd) was added together with the sample, and the gel was run. After running the gel, the gel was stained with Caumas Brilliant Blue staining solution for 1h and destained with destaining solution for about 2h.

10. Protein molecular weight and concentration were estimated visually from the manufacturer's gel run results.

11. The collected fusion proteins were stored in a -20°C refrigerator and the location number was recorded.

List of reagents used

Extraction solution (washing solution) PBST/PBSS PH7.4

PBS 

LB Medium

IPTG

1M IPTG in water

 11.33g IPTG in 40ml ddH20

PMSF

100mM in isopropyl alcohol

1g PMSF in 57ml isopropyl alcohol

EDTA

0.5M in water

55.8g EDTA in 300ml ddH20

GST Protein Eluent  PH 8.0

100mM GSH in PBS/T

3.07g GSH in 100ml PBS/T

His Protein Eluent   PH 7.0

300mM imidazole in PBSS(or PBS)

His Protein Wash   PH 7.0 

20mM imidazole in PBSS