Protein Purification

3.3.1. Cell Disruption

1. Sonicate four times the resuspended pellet in bursts of 30 s at maximum power using a microtip probe. The Falcon tube should be immersed on ice. Special care should be taken to avoid heating the preparation.

2. Centrifuge the lysate at 14,000g for 15 min. Keep the supernatant, containing the soluble T7 RNAP fraction of interest, on ice for subsequent treatment.

3.3.2. Protein Purification

1. Add 5 mL suspended iminodiacetic acid-Sepharose beads to a disposable 10-mL polypropylene column with a final bed volume of 2.5 mL.

2. Wash the column with 3 bed volumes of water.

3. Charge the column with 5 bed volumes of activation buffer (50 mM NiSO4).

4. Equilibrate the column with 3 bed volumes of loading buffer.

5. Load the supernatant bacterial preparation of T7 RNAP onto the column.

6. Wash the column with 20 mL loading buffer.

7. Elute the immobilized T7 RNAP by adding 15 mL elution buffer and collect 1-mL fractions.

3.3.3. Detection of T7 RNAP

(Coomassie Blue Staining and Western Blotting)

The quality of the purification procedure can be assessed by gel electrophoresis and immunoblotting as shown in Fig. 2. The purity and amount of T7 RNAP are analyzed as described next:

1. Load 3 ||L of each eluted fraction on a 5-15% gradient SDS-PAGE.

2. Stain the gel with Coomassie blue staining solution (Bio-Rad).

3. Destain the gel with destaining solution and check for a protein band with a molecular weight of 98 kDa, corresponding to purified T7 RNAP. Determine the most concentrated elution fractions and analyze them by Western blotting. In our experience, the two most concentrated fractions correspond to elution fractions 3 and 4.

4. For immunodetection, first transfer the proteins from the gel to a nitrocellulose membrane (Hybond ECL). Perform the transfer during 1 h 30 min at 400 mA in transfer buffer.

5. Block the membrane overnight at 4°C in blocking solution.

6. Incubate the membrane for 30 min at 37°C with an anti-T7 monoclonal antibody (from Novagen) previously diluted 5000-fold in the same blocking solution.

7. After four 10-min washing steps in TBS, incubate the membrane for 30 min with the secondary antibody conjugate (anti-mouse immunoglobulin G coupled to peroxidase from Zymed diluted 1/5000).

8. Wash the membrane again four times with TBS.

9. Reveal the peroxidase activity by a 1-min incubation of the membrane with the chemilu-minescent reagent (Perkin-Elmer) and its exposure to a Hyperfilm ECL film (Amersham Biosciences). Figure 2 shows a representative example of immunodetection of purified T7 RNAP.

3.3.4. Dialysis of Purest and Most Concentrated Fractions

1. According to a visual examination of SDS-PAGE analysis, pool together the purest and most concentrated elution fractions for dialysis and buffer exchange (up to 4-6 mL in volume).

2. Load the pooled eluted T7 RNAP fractions into a dialysis tube (Spectra/Por membrane, Biovalley) with a molecular weight cutoff of 10,000 Da.

3. Dialyze the fractions overnight at 4°C against 1 L TBS. Repeat the operation once for 2 h by replacing the external TBS solution.

4. Recover the dialyzed sample, which is ready for protein dosage.

3.3.5. Yield Analysis

Determine the protein concentration of the dialyzed T7 RNAP sample using a Bradford assay following manufacturer's instructions (Bio-Rad commercial reagents). Expect total T7 RNAP quantities to be in the range of 20-40 mg.

3.3.6. Aliquoting and Storage

Purified T7 RNAP is stable for 2 wk at 4°C and can be used as such without further aliquoting. For long-term storage, it is recommended to adjust the concentration of the sample to 10 mg/mL with TBS and by adding 40% glycerol. Small-volume aliquots of the sample (10-100 |L) should be produced and kept at -20°C.

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