Editing Purification of Benzonase, SM2 Nuclease (section)

== Wild-Type Secreted Benzonase  ==

<br> 

SM2 nuclease in periplasm can be released from spheroplast using a protocol of Randall and Hardy (1986)<br> 

1. Cells are harvested by centrifugation at 10,000 x g for 4 minutes. 

2. The pellet is resuspended in a volume equal to the cell pellet of 10 mM Tris hydrochloride (Tris-HCl, pH 8.0) buffer <br> 

3. The cell suspension is centrifuged again at the same settings. Discard the supernatant. 

4. The cell pellet is suspended in an equal volume of spheroplasting buffer, containing 20% sucrose, 30 mM Tris-HCI (pH 8.0) 

5. The cells are then converted to spheroplasts by incubation at 0°C for 30 min in the presence of 1/10 the cell pellet volume of 1mg/ml lysozyme freshly dissolved in 0.1 M EDTA (pH 7.5). 

6. Spheroplast and periplasm are separated by centrifugation at 10,000 x g for 10 min. 

7. The supernatant contains proteins that are secreted into the periplasm.&nbsp; <br> 

<br> 

Culture medium<br>1. Concentrate culture medium using ultrafiltration membrane with a cut off at a molecular weight of 10,000 (Millipore type PTGC0005). <br> 

LB medium: 10 g NaCl/L--&gt; 0.17 M

2. Add solid(NH4)2SO4 to cleared lysate to conc. of 80% (saturation)<br>3. Collect precipitate by spinning for 30 min at 14,000xg.<br>4. Dissolve pellets in l0 mM Tris-HCl pH 8.3.<br>5. Dialyse against the same buffer overnight.<br>6. Load to a <strike>DE-52 (Whatman) column (5 cmx5 cm),</strike>&nbsp; 30 ml of Q FF equilibrated with the same buffer.&nbsp;<br>7. Elute the adsorbed protein with the buffer containing a linear gradient of NaCl from 0 to 0.3 M. <br>8. Pool the nuclease active fractions.<br>9. Adjust to pH 5.6 with acetic acid.<br>10.Load to a <strike>CM-Sepharose CL-6B column (2.6 cmx l0 cm)</strike>&nbsp;15~ 20 ml of SP-FF equilibrated with 20 mM sodium-acetate pH 5.6. (benzonase's pI: 6.23)

11. Elute the adsorbed nuclease with the buffer containing a linear gradient of NaCl from 0 to 0.5 M. <br>12. Pool the active fractions.<br>13. Dialyze against distilled water<br>14. Lyophilize<br> 

<br> 

Alternatively: 

or P11 phosphate cellulose (cation exchange) 2.5 X 20 cm column, equilibrated with 50 mM NaAC, pH 5.2 buffer, wash with 200 ml the same buffer, elute with 500 ml linear gradient from pH 5.2 to pH 7.5, NaCl from 0 to 3 M, then dialyse pooled protein vs 10 mM Tris-HCl, pH8.2, 20 mM NaCl, load to DE52 (anion exchange, or QFF) column, Wash with 200 ml 10 mM Tris-HCl, pH 8.2, elute with 10 mM Tris-HCl, pH 8.2, 500 mM NaCl. 

DE52: can be replaced by QFF