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1. Prepare LB media, ampicillin, IPTG and lysozyme stock solutions and Nickel binding buffer (20 mM Tris, 500 mM NaCl, 5 mM Imidazole) pH 8.0.
2) Inoculate a few mL from a glycerol stock into 5 mL LB with 100 mg/mL Amp. Incubate with shaking at 37 °C ON.
1) Grow cells to a high enough concentration to make a large quantity of protein.
Dilute E. coli BL21 with pET-portal ON in LB/Amp to an OD600 less than 0.1.
Place at 37 °C with shaking and grow to OD600 ~ 0.4.
At this point decrease the temperature to 28 °C and continue incubation with shaking until the OD600 ~ 0.7
2) Turn on the protein production.
Induce culture with IPTG to a final concentration of 1 mM and continue incubation (28 °C) with shaking for 5-6 hours.
3. Harvest the cells by centrifugation at 5000 X g for 15 minute at 4 °C.
4. Resuspend the cells in Nickel Binding buffer (1 mL buffer for every 10 mL of culture that you started with) and add lysozyme (0.25 mg lysozyme for every 1 mL of culture that you started with). Freeze at -80 °C.
1. Prepare reagents:
- Nickel Binding Buffer (500 mM NaCl, 20 mM Tris-Cl, 5 mM imidazole, pH 8.0).
- Nickel Wash Buffer (same as Binding Buffer but with 65 mM imidazole)
- Nickel Elute Buffer (same as Binding Buffer but with 500 mM)
- Q-Buffer, for dialysis (30 mM Tris-Cl, 75 mM NaCl, 2 mM EDTA, 20 mM b-mercaptoethanol, pH 7.5).
- NaCl Buffers (30 mM Tris-HCl, either 0.0 or 1.0 M NaCl (make one of each), 1 mM EDTA pH 7.5).
1. Thaw the frozen culture. Add a protease inhibitor cocktail, which is free of EDTA and add 1/200 volume of 100 mM PMSF.
2. Sonicate the lysed cells.
3. To pellet the cell debris, spin the lysed cells at 5000 X g for 45 minutes at 4 °C. The supernatant contains the P22 His6-Tagged Portal Protein.
4. Charge the column with Nickel solution and equilibrate the column.
5. Load the supernatant (protein solution) through the charged column.
6. Wash with Binding Buffer.
7. Wash with Washing Buffer.
8. Elute protein with 500 mM imidazole.
9. Add an EDTA solution to the eluted protein solution so that the final concentration of EDTA is ~10 mM.
10. Clean the column. Store the column with Storage Buffer in it.
11. Dialyze portal against Q-Buffer for at least 3 hours.
12. Prepare and equilibrate the Q-column with 30 mM Tris-HCl, 1 mM EDTA pH 7.5 (no NaCl).
13. Load portal onto Q-column.
14. Wash with a 0-1.0 M NaCl, 30 mM Tris-HCl, 1 mM EDTA pH 7.5 Buffer. Contaminants are removed at 260 mM NaCl, monomer at 340 mM NaCl, intermediate assemblies at 465 mM NaCl, and rings at 660 mM NaCl . (These rings appear to contain some aberrant forms.)
15) Strip column with Gdn-Cl Buffer. Store column in storage buffer.
16. Dilute the monomeric portal with Tris Buffer so that the final NaCl concentration is 100 mM.
17. Make the final concentration of protein ~0.5 mg/mL, by diluting with 100 mM NaCl.
18. Aliquot this monomer into tubes.
19. Freeze these aliquots at Ð80 °C.
To make rings from monomer
20. Concentrate portal monomer to ~50 mg/mL (a high concentration). Leave the monomeric portal (50 mg/ml) at room temperature to allow oligomerization to occur, about 24 h. Purify using the Q-column as described above with a 0-1.0 M NaCl, 30 mM Tris-HCl, 1 mM EDTA pH 7.5 Buffer. Collect the rings after elution with 660 mM NaCl.
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