Editing TEV Purification (section)

=== Purification<br>  ===

Buffers:<br>Buffer A: 20 mM phosphate, pH 7.5, 500 mM NaCl and 0.3 mM &lt;span style="background-color: navy; color: white;" /&gt;beta-ME. <br>Buffer B: 20 mM phosphate, pH 7.5, 350 mM NaCl, 500 mM imidazole and 0.3 mM &lt;span style="background-color: navy; color: white;" /&gt;beta-ME.<br>Buffer C: 10 mM Tris, pH 7.5, 0.3 mM &lt;span style="background-color: navy; color: white;" /&gt;beta-ME. <br>Buffer D: 10 mM Tris, pH 7.5, 1 M NaCl and 0.3 mM beta-ME. <br> 

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1. Suspend cell paste in 50 mM phosphate, pH 7.5, containing 300 mM NaCl, 20% ethylene glycol, 1 mM EDTA and 0.3 mM beta-ME at a ratio of 6 mL of buffer per g of wet cell paste. (34 g cells + 200 ml buffer) 

2. Sonicate cell suspension for 6 min at 4 °C. 

3. Spin for 25 min at 95,000 × g. 

4. Load to 2 X 5 ml Ni HisTrap HP column pre-equilibrated with buffer A at 3 mL/min. 

5. Was columns with eight volumes of a mixture of 85% buffer A and 15% buffer B (7.5 mM imidazole) at 5 mL/min. 

6. Elute TEV with 100% buffer B at 0.7 mL/min. 

7. Eluate is loaded to the second purifier with two 5 mL '''SP Fast Flow''' columns arranged in series at 0.7 mL/min. 

The columns were equilibrated with buffer C. The sample from the first purifier was injected into the mixing chamber mixed with 100% buffer C at 10 mL/min and loaded onto the columns of purification system 2 at a total flow rate of 10.7 mL/min. The resultant ~15-fold dilution of the sample prior to application to the cation exchange columns ensured that the ionic strength was low enough allow tight binding of the protease to the column.Upon completion of the IMAC elution, the flow through purifier system 1 was increased to 5 mL/min and directed to waste for column wash and reequilibration with buffer A prior to the injection of the next aliquot of lysate. The waste sample was collected so that possible losses of TEV protease could be determined. 

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8. flow through purifier system 2 was decreased to 5 mL/min and a six column volume gradient from 100% buffer C to a mixture of 40% buffer C and 60% buffer D was started. 

Yield: ~400 mg per L of expression culture with &gt;99% purity<br><br>