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| This protocol uses an Ammonium Sulfate cut and chromatography over a Mono Q column to purify bacterially produced p50/dynamitin. |
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1. Clone the full-length p50/dynamitin cDNA into a T7 expression vector by PCR (see Protocol and Hint #1).
2. Grow a 125 ml overnight culture of the transformed E. coli cells at 37°C in Supplemented LB.
3. The next morning, dilute the bacterial culture 8-fold (to 1 liter) in fresh Supplemented LB and allow the culture to grow at 20°C until its optical density at 600 nm is between 0.4 and 0.6 (OD600= 0.4 to 0.6).
4. Add IPTG to a final concentration of 0.1 mM to the bacterial culture to induce protein expression and allow the culture to grow overnight at 20°C (see Hint #2).
5. Harvest the bacteria from the culture by centrifugation at 5,000 X g for 30 min at 4°C.
6. Remove the supernatant and resuspend the cells in a small volume of PBS and centrifuge the cells at 5,000 X g for 30 min at 4°C.
7. Remove the supernatant and freeze the bacterial pellet in Liquid Nitrogen (see Hint #3).
8. Perform all of the subsequent steps at 4°C. Thaw the bacterial pellet from the previous step and resuspend the cells in 10 ml of Lysis Buffer.
9. Sonicate the bacteria using 3 repetitions of 30 seconds of sonication in the presence of 1 mg/ml Lysozyme. Keep the cell suspension on ice during the sonication (see Hint #4).
10. If the lysate is very viscous due to bacterial DNA contamination, add 10 μg/ml of DNase I and incubate the lysate on ice for 15 min.
11. Dilute the extract to 20 ml in fresh Lysis Buffer.
12. Preclear the lysate by centrifugation at 30,000 X g for 15 min at 4°C.
13. Transfer the precleared lysate to a beaker containing a magnetic stir bar in an ice bath over a magnetic stirring plate.
14. While stirring the lysate, slowly add finely-ground solid Ammonium Sulfate to a saturation of 20% (2.12 g of Ammonium Sulfate per 20 ml of lysate).
15. Allow the solution to incubate on ice for about 1 hr with gentle agitation.
16. Centrifuge the solution for 10 min at 20,000 X g at 4°C to recover the precipitate (see Hint #5).
17. Remove the supernatant and dissolve the precipitate in Mono Q Buffer with Protease Inhibitors over 30 minutes at 4°C with gentle agitation (see Hint #6).
18. Centrifuge the solution for 10 min at 30,000 X g at 4°C to remove any remaining particulate matter and filter the supernatant through a low protein binding filter unit.
19. Apply 20 ml of the protein solution to a 1 ml Mono Q FPLC column (Pharmacia) that has been equilibrated with Mono Q Buffer (see Hint #7).
20. Wash the column extensively with Mono Q Buffer until the absorbance at 280 nm (A280) returns to zero.
21. Elute the p50/dynamitin protein with a 20 ml linear gradient of 0 to 500 mM KCl in Mono Q Buffer. Collect 1 ml fractions. The peak of the p50/dynamitin elutes at about 200 mM KCl (see Hint #8).
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| PBS |
| 2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
137 mM NaCl
pH 7.4
1.4 mM Potassium Phosphate Monobasic (KH2PO4)
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| Supplemented LB |
| 5 g/liter NaCl
5 g/liter Yeast Extract
10 g/liter Tryptone
Supplement with 100 μg/ml Ampicillin and 25 μg/ml Chloramphenicol
1 ml/liter 1.0 M NaOH
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| Mono Q Buffer with Protease Inhibitors |
| 10 μg/ml Leupeptin
1 mM DTT
0.01% (v/v) Tween-20
10 μg/ml Pepstatin
10 μg/ml Aprotinin
40 mM Bis-Tris Propane, pH 7.0
1 mM PMSF
1 mM EDTA
10% (v/v) Glycerol
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| Mono Q Buffer |
| 40 mM Bis-Tris Propane, pH 7.0
1 mM DTT
0.01% (v/v) Tween-20
10% (v/v) Glycerol
1 mM EDTA
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| Lysis Buffer |
| Add the PMSF fresh before use from a stock solution in an anhydrous solvent (Ethanol) stored at -20°C.
10 μg/ml Leupeptin
0.1% (w/v) 2-Mercaptoethanol
1 mM EGTA
10 μg/ml Pepstatin
10 μg/ml Aprotinin
1 mM PMSF (CAUTION! see Hint #9)
1 mM EDTA
Make up this solution in PBS
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Glycerol
Lysozyme
Sodium Hydroxide
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Leupeptin
EDTA
PMSF
Yeast Extract
Tryptone
Chloramphenicol
Bis-Tris Propane
Nitrogen, Liquid
Ethanol
2-Mercaptoethanol
Potassium Chloride
Sodium Chloride
Aprotinin
DNase I
DTT
EGTA
IPTG
Ampicillin
Pepstatin
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1. For protein expression, transform the plasmid into BL21(DE3)pLysS E. coli cells.
2. Sometimes the expression of p50/dynamitin is induced already before the addition of IPTG, leading to very slow growth of the bacteria. However, it does not affect the high level of expression. You can also keep a glycerol stock of p50/dynamitin-overexpressing BL21 strain without any reduction in the expression level.
3. At this point, you can store the bacterial pellet at -70°C for several months.
4. Alternatively, you can lyse the bacteria using a French Press with comparable results.
5. Under these conditions, most of the p50/dynamitin is recovered in the precipitate while most of the bacterial proteins remain soluble.
6. This volume of buffer sufficiently reduces the salt concentration to allow binding of p50/dynamitin to the Mono Q resin.
7. More than 20 ml tends to overload the column and a substantial amount of protein is lost in the flowthrough.
8. The Mono Q peak fractions usually contain more than 10 mg/ml of p50/dynamitin, making further concentration of the protein unnecessary for most applications. If required, the protein can be further purified on a Superose 12 gel filtration column.
9. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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1. Wittmann T, Hyman T. Recombinant p50/dynamitin as a tool to examine the role of dynactin in intracellular processes. Methods Cell Biol 1999; 61:137-43
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